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Dissection of the interaction between FLT3 ligand and the extracellular domains of its receptor
Mariska JANUAR
Master’s dissertation submitted to obtain the degree of Master of Biochemistry and Biotechnology Major Biochemistry and Structural biology
Academic year 2009-2010
Promoter(s): Prof. Dr. Savvas Savvides Scientific supervisor: Drs. Kenneth Verstraete Department of Biochemistry and Microbiology Laboratory for Protein Biochemistry & Biomolecular Engineering
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Acknowlegdment Many years ago my father said to me, “Now you have the chance to study abroad and to see the other side of the world, things I couldn’t do when I was young. So go, don’t let the wind breaks your wings”. Here I am today with wings full of scars, writing the last page of this master thesis. I want to express my gratitude to these persons without whom this journey would have been impossible. I thank Prof. Savvides who opened the door of structural biology for me and allowed me to do my master thesis in his lab. If Experimental Structural Biology course was given by someone else, then I wouldn’t have wrote this thesis. Without him, structural biology wouldn’t be as interesting and as fun. I especially want to thank Kenneth, the faithful supervisor, for his guidance during the progress of my research and for having faith in me more than I have in myself. His passion and enthusiasm in research are surely contagious and got into me. And also for revising this thesis carefully. This book wouldn’t be as it is without him. These people also deserve my gratitude, Bjorn for his assistance in ITC and SPR experiments, Jonathan, Nathalie, Ester, Géraldine, Ann, Kedar, Ruben for the practical advices in the lab. Ellen, Nienke and Tien for sharing all the up and down as master thesis students on the 5th
floor Ledeganck, for the lunches and the companion during the “night shifts”.
I sincerely thank my brother Reynard and cousins Edward and Evelien for their silly jokes that make me forgot how tired I was when I got home and for always reminding me there’s a life outside the lab. My aunt and uncle Eveline and Luc deserve my utmost gratitude for their love and support. Without them this road less travelled wouldn’t have been possible. Surely I must thank my parents Marcus and Lanny for giving me this life, raised me and let me live my life in my own way. Their unconditional love kept me warm in the winter and their faith kept me going. More than anything, for providing me a home full of warmth and laughter where I can go to recharge my battery. -And in the end, the love we take is equal to the love we make-. The Beatles.
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List of abbreviations Abbreviations
(NH4)2SO ammonium sulfate 4 ∆H enthalpy ∆S entropy A Alanine AML Acute Myeloid Leukemia BB Binding Buffer BM Bone Marrow bp basepairs C lobe C-terminal CHO Chinese Hamster Ovary cell CIP Capture, Intermediate purification and Polishing CMV Cytomegalovirus CoCl cobalt chloride 2 CSF Colony Stimulating Factor CV Column Volume DC Dendritic Cell DMEM Dulbecco's Modified Eagle's Medium E Glutamine acid EB Elution Buffer EGF Epidermal Growth Factor endo H endoglycosidase H ER Endoplasmic Reticulum Fc Flow channel FCS Fetal Calf Serum FGF Fibroblast Growth Factor FLK-2 Fetal Liver Kinase 2 FLT3 FMS-like receptor kinase 3 FLT3 ligand FMS-like tyrosine kinase 3 ligand G Glycine GM-CSF Granulocyte-macrophage colony stimulating factor GnTI N-Acetylglucosamine transferase I GuHCl Guanidium Hydrochloride H Histidine H8R Histidine 8 Arginine H8Y Histidine 8 Tyrosine HEK Human Embryonic Kidney cell HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid His Hexahistidine 6 HSC Haematopoietic Stem Cell IB Inclusion Bodies IFN Interferon
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IgG Immunoglobuline G Ig-like Immunoglobuline-like IL-3 Interleukin 3 IL-7 Interleukin 7 ILN Interleukin IMAC Immobilization Metal Affinity Chromatography IPTG isopropyl β-D-1-thiogalactopyranoside ITC Isothermal Titration Calorimetry ITD Internal Tandem Duplication JM Juxtamembrane JM-B JM binding motif JM-S JM switch motif JM-Z JM zipper linker segment K Lysine KA affinity constant kb kilobases KD dissociation constant kDa kilodalton KL Kit Ligand L Leucine MCS Multi Cloning Site M-CSF Macrophage-colony stimulating factor MES 2-(N-morpholino)ethanesulfonic acid MI α-mannosidase I MOPS 3-(N-morpholino)propabesulfonic acid N reaction stoeichiometry N lobe N-terminal NaCl sodium Chloride NaPO sodium phosphate 4 N-glycosylation Asparagine-linked glycosylation NK Natural Killer Cell ORF Open Reading Frame P Proline PBS Phosphate-saline buffer PCR Polymerase Chain Reaction PDGF Platelet-Derived Growth Factor PDGFR Platelet-Derived Growth Factor Receptor PEI Polyethyleneimine poly-A polyadenylation Q Glutamine R Arginine rhFL recombinant human FLT3 ligand rhFL3 recombinant human FLT3 ligand without His6
rhFL-AVI-B tag
recombinant human FLT3 ligand with AVI tag and biotinylated rhFL-AVI-B-His recombinant human FLT3 ligand, biotinylated, with AVI and His6 6
rhFL-His tag
6 SeMet incorporated recombinant human FLT3 ligand -SeMet
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rhFL-SeMet SeMet incorporated recombinant human FLT3 ligand without His6
rhFLT3d1 tag
recombinant human FLT4 domain 1 rhFLT3d12 recombinant human FLT4 domain 1 to 2 rhFLT3d123 recombinant human FLT4 domain 1 to 3 rhFLT3d1234 recombinant human FLT4 domain 1 to 4 rhFLT3d12345 recombinant human FLT4 domain 1 to 5 rhFLT3d1234-SeMet SeMet incorporated recombinant human FLT3 receptor domain 1 to 4 RTK Receptor Tyrosine Kinase RU Respons Units S Serine SD Superdex SeMet Selenium-Methionine SPR Surface Plasmon Resonance SV40 Simian vaculoting Virus 40 T Threonine Tet Tetracycline TGF Transforming Growth Factor TIR Total internal reflection TNF Tumor Necrosis Factor V Valine VEGF Vascular Endothelial Growth Factor W Tryptophane Y Tyrosine αC α-helix
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Abstract
Three-dimensional structural information obtained from crystallographic studies has already been used to understand molecular mechanism of receptor activated signal transductions. Nowadays it is increasingly being used to design clinically useful cytokine antagonists and agonists. However, only a handful of publications shed the light on the interactions between class III RTK cytokine receptors and their ligands, indicating that there is still much more to be done to gather these important knowledge.
The interaction of human FLT3 (hFLT3) receptor and human FLT3 ligand (hFL) generates a lot of interest and many questions about how the ligand binds and how ligand binding leads to receptor activation remain to be answered. FLT3 ligand is a cytokine that is capable to stimulate the proliferation of hematopoietic progenitor cells of both lymphoid and myeloid origin by interactions with its receptor. The threedimensional structure of the FLT3 receptor and the architecture of the FLT3 ligand-receptor complex are still unknown today.
This master thesis was conducted in a framework that aim to elucidate the architecture of the FLT3 complex by crystallography and characterize the contribution of the various Ig-like domains of the receptor by methods as analytical gel filtration, SRP and ITC.
In this work, we found out that rhFLT3d1 and rhFLT3d12 can be produced as secreted proteins by an inducible stable HEK293 GnT-/-
ITC and SPR were performed to study the interactions of hFL and various constructs of it receptor. The results led us to the conclusion that domain 5 stabilizes the complex possibly by mediating homotypic interactions, and that domain 4 does not form such contacts in contradiction with the other class III RTK members.
cell line. We also observed that neither rhFLT3d1 and rhFLT3d12 shows a high affinity complex with the ligand meaning that domain 3 is necessary to for the binding site, which was later confirmed by X-ray studies (personal communication with K. Verstraete). We also aimed to crystallize domain 1 since a high resolution of domain 1 could be very helpful in determining the structure of the complex rhFL3d1234-FL (available data set of 4.2 Å).
In addition, receptor and ligand recombinant proteins were labeled with SeMet. The labeled receptor-ligand complex can be used to solve the Phase problem in the structure determination using SAD or MAD.
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Samenvatting Driedimensionale structuur informatie verkregen uit kristallografische studies werd reeds gebruikt voor het begrijpen van de moleculaire mechanisme van de receptor geactiveerde signaaltransductiecascade. Tegenwoordig wordt deze meer en meer aangewend voor het design van klinisch bruikbare cytokine antagonisten en agonisten design. Slechts een handvol publicaties werpen licht op de interactie tussen klasse III RTK cytokine receptoren en hun liganden, wat aangeeft dat er nog heel wat moet gebeuren om belangrijke kennis te vergaren.
De interactie van de humane FLT3 (hFLT3) receptor en de human FLT3 ligand wekt heel wat belangstelling op. FLT3 ligand is een cytokine dat in staat is om de proliferatie van hematopoëtische voorlopercellen van zowel lymfoïde en myeloïde oorsprong te stimuleren door zijn interactie met de receptor. De driedimensionele structuur van FLT3 receptor en de architectuur van het FLT3 ligand-receptor complex zijn steeds onbekend tot vandaag.
Dit masterthesis werd uitgevoerd in het kader van de determinatie van de architectuur van het FLT3 complex door X-stralen crystallografie, naast de karakterisering van de contributie van de verschillende Ig-like domeinen van de receptor door het gebruik maken van SPR en ITC.
In dit werk stellen we vast dat rhFLT3d1 en rhFLT3d12 kunnen geproduceerd worden als gesecreteerde eiwitten door gebruik maken van stabiele HEK293 GnT-/- cellijn. We stellen ook vast dat rhFLT3d1 en rhFLT3d12 geen hoog affiniteit complex vertonen met het ligand, wat betekent dat domein 3 noodzakelijk is voor de bindingplaats. Dit wordt bevestigd door X-straalstructuur van het complex waaruit blijkt dat domein 3 de belangrijkste (mogelijks de enige) site voor interactie met het ligand is (persoonlijke communicatie met K. Verstraete). We trachtten om rhFLT3d1 te kristallizeren aangezien dat een hoge resolutie van domein 1 zeer belangrijk kan zijn in de structuurdeterminatie van het rhFLT3d1234-FL complex tot 4.2 Å.
ITC en SPR werden uitgevoed om de interacties van het ligand met verschillende constructen van zijn receptor te bestuderen. We namen het besluit dat domein 5 van FLT3 receptor het complex stabiliseert, vermoedelijk door homotypishe interacties en dat domein 4 geen interacties vertonen met het ligand wat een contradictie is met andere klasse III RTKs.
Daarnaast werden recombinante receptor en ligand gelabeld met SeMet. Het receptor-ligand complex kan worden gebruikt om het Phase probleem op te lossen in de structuur bepaling met SAD of MAD.
<Table of Contents
Table of Contents
Literature study ........................................................................................................................ 1
Chapter 1 Haematopoiesis ...................................................................................................... 2
1.1 Introduction ................................................................................................................. 2
1.2. Cytokines ..................................................................................................................... 3
Chapter 2 FMS-like tyrosine kinase 3 ligand ......................................................................... 5
2.1. The four-α-helical bundle ............................................................................................ 5
2.2. FLT3 ligand and its role in hematopoiesis .................................................................. 7
2.3. Discovery of FLT3 ligand ........................................................................................... 7
2.4. FLT3 ligand structure .................................................................................................. 9
Chapter 3 FMS-like tyosine kinase 3 receptor ..................................................................... 14
3.1. Receptor Tyrosine Kinase ......................................................................................... 14
3.2. Discovery of FLT3 receptor ...................................................................................... 17
3.2.1. Murine FLT3 receptor (or Fetal Liver Kinase 2) ............................................... 17
3.2.2. Human FLT3 receptor ........................................................................................ 20
3.3. Expression of FLT3 receptor ..................................................................................... 22
3.4. FLT3 receptor expression in human leukemia .......................................................... 22
3.4.1. Internal tandem duplication (ITD) ..................................................................... 22
3.4.2. JM domain and its autoinibitory mechanism ..................................................... 23
3.4.3. Activation loop and Asparagine 835 mutation ................................................... 26
Objectives of the study ........................................................................................................... 27
Chapter 4 Objectives of the study .......................................................................................... 28
Results and discussions .......................................................................................................... 29
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Chapter 5 Overexpression of recombinant human FLT3 receptor construct in a human cell line, their purification and binding studies ..................................................................... 30
5.1. Introduction ............................................................................................................... 30
5.2. Expression of recombinant human FLT3 receptor domain 1 and domain 1 to 2 in eukaryotic expression system .................................................................................... 32
5.2.1 Cloning of the extracellular domains of rhFLT3 receptor ................................. 32
5.2.2. Expression test of human FLT3 receptor domain 1 and domain 1 to 2 in HEK293T cells ................................................................................................... 33
5.2.2. Large scale production of recombinant human FLT3 receptor domain 1 in HEK293S cells and purifications ....................................................................... 34
5.2.3. Large scale production of recombinant human FLT3 receptor domain 1 to 2 in HEK293T cells and purifications ....................................................................... 36
5.3. Complex formation study of human recombinant FLT3 ligand and human recombinant FLT3 receptor domain 1 ....................................................................... 38
5.4. Complex formation study of human recombinant FLT3 ligand and human recombinant FLT3 receptor domain 1 to 2 ................................................................ 39
5.5. Conclusion ................................................................................................................. 40
Chapter 6 Crystallization of recombinant human FLT3 receptor domain 1 ...................... 41
6.1. Introduction ............................................................................................................... 41
6.2. Recombinant human FLT3d1 crystallization ............................................................ 42
6.3. Crystal tests ................................................................................................................ 43
6.4. Recombinant human FLT3 receptor domain 1 crystals diffraction ........................... 44
6.5. Conclusion ................................................................................................................. 45
Chapter 7 Overexpression of recombinant human FLT3 ligand construct in E. coli, its refolding and purification ....................................................................................................... 46
7.1. Introduction ............................................................................................................... 46
7.2. Overexpression of recombinant human FLT3 ligand, its refolding and purification 47
7.3. Conclusion ................................................................................................................. 49
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Chapter 8 Interaction studies of recombinant human FLT3 ligand with recombinant human FLT3 domain 1 to 3, domain 1 to 4, domain 1 to 5 ................................................... 50
8.1. Isothermal Titration Calorimetry ............................................................................... 50
8.2. Surface Plasmon Resonance ...................................................................................... 53
8.3. Conclusion ................................................................................................................. 57
Chapter 9 Selenium-Methionine incorporated recombinant human FLT3 receptor and recombinant human FLT3 ligand .......................................................................................... 58
9.1. Introduction ............................................................................................................... 58
9.2. Overexpression of SeMet incorporated recombinant human FLT3 ligand and its purification ................................................................................................................. 58
9.3. Overexpression of SeMet incorporated recombinant human FLT3 domain 1 to 4 and its purification ............................................................................................................ 59
9.4. Complex formation of SeMet incorporated recombinant human FLT3d1234 and SeMet incorporated recombinant human FL and its purification. ............................. 61
9.5. Conclusion ................................................................................................................. 62
Materials and methods ........................................................................................................... 63
Chapter 10 Cloning of recombinant human FLT3 domain 1 and domain 1 to 2, small and large scale expression, purification, crystallization trials and complex formation study .... 64
10.1. Cloning of recombinant human FLT3 domain 1 and domain 1 to 2 ..................... 64
10.1.1. Cloning to pCR2.1-TOPO vector ....................................................................... 64
10.1.2. Cloning to pcDNA4-TO vector .......................................................................... 66
10.2. Small scale expression of recombinant human FLT3 domain 1 and domain 1 to 2 .. 67
10.3. Large scale expression and purification of recombinant human FLT3 domain 1 and domain 1 to 2 ............................................................................................................. 68
10.4. Crystallization (trials) of recombinant human FLT3 domain 1 ................................. 70
10.5. Complex formation studies ........................................................................................ 71
Chapter 11 Recombinant human FLT3 ligand refolding and purification ........................ 72
11.1. Expression of recombinant human FLT3 ligand in E. coli .................................... 72
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11.2. Isolation of rhFL inclusion bodies and its purifications under denaturing conditions . ……………………………………………………………………………72
11.3. In vitro refolding of rhFL and its purification ....................................................... 73
Chapter 12 Interaction studies of recombinant human FLT3 ligand with recombinant human receptors ...................................................................................................................... 74
12.1. Isothermal Titration Columetry ............................................................................. 74
12.1.1. Introduction ...................................................................................................... 74
12.1.2. Sample preparations and interaction studies ...................................................... 75
12.2. Surface Plasmon Resonance .................................................................................. 76
12.2.1. Introduction ........................................................................................................ 76
12.2.2. Immobilization of rhFL and interaction studies ................................................. 77
Chapter 13 Large scale expression of Selenium-Methionine incorporated recombinant human FLT3 receptor and recombinant human FLT3 ligand and purification ................. 78
13.1. Large scale expression of Selenium-Methionine incorporated recombinant human FLT3 receptors ....................................................................................................... 78
13.2 . Large scale expression of Selenium-Methionine incorporated recombinant human FLT3 ligand ............................................................................................................ 78
13.3. Complex formation ................................................................................................ 79
General conclusion ................................................................................................................. 80
List of reference ...................................................................................................................... 82
Samenvatting .......................................................................................................................... 86
Addendum ............................................................................................................................... 91
<Table of Contents
Literature studies
1
Section I
Literature studies
Chapter 1
2
Chapter 1
Haematopoiesis
1.1 Introduction
Haematopoiesis is the proces whereby the cellular blood components are being formed. All the cellular components of blood, such as the red blood cells that transport oxygen, the platelets that induce blood clotting in damaged tissues and including the white blood cells of the immune system are derived from haematopoietic stem cells (HSC) that reside in the bone marrow (BM).
Hematopoiesis is a hierarchical process whereby starting from a limited pool of HSC, all mature blood cell types are produced. Based on their developmental pathway, two major lineages can be discerned. The lymphoid lineage consist of T lymphocytes, B lymphocytes and natural killer (NK) cells while the myeloid lineage includes granulocytes, monocytes, macrophages, erythrocytes, megakaryocytes and mast cells. Interestingly, the dendritic cells (DCs) can develop from either the myeloid or the lymphoid pathways. The hierarchy is given in Figure 1.1.
Chapter 1
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Figure 1.1. Diagram of hematopoietic developmental hierarchy. Self-renewing HCSs are at the top of the hierarchy, giving rise to a number of multipotent progenitors. In turn, multipotent progenitors give rise to oligopotent progenitors Common Lymphoid Progenitors (CLP) and Common Myeloid Progenitors (CMP). CLP gives rise to mature B lymphocytes, T lymphocytes and natural killer (NK) cells. CMP gives rise to gives rise to granulocyte-macrophage progenitors (GMP), which differentiate into monocytes/macrophages and granulocytes, and megakaryocyte/erythrocyte progenitors (MEP). MEP differentiates into megakaryocytes/platelets and erythrocytes. Both CMPs and CLPs give rise to dendritic cells. Figure is adapted for Bryder et al., 2006.
Haematopoeitic growth factors, also known as cytokines, play a crucial role in the HSC development by synergy or activation of their cognate receptors.
1.2. Cytokines
Cytokine is a term used for a large group of molecules, proteins or glycoproteins, that act as a chemical messenger important in intercellular communication. These molecules regulate the differentiation, proliferation, activation and death of many cell types. Cytokines are particularly involved in the regulation of the circulatory system and production of immunity and inflammatory responses. They are divided into different categories based on their functions, namely Interferons (IFNs), Interleukins (ILs), Colony stimulating factors (CSFs), Chemokines, Tumor necrosis factors (TNFα and TNFβ), Transforming growth factor-β (TGFβ), etc.
IFNs are very important in limiting the spread of certain viral infection and they induce a state of antiviral resistance in uninfected cells. They are produced early in infection and are important in delaying the spread of a virus until the adaptive immune response has developed.
Chapter 2
4
ILs are mainly produced by T cells although some are also produced by mononuclear phagocytes or by tissue cells. They have different kinds of functions, many interleukins cause other cells to divide and differentiate.
CSFs are primarily involved in directing the division and differentiation of bone marrow stem cells, and the precursor of blood leukocyte. The balance of different CSFs is partially responsible for the proportion of different immune cells produced. Some CSFs promote further differentiation of cells outside the bone marrow. FMS-like tyrosine kinase 3 ligand (FLT3 ligand) cytokine is widely mentioned in literatures. Apparently, FLT3 ligand is a growth factor which is capable to stimulate the proliferation of haematopoietic progenitor cells of both lymphoid and myeloid origin (Lyman et al., 1993; Hannum et al., 1994; McKenna et al., 2000). FLT3 ligand is also an important growth factor for dendritic cells (DCs) and Natural Killer cells (NKCs) homeostasis in vivo (McKenna et al., 2000).
Chemokines direct the movement of leukocytes around the body, from the blood stream into the tissues and to the correct place within each tissue. TNFα, TNFβ and TGFβ have a variety of functions, however they are particularly important in mediating inflammation and cytotoxic reactions.
Chapter 2
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Chapter 2
FMS-Like Tyrosine Kinase 3 Ligand
2.1. The four-α-helical bundle
The most simple and most frequent α-helical domain is known as the four-α-helix bundle. It consists of four α-helices arranged in a bundle with the helical axes parallel or anti-parallel to each other. The side chains of each helix in the four-helix bundle are arranged such that hydrophobic side chains are buried between the helices and hydrophilic side chains are found on the outer surface of the bundle. This kind of arrangement creates a hydrophobic core in the middle of the bundle along its length, where the side chains are tightly packed.
The helix-bundles are categorized into two general types, based on the connectivity that exists between helical segments. The first type is a plain or adjacent connection, where the C terminus of one helix is adjacent in space to the N terminus of the next helix, but the direction vectors changes orientation by 180° within the connecting loop of polypeptide backbone (Figure 2.1.1) (Presnell and Cohen, 1989). The second type, also known as “overhand” connection, the chain passes back over the length of the first helix to enter the second helix with approximately the same directional vector as the entry to the first helix (Figure 2.1.1) (Presnell and Cohen, 1989).
Figure 2.1.1. Side view of two left-handed bundles. On the left side is the first type helix bundle where overhand connection is absence. On the right side is the second type helix bundle where there is one overhand connection. Figure is taken from Presnell and Cohen 1989.
Chapter 2
6
In some cases, proteins have convincing structural, functional and genetic evidence of a common evolutionary origin but they don’t show significant sequence similarities. This is possible because they have evolved to a point where there is no significant sequence similarity, even though they share common origin, except for those residues needed to maintain the hydrophobic core and hydrophilic exteriors. Another reason could be because there is sequence conservation in regions that are important for function, but it is hidden by the regions where large sequence changes has occurred.
The four-helical cytokines show good evidence for their evolutionary relationship because in addition to unique structural similarities, they also perform similar functions, namely bind a subset of homologous receptors and they have a similar exon-intron exon-intron structure (Bazan, 1990; Betts et al., 2001). But in most of the cases, their sequences cannot be matched by sequence comparison methods.
The orientation of the four-helical bundle cytokines is different from the conventional four-helical up-and-down bundle, instead they have an up-up-down-down topology with long overhand connections between helices A-B and C-D (fig a) (Hill et al., 2002). The four-helical bundle cytokine superfamily is sub-classified in SCOP (Murzin et al., 1995) into three families, the long and short chain cytokines and the interferons/interleukin 10 family. The overall topology of the four-helical cytokines is conserved between the families but there are some differences in secondary structure, chain length and topological details (Hill et al., 2002).
The long chain cytokines are on average 180 amino acids long and their helices are between 20 and 30 amino acids long (Figure 2.1.2). On the other hand, the short chain cytokines are on average 140 amino acid long with helices between 10 and 20 amino acids long (Figure 2.1.2).
Figure 2.1.2. Cartoon (a) and ribbon (b), (c) diagram of four-helical cytokines. (b) is the long-chain cytokine, (c) is the short-chain cytokine. Red, helix A; yellow, helix B; green, helix C; blue, helix D. Figure is taken from Hill, et al 2002.
Chapter 2
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2.2. FLT3 ligand and its role in hematopoiesis
In early studies it was found that FLT3 ligand works in synergy with interleukin 3 (IL-3) and with macrophage colony-stimulating factor (M-CSF) to enhance the response of stem and primitive progenitor cells to generate all myeloid lineages but the erythroid cells (Lyman et al., 1993a). In addition to this, FLT3 ligand in combination with interleukin 7 (IL-7) induces proliferation of T-cell precursors which indicates a role for FLT3 ligand in T lymphopoiesis (Hannum et al., 1994).
In the more recent studies, FLT3 ligand was shown to be a crucial multipotent cytokine in the development of hematopoietic progenitors. Mice that are deficient for the gene encoding the FLT3 ligand have reduced numbers of myeloid-related and lymphoid-related dendritic cells (DCs) in the spleens, lymph node and thymus which indicates the important role of FLT3 ligand for the production of DCs (McKenna et al., 2000). This finding is in agreement with an in vitro study that showed administration of FLT3 ligand to a combination of cytokines can increase the generation and expansion of myeloid-related (Strobl et al., 1997; Rosenzwajg et al., 1998) and lymphoid-related DC (Saunders et al., 1996). In addition to this, FLT3 ligand is remarkably important in the generation of natural killer (NK) cells (McKenna et al., 2000) and plays an important role in synergy with IL-11 (Ray et al., 1996) and IL-7 (Veiby et al., 1996; Ray et al., 1996) in the generation of B-cell progenitors in the bone marrow (BM) (Hunte et al., 1996; McKenna et al., 2000). However, there is no expression of FLT3 receptor nor respond to FLT3 ligand observed in mature NK cells in vivo (Yu et al., 1998). The role of FLT3 ligand for NK cells homeostasis is set by the observation that it can induce the NK cell expansion in vivo only if it is mediated by DC population expressing IL-15 (Guimond et al.).
2.3. Discovery of FLT3 ligand
Lyman, James and co-workers succeeded to clone mouse cDNA that encodes the FLT3/FLK-2 ligand. In order to do this, they designed a fusion protein that contains the extracellular domain of FLT3 receptor and the Fc portion of human immunoglobuline G (IgG). This fusion protein was used as a probe to screen many mouse and human cell lines for their binding capacity. The occurring binding might indicate binding of extracellular region of FLT3 to its ligand. The binding was confirmed using 125
The open reading frame (ORF) of FLT3 ligand spans over 696 basepairs (bp) where 31 bp of non-coding sequence is at 5´ and 102 bp at 3´. In the 3´ noncoding sequence, apolyadenylation (poly-A) addition signal, mRNA instability motifs (ATTTA) and a polyadenylation tail are absence. The ORF encodes a 231 amino acids long type I transmembrane protein. This protein contains a 27 amino acid N-terminal signal peptide that is followed by a 161 amino acid extracellular domain, a 22 amino acid transmembrane domain and a 21 amino acid cytoplasmic domain. In the extracellular region, there are two
I-labeled mouse anti human IgG antibody againts Fc portion of IgG which bind to the membrane-bound FLT3 ligand.
Chapter 2
8
potential asparagines-linked glycosylation (N-glycosylation) sites. Without the signal sequence, the predicted molecular weight is 23164 daltons and the estimated pI is 8.17.
This group suggested FLT3 ligand to be similar to Kit ligand (KL) and macrophage colony-stimulating factor (M-CSF) because of sequence similarity and all three are type I transmembrane proteins. In addition, the conserved cysteine residues between KL and M-CSF are likewise conserved in the FLT3 ligand, their receptors and functions are similar. Putting all these together indicates the overall structure similarity of these proteins and suggest that FLT3 ligand is also a four helix bundle protein like M-CSF (Pandit et al., 1992).
Alternative splicing of transmembrane bound FLT3 ligand gives rise to soluble form of the ligand. The recombinant soluble form of FLT3 ligand is shown to be likewise active and binds the FLT3-Fc fusion protein. The membrane bound as well as the soluble form of FLT3 ligand are suggested to play an important role in the proliferation of primitive hematopoietic cells (Lyman et al., 1993a).
Another group that identified FLT3 ligand in mouse and also in human is the group of Hannum et al. in 1994. They transfected pro-B cell line with a cDNA clone that coded for the mouse FLT3 receptor. The stable transformants were used as target cells in a proliferation assays with conditioned medium from different mouse stromal lines. In the thymic stromal cell line TA4 they found that FLT3 ligand activity posses the characteristics expected for the ligand, namely specificity for cells that express the receptor, because only in the transformants stimulatory activity was found, and the ability to induce receptor phosphorylation.
Further, they used an affinity column made with the mouse FLT3 receptor extracellular domain to purify FLT3 ligand from the TA4 conditioned medium. The result of the purification showed a large band of 30 kDa and the elution pattern corresponded with the peak of biological activity. N-terminal sequencing of the purified protein resulted in short amino acid sequence, which was then used to design degenerate oligonucletotide primers to amplify an FLT3 ligand cDNA of sequence corresponding to the peptide sequence by polymerase chain reaction (PCR). Besides this finding, a longer PCR product was also isolated by using primers that were derived from a non-contiguous downstream peptide.
These cDNAs were used as probes to screen TA4 cDNA libraries by hybridization. Their screening method enabled to isolate two TA4 clones, namely T118 and T110. The first 163 codons of both clones ORF are identical but then the sequences deviate whereby two different C terminals are encoded. The T110 polypeptide contains a potential 22 hydrophobic amino acids transmembrane segment that is followed by a cytoplasmic tail while the T118 polypeptide, although it is shorter, it is hydrophobic. So even though the polypeptides of T118 and T110 have different length, both are membrane associated.
The group of Hannum et al. 1994 also isolated the human FLT3 ligand cDNAs by hybridization with the mouse cDNA probes. Here they also found two clones that had identical ORF for the first 160 codons and then the sequence deviate to encode different C-terminal. Similar to the mouse T110, clone S86 of human FLT3 ligand polypeptide has a
Chapter 2
9
transmembrane segment. On the other hand, clone S109 is rather different, the polypeptide is 245 amino acid long and contains a distinct, mostly hydrophilic amino acids at the C-terminal. This is likely to be a soluble form of FLT3 ligand in human stromal cell line.
By comparative sequencing, they identified that the identical part of the FLT3 ligand variants is structurally related to KL and to M-CSF which are already known to adopt the α-helical cytokine fold and have a disulphide bridge network (Figure 2.3.1) (Bazan, 1991; Pandit et al., 1992; Bazan, 1993). The mouse and human FLT3 ligand display a broad expression pattern in adult and fetal tissues, being the highest in spleen and lung (Hannum et al., 1994).
Figure 2.3.1. Picture of FLT3 ligand predicted structure and chain variants. C1-C3, C2-C4, C5-C6 are the predicted KL/M-CSF-like disulphide bridge. The wavy FL tails belongs to the mouse T118 clone. Cilinder, α-helices; arrow, β-sheets; diamonds, potential asparagines-linked glycosylation; SP, signal peptide. Figure is taken from Hannum, et al. 1994.
It is interesting that FLT3 ligand was discovered by two different research groups within a short time span. Although they performed different methods, both used FLT3 receptor to isolate FLT3 ligand.
2.4. FLT3 ligand structure
As already mentioned above, FLT3 ligand is biologically active as a transmembrane and as well as soluble form, and it is predicted to be a helical cytokine that is homologue to KL and M-CSF (figure). Interesting about FLT3 ligand, KL, and M-CSF is that they bind to class III RTKs from the platelet-derived growth factor receptor (PDGFR) subfamily, while this receptor family usually binds the cysteine-knot growth factor such as PDGF and vascular endothelial growth factor (VEGF) (van der Geer et al., 1994).
Structure-activity study was performed by Graddis and co-workers where they screened randomly mutagenized FLT3 ligand using a FLT3 receptor-Fc fusion protein. The fusion protein was used to probe the relative binding activities of mutated ligand. FLT3 ligand is predicted to be a homodimer, where L27P as well as A64T mutations shift the monomer-dimer equilibrium to monomer and reduce the biological activity (Graddis et al., 1998). Graddis and co-workers elucidated 31 amino acid substitutions at 24 sites that alter the biological activity of FLT3 ligand. There are three hot spots of high amino acid substitution
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frequency which are clustered in a small surface patch of the tertiary structure, namely at position 8-15, 81-87, and 116-124 (Figure 2.4.1). Substitution at position 8, 84, 118, and 122 improve the binding and biological activity. However, the H8R instead of H8Y substitution led to a diminished FLT3 ligand activity.
Figure 2.4.1. Stereo diagram of an α-carbon ribbon trace of FLT3 ligand. One subunit is shown in gray and the other subunit is shown in gold. Note that at the lower subunit A and D α-helices (front helices) are in yellow, B and C α-helices (back helices) are in gold. The specific residues are shown in balls. Yellow, cysteine residues; red, residues where mutation caused activity reduction; blue, residues where mutation caused activity increase. Figure is taken from Graddis, Brassel et al. 1998.
The structure of FLT3 ligand is fully elucidated by the group of Savvides et al. in 2000 using multiple isomorphous replacement (MIR) at 3.2 Å resolution which then refined at 2.2 Å resolution. They confirmed that FLT3 ligand is indeed a homodimer subunits that adopt the short chain helical cytokine fold (Figure 2.4.2).
The FLT3 ligand has a 310-helix fold that is continuous with αC, and a proline residues triplet (P89, P90, P91) in the middle of the loop connecting the 310-helix to β2. There are three intramolecular disulfide bridges found in FLT3 ligand where two of them (C4-C85 and C44-C127) that are required for bioactivity are buried, and one is solvent accessible. This finding indicates that two of these disulfide bridges are not involved in receptor recognition but they certainly have a structural role.
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Figure 2.4.2. The homodimer transmembrane form of FLT3 ligand structure, oriented with respect to the cell surface based on the position of C-terminal. One monomer is represented in green and the other monomer is represented in red. Figure is taken from Savvides, Boone et al. 2000.
Based on correlation of the structure and the mutational studies carried out by Graddis and co-workers, Savvas and team identified were able to identify nine surface residues where mutation of these alters ligand bioactivity (Figure 2.4.3). The nine residues are clustered around the poles of FLT3 ligand dimer ~45Ų apart and on a surface that is hypothesized to face away from the cell membrane. Each cluster contains three segments of the FLT3 ligand scaffold, namely the flexible N-terminal loop prior to αA (H8, S9 and S13), the C-terminal of αC (K84), and the C-terminal of αD (K116, W118, Q122). It was suggested that the identified surfaces, which is accounts for ~12.000 Ų surface area, on each FLT3 ligand monomer cover for the receptor binding epitope.
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Figure 2.4.3. Molecular mapping of solvent accessible residues of Flt3L that are important for bioactivity as reported by Graddis en co-workers. Shown in red are sites that when mutated reduced or otherwise increased (shown in blue) the bioactivity: H8R 0 %, S9G 14 %, S13F 0 %, R55L 30 %, K116E 2 %, H8Y 200 %, K84E 300 %, K84T 160 %, W118R 200 %, Q122R 200 %. Figure is taken from Savvides, Boon et al. 2000.
As stated above FLT3 ligand, KL, M-CSF, PDGF and VEGF bind to the PDGFR subfamily receptors (Figure 2.4.4). A crucial question is, do they bind to their respective receptors involving the same features. Answering this question, there are three similarities identified between these structurally distinct molecules (Savvides et al., 2000). First, all the five ligands have nearly identical overall dimension, that is ~70 Å x ~35 Å x ~25 Å. Second, all five are biologically active as homodimers and consist of two-fold axes of symmetry perpendicular to the longest dimension of the dimeric scaffold. Third, based on structure-function studies for the five ligands, it is suggested that they bind to their respective receptors bivalently with the equivalent receptor binding sites separated by ~45 Å. Thus, in spite of the diverse folds, these two family of ligands bind to and activate PDGFR-like receptors in an equivalent manner, providing symmetric sites to bind receptor domains 2 and 3, and allowing domains 4 and higher to form receptor-receptor contacts (Wiesmann et al., 1997).
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Figure 2.4.4. PDGRF-like receptors associated ligand and PDGRF-like receptor cartoon. A) until E) are the comparison of ligands that associate with PDGFR-like receptors. Note that SCF is marked with an asterisk because the model presented is based on the structure of Flt3 ligand. Red represents the receptor binding sites, green respresents the two-fold axes symmetry. F) is a cartoon of PDGFR-like receptor where green box represents general bivalent mode of ligand binding to class III (five Ig-like domains, n = domain 5) and class V (seven Ig-like domains, n = domains 5, 6, and 7) RTKs through Ig-like domains 1, 2, and 3 of the receptor. Figure is adapted from Savvides, Boon et al. 2000.
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Chapter 3
FMS-Like Tyrosine Kinase 3 Receptor
3.1. Receptor Tyrosine Kinase
Various polypeptides that play an important role in cell proliferation, differentiation, migration and metabolism, perform their actions by binding to cell surface receptors with tyrosine kinase activity and thereby activate them. These cell surface receptors are known to be the receptor tyrosine kinases (RTKs). All RTKs are glycoproteins consisting of glycosylated extracellular domains, a single hydrophobic transmembrane helix and cytoplasmic kinase domains (Hanks et al., 1988; Yarden and Ullrich, 1988; Schlessinger, 1988; Williams, 1989b). The extracellular domains possess the sites for ligand-binding while the cytoplasmic kinase domains feature catalytic activity and possess the sites for protein-protein interaction.
Based on their sequence similarity and different structural analysis, the RTKs were modestly classified into four classes by Ullrich and Schlessinger in 1990. The first class included those with two cysteine-rich repeat sequences in the extracellular domain and belonging to the second class are those with disulfide-linked heterotetrameric α2β2
Figure 3.1.1
structures with similar cysteine-rich sequences. Those with five and three immunoglobuline-like (Ig-like) repeats in the extracellular domains were classified into the third and fourth class respectively. However, more RTKs are identified throughout the years which add the number of classes of RTKs, illustrated in . The classification is based on structure of extracellular domain.
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Figure 3.1.1. Cartoon showing the architecture and domain organization of receptor tyrosine kinases. The extracellular domain of the receptor is on top and the cytoplasmic domain is on bottom. Figure is adapted from Hubbard. S, 1999.
The tyrosine kinase domain is highly conserved in all RTKs. It contains the consensus sequence GXGXXGX(15-20)K that serves as part of the binding site for adenosine triphosphates (ATP) (Yarden and Ullrich, 1988; Schlessinger, 1988; Hanks et al., 1988). Replacement of the consensus lysine residue of the ATP binding site in the EGF, insulin, and PDGF receptors completely abolish their kinase activities in vitro as well as in vivo (Chou et al., 1987; McClain et al., 1987; Russell et al., 1987; Honegger et al., 1987; Chen et al., 1987; Williams, 1989a). Although the kinase activity is obligatory for signal transduction and induction of early as well as delayed cellular responses, including mitogenesis and transformation, however it is not necessary for expression and cell surface targeting (Ullrich and Schlessinger, 1990).
RTKs comprise a large family of receptors that include the insulin receptor, the epidermal growth factor (EGF) receptor, platelet-derived growth factor (PDGF) receptor, fibroblast growth factor (FGF) receptor, and vascular endothelial growth factor (VEGF) receptor. Based on the structural characteristics, the largest families are given in Table 3.1.1.
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Table 3.1.1. RTKs large families classification. R, receptor; PDGF, platelet-derived growth factor; SCF, stem cell factor; CSF, colony stimulating factor; EGF, epidermal growth factor; FGF, fibrobalst growth factor; IGF,
insulin-like growth factor; HGF, hepatocyt growth factor; MSP, macrophage-stimulating protein; VEGF, vascular endothelial growth factor; FN, fibronectin; GH, growth hormon; EPO, erytrhopoietin; PRL, prolactin; IL, interleukin; LIF, leukemia inhibitory factor; CNTF, ciliary neurotrophic factor; IFN, interferon; TNF, tumor necrosis factor, LNGFR, low affinity nerve growth factor receptor; TCR, T cell receptor; BCR, B cell receptor; TGFβ, transforming growth factor β; Act, activin; BMP, bone morphogenic protein. Table is taken from Heldin,
1995.
The receptors are oligomerised upon ligand binding and the succeeding conformational change of the extracellular domain, which stabilizes interactions between adjacent cytoplasmic domains and leads to activation of kinase function (Schlessinger, 1988). Receptor oligomerization can be induced by monomeric ligands such as EGF that induce conformational changes resulting in receptor-receptor interactions (Greenfield et al., 1989) or by homodimeric ligands such as PDGF, CSF-1, and VEGF that dimerize neighboring receptors (Seifert et al., 1989; Heldin et al., 1989; Hammacher et al., 1989; Wiesmann et al., 1997). These ligands simultaneously bind two receptor molecules whereby a stable noncovalent receptor dimers are formed and it is possible that direct interaction between the receptors, involving epitopes located outside the ligand-binding domains, are important for
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stabilization of the receptor dimer (Heldin, 1995). The receptor dimerization is thus further stabilized by additional receptor-receptor interaction (Schlessinger, 2000).
When the RTKs are active, they catalyze the phosphorylation of exogenous substrates as well as tyrosine residues within their own polypeptide chain, also known as autophosphorylation, where one receptor molecule phosphorylating the other in the dimer (Ullrich and Schlessinger, 1990). Autophosphorylation occurs mainly on two different classes of tyrosine residues, namely on the conserved tyrosine residue in the activation loop of the kinase domain, or on a tyrosine residue outside the kinase domain which serve as a docking site for downstream signal transduction molecules containing Scr homology 2 (SH2) domains (Heldin, 1995). Autophosphorylation of PDGF receptor resulted in the phosphorylation of a consensus tyrosine residue (Y857) present in the catalytic domain of all tyrosine kinases and also Y571, which resides in the kinase insert and seems to be involved in modulating the interaction between activated receptor with certain cellular substrate and effector proteins (Kazlauskas and Cooper, 1989).
Experiments with chimeric receptors were performed by several research groups which all demonstrated that the identity of the transmembrane (TM) domains had no influence on RTKs signaling capacity (Riedel et al., 1989; Lammers et al., 1989; Lee et al., 1989). However, it is now proven that TM domains play an active role in signaling. TM domains give contribution to the stability of full-length receptor dimmers and maintain a signaling-competent dimeric receptor conformation (Li and Hristova).
The juxtamembrane domain is a short region which connects the transmembrane domain and the cytoplasmic domain. The sequences of this region are divergent between receptor subclasses but conserved between members of the same subclasses, and it is involved in modulation of receptor functions.
The kinase insert domain is observed on class III, IV and V RTKs (Figure 3.1.1), where it divides the kinase domain in two halves. This insertion is up until 100 amino acid residues where most of them are mostly hydrophilic. The kinase inserts of the various receptor differ in length and show only marginal similarity, but for a specific receptor the sequences of kinase insertion are highly conserved between species which implies an important role in receptor function (Ullrich and Schlessinger, 1990).
3.2. Discovery of FLT3 receptor
3.2.1. Murine FLT3 receptor (or Fetal Liver Kinase 2)
Research group of Matthews in 1991 used degenerate oligonucleotides, based on conserved regions within the kinase domain of tyrosine kinase receptors, in a PCR-based strategy to isolate a novel receptor fragment from highly purified mouse fetal liver stem cells. This fragment was then used to isolate full-length receptor clone that was named fetal liver kinase
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2 (FLK-2). FLK-2 was actually the same protein as FLT3 receptor discovered by the research group of Rosnet in 1991 (read further).
The sequence of FLK-2 cDNA extends over 3453 nucleotides. A polyadenylation signal is found 11 nucleotides upstream of the poly-A sequence at the 3’ end of the cDNA. This cDNA is able to code for a primary translational product of 990 amino acids which contains several distinct features that are characteristic of RTKs.
The extracellular region is 542 amino acids long which contains 22 cysteine residues and it is terminated by a sequence of 20 hydrophobic amino acids. They also identified nine potential N-glycosylation sites within the region. Following the extracellular region, a basic region is identified that suggests the junction between the transmembrane and the cytoplasmic region.
The cytoplasmic domain contains the conserved protein kinase domain that are responsible for catalytic function. The tyrosine kinase domain of FLK-2 is interrupted by a long hydrophilic kinase insert of 77 amino acid residues that are predominantly hydrophilic. This kinase insert domain is unique for each member of class III and IV RTKs and it is thought to play a role in the kinase substrate binding specificity. Although the kinase insert is unique and the C-terminal segment is different, the two flanking subdomains are highly homologous at the amino acid level with KIT and FMS.
The extracellular domain protein sequence of FLK-2, KIT and FMS were compared. The result showed approximately 30% overall homology based on consensus amino acids and conservative amino acid substitution. Nine of the ten conserved cysteine residues of the receptor family are identically conserved in FLK-2. However, they showed that the extracellular domain contains an additional 12 cysteine residues that are not seen in KIT nor FMS. In addition, the FLK-2 contains two regions of intragenic homology characterized by a conserved spacing of cysteine residues (Matthews et al., 1991). This duplicated region shares significant homology with the region close to the transmembrane domain in KIT and FMS and contains four conserved cysteine residues. This homology implies that the extracellular domain of FLK-2 may have arisen by an ancient duplication event of highly conserved Ig-like domains.
FMS-like receptor tyrosine kinase 3 (FLT3 receptor) was discovered and characterized in the mouse by the group of Rosnet in 1991. They identified this by using low stringency DNA hybridization on mouse testis and placenta. In a search for new RTKs, several cDNAs with homology to FMS were isolated. The receptors they encoded were named FMS-like RTKs. The FLT3 cDNA sequence extends over 3520 nucleotides and terminates at a polyadenylation (poly-A) stretch. The largest open reading frame (ORF) starts at ATG codon at position 82-84 and terminates at a TAG codon at position 3082-3084, which makes it able to code for a primary translational product of 1000 amino acids with an theoretical molecular weight of 132 to 155 kilodalton (kDa) (Maroc et al., 1993). The deduced FLT3 amino acid sequence is closely related to the sequence of class III RTKs. Based on sequence analogy, the FLT3 proteins was likely to be composed of an extracellular region that is potentially involved in ligand binding, and a cytoplasmic region separated by a 21 amino acids hydrophobic transmembrane segment (Rosnet et al., 1991).
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The ATG start codon is followed by a sequence that codes for a potential signal peptide, consisting of 23 predominantly hydrophobic amino acids and extending from position 1 to 23. In addition, a putative proteolytic cleavage site between residues 25 and 26, and nine potential asparagines-linked glycosylation (N-glycosylation) sites (NXS/T) were also identified (Rosnet et al., 1991). The extracellular region contains several cysteine residues showing the particular spacing patterns found in members of the immunoglobulin superfamily. It can be divided into five segments that could be organized into immunoglobuline-like (Ig-like) domains (Rosnet et al., 1991; Lyman et al., 1993b).
Rosnet and co-workers also identified the transmembrane region, which extends over 21 amino acids long and is followed consecutively by a sequence of positively charged amino acids and by the juxtamembrane (JM) segment. The catalytic tyrosine kinase part at the cytoplasmic side is interrupted by a long hydrophilic kinase insert of 74 residues. The C-terminal tail is hydrophilic and it is 47 amino acids long. This group also showed that the cytoplasmic region consists of 23 tyrosine residues which two of them are in the kinase insert. Further, they compared the transmembrane and cytoplasmic regions with those of members of the same class. The results showed broad amino acids identities except those in the kinase insert and C-terminal tail, which might point to a potentially specific role for these two regions.
The FLT3 and FLK2 proteins are overall closely identical in amino acid sequence except those at the C-terminal tail. At the C-terminal tail, their sequences differ by 31 amino acids long. Nevertheless, with 98% identical amino acid sequence, it seems that both proteins are encoded by the same gene (Lyman et al., 1993b).
Metabolic labeling studies on mouse FLT3 receptor demonstrated that two glycoforms of FLR3 receptor exist in the steady-state, namely an immature high-mannose N-linked population probably confined to the endoplasmic reticulum (ER)-Golgi transition and a mature population expressed at the cell surface that is glycosylated (Maroc et al., 1993). The lower molecular weight form of FLT3 contains high-mannose structures because it is sensitive to endoglycosidase H (endo H) (Lyman et al., 1993b; Maroc et al., 1993) while the higher molecular weight form is not (Lyman et al., 1993b). The insensivity of the higher molecular weight form to endo H may be due to addition of complex carbohydrate parts during post-translational modification (Lyman et al., 1993b). Both of these biochemical characterization showed that FLT3 protein is indeed N-glycosylated. The two glycoforms do not appear to be O-glycosylated (Maroc et al., 1993).
Another character of FLT3 protein is that it contains a tyrosine kinase activity. The two kinase domains are responsible for the receptor tyrosine phosphorylation and this activity can be abolished by a mutation within the conserved sequence (Maroc et al., 1993). Upon ligand binding, autophosphorylation of the receptor tyrosine occurs at the cytoplasmic region of FLT3 protein.
An isoform of the murine FLT3 receptor has been reported lacking the fifth Ig-like region in the extracellular domain which is caused by alternative splicing (Figure 3.2.1) (Lavagna et al., 1995). This isoform is present at lower level than the wild type receptor, but it is still able to
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bind ligand and is phosphorylated upon binding, which indicates that the fifth Ig-like domain of murine FLT3 is not needed for ligand binding nor phosphorylation (Lyman and Jacobsen, 1998). The physiologic importance of this alternative isoform is still unknown and a soluble version in human serum has not yet been identified.
Figure 3.2.1. Cartoon of FLT3 receptor wild type (above) and the alternative isoform (below) structure. Figure is taken from Lavagna et al. 1995.
3.2.2. Human FLT3 receptor
Human cDNA from a pre-B-cell line was cloned and then characterized by the group of Rosnet, Schiff et all. in 1993. Their work showed that the cDNA sequence of FLT3 receptor protein is similar to mouse FLT3 receptor cDNA. Based on the primary amino acid sequence, the extracellular region of FLT3 is predicted also to fold into five Ig-like domains, which is a characteristic of typical class III RTKs (Figure 3.2.2). The FLT3 protein is observed as a major 140 kDa band and a minor 160 kDa band due to the N-glycosylation and when the protein is not glycosylated nor membrane bound, it is observed as 130 kDa band (Rosnet et al., 1993; Rosnet et al., 1991; Lyman et al., 1993b; Carow et al., 1996).
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Figure 3.2.2. Cartoon showing the architecture and domain organization of FLT3 receptor tyrosine kinase. The extracellular domain of the receptor is on top and the cytoplasmic domain is on bottom. Figure is adapted from Parcells et.al 2006.
In has been found that the human amino acid sequence of FLT3 protein (hFLT3) is homologous to amino acid sequence of the mouse FLT3 protein with 86% identity but not the mouse FLK-2 (Rosnet et al., 1993; Small et al., 1994). This similarity is observed throughout the whole molecule, including in the regions that are naturally less conserved among different class III RTKs, namely the extracellular region, kinase insert and C-terminal. In addition, hFLT3 shows respectively 18% and 19% similarity with KIT and FMS in the extracellular region, and 63% and 64% in the tyrosine kinase domain. However, FMS and KIT are more closely related to each other. Putting FLT3, KIT, and FMS together, they share 57% similarity in their tyrosine kinase domains but only 9% similarity in their extracellular regions. Nevertheless, this similarity includes eight cysteines that are perhaps involved in intramolecular bounds responsible for the Ig-like folding of the second, third and fifth Ig-like domains.
As mentioned above, KIT and FLT3 have five Ig-like domain in their extracellular domains. When the ligand is associated with the receptors, both in dimeric form, signal transduction occurs. Based on mutagenesis studies on KIT, it has been confirmed that the first three domains are both necessary and adequate for binding of ligand and that the fourth domain is required for dimerization of the receptor (Blechman et al., 1995). Whether a similar process is likely to occur with FLT3 ligand and FLT3 still needs to be investigated.
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3.3. Expression of FLT3 receptor
FLT3, KIT, FMS, and platelet-derived growth factor receptor (PDGFR) are member of class III RTKs that have been shown to be important in the growth and/or differentiation of a variety of cells in which they are expressed. In contrast with its ligand, the expression of FLT3 is on more limited number of cells. There is no FLT3 expression observed on mouse myeloid, macrophage, erythroid, megakaryocyte, mast cell lines, T-cell lines, and most of early mouse B-cell lines. However, expression on several mature B-cell lines and pro-T-cell lines has been reported. On the other hand, the expression of FLT3 in human cell lines is found on a high percentage in myeloid and monocytic cell lines. Also a few of megakaryocytic cell lines are FLT3 positive. All human myeloma, erythroid and erytrhoblastic cell lines are FLT3 negative. FLT3 expression on CD34+ stem/progenitor cell fraction of both mouse and human hematopoietic cells indicate that it is likely to be involved in growth and/or differentiation of these cells (Small et al., 1994).
3.4. FLT3 receptor expression in human leukemia
3.4.1. Internal tandem duplication (ITD)
Oncogenesis is by definition, a progression of cytological, genetic, and cellular changes that culminate in a malignant tumor. Self-renewal and growth factor-independent proliferation are two important oncogenesis characteristics. The involvement of FLT3 in proliferation of highly undifferentiated hematopoietic cells suggests the oncogenic potential of this signaling pathway.
Several studies have shown that FLT3 receptor is expressed on the surface of normal and malignant human hematopoietic cells (Turner et al., 1996; Rosnet et al., 1996). In the malignant human hematopoietic cells, particularly the acute myelogenous leukemia (AML) patient cells, it is observed that the FLT3 transcript contained abnormal longer PCR products in addition to the germline products at the JM region through the first tyrosine kinase domain (TK1) while the extracellular domain and the transmembrane domain transcript length showed no length alteration (Nakao et al., 1996). By sequence analysis it was also demonstrated that partial sequences are tandemly duplicated and the altered transcripts are in-frame (Nakao et al., 1996).
The JM region contains a tyrosine (Y)-rich stretch 589YFYVDFREYEY599, which is associated with signal transduction. In the aberrant FLT3 transcript, some of the Y were duplicated, most frequently Y589/Y591/Y597/599 followed by Y597/Y599, while the duplication at the fragment upstream Y589 is not observed (Kiyoi et al., 1998).
The study of Kiyoi, Towatari et al. in 1998 revealed several important characteristics of the FLT3-ITD mutant. Firstly, the mutant FLT3 has a conformational change of it C-terminus region. This conclusion was drawn because the mutant FLT3 precipitated to a lesser degree than the wild type, or even not precipitated, by anti-C-terminal FLT3 antibody
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immunoprecipitation experiment. Second, the FLT3-ITD mutants were constitutively phosphorylated in a ligand-independent manner regardless of the ITD location and length or in another words, the mutants were constitutively activated. Third, the wild type of FLT3 was phosphorylated by the mutant that is expressed in the same cell or in another words, the FLT3 mutant is physically associated with the FLT3 wild type. Fourth, the ITD or insertion at the JM domain induced the extracellular dimerization without FLT3 ligand stimulation, because the same sized band (260 kDa) was detected in the mutant FLT3 without FLT3 ligand stimulation as the wild type FLT3 with ligand stimulation (Figure 3.4.1). This phenomena indicated that the constitutive dimerization is a key mechanism for activating mutation of RTK. The JM domain mutations induce an extracellular conformational change which favors dimerization. Lastly, the increase of Y residues is not necessary for constitutive phosphorylation, rather the elongation of the JM domain may be more important.
Figure 3.4.1. Immunoblotting of cross-linking experiments of wild type FLT3 with and without FLT3 ligand stimulation and mutant FLT3 without FLT3 ligand stimulation. Arrow indicates homodimer band. Wild, FLT3 wild type; Mt 2-6, FLT3 mutant type 2-6, FL, FLT3 ligand. Figure is taken from Kiyoi, Towatari et al. 1998.
3.4.2. JM domain and its autoinibitory mechanism
The crystallography structure of juxtamembrane (JM) domain of hFLT3receptor was already determined by Griffith and co-workers in 2004. The autoinhibited structure of FLT3 consists of the bilobal kinase fold, the activation loop and the JM domain, where the N-terminal and the C-terminal domains constitute the standard kinase fold (Griffith et al., 2004). The N-terminal (N lobe) contains a twisted five-stranded anti-parallel β-sheet adjacent to an α-helix (αC) while the C-terminal (C lobe) consist of seven α helices and three β sheets (Griffith et al., 2004).
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Figure 3.4.2. FLT3 diagram with the spatial arrangement of the different structural components of the molecule. The N lobe (in red) and the C lobe (in blue) constitute the standard kinase fold, the activation loop (in green) is folded up between the two kinase domains and the JM domain (in yellow) spans the length of the molecule. N lobe, N-terminal kinase domain; C-lobe, C-terminal domain; JM, juxtamembrane domain. Figure is adapted from Griffith et al. 2004.
What is typically observed in available kinase structures is that the two kinase fold of FLT3 are connected to each other by a flexible polypeptide stretch which allows rotational movement of the two domains (Huse and Kuriyan, 2002). The kinase is in catalytically inactive form when the N lobe is rotated away from the C lobe. Inversely, it is in active form when the N lobe is rotated toward the C lobe, because by doing so, the important catalytic residues from both lobes are able to align. Griffith, Black et al. 2004 found that the crystal structure of autoinhibited FLT3 corresponds to the prototypical conformation common to other inactive kinases that have a closed activation loop, folded between the two lobes. However, a distinct feature of FLT3 is the presence of the complete JM domain which adopts an autoinhibited conformation and interacts with all important features of FLT3.
The group of Griffith, Black et al. 2004 divided the JM domain into three different topological components, namely the JM binding motif (JM-B), the JM switch motif (JM-S) and the zipper linker segment (JM-Z) (Error! Reference source not found.).
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Figure 3.4.3. Diagram of FLT3, focusing on the JM-B position.
The JM-B is short, only 7 residues, but it makes contacts with every structural component that are involved in the activation/inactivation cycle of the FLT3 cytoplasmic domain, including the glycine-rich loop, the activation loop and the catalytically important αC (Figure 3.4.3). The JM-B it is nearly buried in the autoinhibited FLT3 structure and stabilizes the inactive kinase conformation by preventing the N lobe from rotating toward the C lobe. The JM-S is a two-stranded anti-parallel β-twist and is located externally on the C lobe. It contains two conserved tyrosine residues (Y589 and Y591) that state of phosphorylation is implicated in the activation and regulation of the receptor enzymatic activity (Mol et al., 2003). JM-S interacts with the rest of the molecule in a less extensive and less complementary way in comparison to the JM-B interaction. It does not provide a significant amount of binding interface between JM-S and the C lobe that in turn can enhance the attachment of the JM-B. The last part of JM, JM-Z is located at the C-terminal of the JM domain and is connected mostly with the αC of the N lobe. The role of JM-Z is to precisely align and maintain the JM-S in the proper manner during and also after the transition between active and inactive state of FLT3.
The activation of FLT3 receptor starts with the binding of FLT3 ligand with the extracellular domain of FLT3. This binding support the dimerization of FLT3 and the subsequent juxtapositioning of the cytoplasmic domains. After the dimer is formed, transphosphorylation of the specific tyrosine residues occurs on the JM domain which is then followed by activation of the catalytical kinase. The active kinase promotes downstream multiple signaling pathways. The FLT3 kinase activity is negatively by tyrosine phosphatases that in turn dephosphorylate the tyrosine on the unattached JM domain.
The ITD insertions occurs commonly in the JM-Z near the JM hinge region. This ITD displaces the position of the JM-S that disturbs or prevents the optimal orientation of JM-S when it tries to position JM-B in its binding site. Moreover, the ITD interrupt the normal
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complementary interaction between JM-Z and N lobe in such a manner that the abnormal JM-Z remains permanent in the unbound conformation.
3.4.3. Activation loop and Asparagine 835 mutation
The kinase activation loop generally consist of one to three tyrosine residues that are able to serve as phosphorylation sites. When these tyrosines are not phosphorylated, the activation loop preserves the closed conformation by folding into the cleft between N and C lobes and thus the access to the peptide substrate and the ATP binding site is blocked. But when these tyrosines are phosphorylated, the activation loop adopts the open conformation and thereby release the access to peptide substrate and ATP binding site (Huse and Kuriyan, 2002; Schlessinger, 2000).
It has been found that point mutations of residues in the activation loop also generate constitutively activated forms of FLT3 in some of the AML cases (Abu-Duhier et al., 2001; Yamamoto et al., 2001). The most occurring mutation is D835T (Abu-Duhier et al., 2001; Yamamoto et al., 2001), followed by mutations of D835V, D835H, D835E and D835N (Yamamoto et al., 2001). This point mutations occur independently of the FLT3/ITD and are not significantly correlated to the oncogenesis, although they tend to worsen disease-free survival (Yamamoto et al., 2001).
Objectives of the study
27
Section II Objectives of the study
Chapter 4
28
Chapter 4
Objectives of the study
Three-dimensional structural information obtained from crystallographic studies has already been used to understand molecular mechanism of receptor activated signal transductions. Nowadays it is increasingly being used to design clinically useful cytokine antagonists and agonists. A search in PubMed features more than 4000 citations in the last ten years that report the studies of one of the class III RTK cytokine receptors and their ligands. However, only a handful of publications shed the light on the interactions between these molecules, indicating that there is still much more to be done to gather these important knowledge. With the significant improvements in molecular biology, in crystallographic methods and advanced equipments, the time needed for structure determination has been drastically reduced.
The interaction of human FLT3 (hFLT3) receptor and human FLT3 ligand generates a lot of interest and many questions yet to be answered. Questions like what is the structure of FLT3 receptor, what are the interactions between the receptor and its ligand, which extracellular domains of the FLT3 receptor involved in binding to FLT3 ligand, what is the kinetic and the affinity of the binding remain to this day unanswered
While the structure of FLT3 ligand is already solved, the structure of FLT3 receptor is yet to be unraveled. The first objective of this study is to produce recombinant hFLT3 receptor domain 1 and domain 1 to 2 by using HEK293S-GnTI-/-
stable cell lines. These construct will be used to elucidate hFLT3 receptor structure by crystallographic studies and to investigate their interactions with recombinant human FLT3 ligand. Other objective is to the receptor-ligand interactions by using SPR and ITC.
29
Section III Results and discussions
Chapter 5
30
Chapter 5
Overexpression of recombinant human FLT3 receptor construct in a human cell line, their purification and binding studies
5.1. Introduction
Until today Escherichia coli (E. coli) is still the most commonly used and the cheapest strategy for the expression of proteins in large amount. However, the disadvantage of prokaryotic cells for recombinant protein production is that they lack the machinery for glycosylation and the quality control mechanisms present in eukaryotic cells. As a consequence, it is often found that eukaryotic proteins which are N-glycosylated and contain disulfide bridges are produced in E. coli as insoluble products. The eukaryotic expression systems such as yeast, insect and mammalian cells expression systems are now often being used.
The mammalian expression systems, other than the fact that they are high eukaryotic systems, they have an efficient post-translational processing machinery in the secretory pathway which is essential for protein folding. This property makes them the most ideal system in term of protein quality and yield to produce human cell surface receptors and secreted proteins. There are two high eukaryotic expression systems commonly being used, the Chinese Hamster Ovary (CHO) cells and the Human Embryonic Kidney cells (HEK). As nothing is perfect in this world, each of these systems have advantages and disadvantages. A modified CHO system has a reduced N-glycosylation and it is suitable for Selenium-Methionine (SeMet) incorporation which is useful for structural studies (Jones et al., 1992; Butterrs et al., 1999; Davis et al., 2001). However, it takes a long time to generate a stable clones that produce sufficient amount of proteins. Aricescu and co-workers have developed a simple, fast and cheap HEK expression system in 2006, which is known as HEK293T system. With this system it is possible to do a rapid small scale expression test and then to extrapolate it to large scale expression. Moreover, HEK293 system offers the possibility for SeMet incorporation and controlled N-glycosylation (Aricescu et al., 2006). SeMet incorporation is often used X-ray crystallography for multiwavelenght selection for anomalous signal.
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The HEK293 cells were generated by transformation of cultures of normal human embryonic kidney cells, cultivated from an healthy aborted fetus, with sheared adenovirus 5’ DNA in the laboratory of Alex Van der Eb in Leiden (Graham et al., 1977). The transformation was brought about by an insert consisting of ~4.5 kb which became incorporated into human chromosome 19 (Louis et al., 1997). An important variant is the HEK293T cell line which contains Simian vaculoting virus 40 (SV40) large T antigen. This allows episomal replication of a transfected plasmid which contains SV40 ori. In this way, an extended temporal expression of the desired gene product is obtained.
Earlier experiments with E. coli expression systems were unsuccessful to deliver native recombinant human FLT3 ectodomains and therefore the HEK293T expression system is now being used. The recombinant human FLT3 domain 1 and domain 1 to 2 were cloned into pcDNA4/TO vector. This vector fuses a C-terminal hexahistidine (His6) tag at the recombinant proteins which facilitates their purification. In addition, a N-terminal secretion signal sequence that is presence in the vector is fused to the recombinant proteins. Subsequently, a small expression test is performed in HEK293T cells, grown in six-wells plates to check if the recombinant protein is formed and what the expression level is. If the expression level is high, transient expression can be used for large scale production. On the other hand stable cell line must be generated in case of low expression level.
The preparative purification of the recombinant protein is based on Capture, Intermediate purification and Polishing principle (CIP) (Figure 5.1.1
). The capture is the first separation of the recombinant protein in the medium by using immobilized metal affinity chromatography (IMAC) Talon column. The intermediate purification is the size exclusion gel filtration with Superdex 200 or Superdex 75 column performed on a semi-automated system. This system made it possible to separate the protein in real time by following the 280 nm absorption. The aromatic amino acids absorb light with 280 nm wavelength. The polishing step is performed in order to obtain protein with a sufficient degree of purity using ion exchange chromatography e.g. MonoQ column. An additive gel filtration can be performed also on the same semi-automated system.
Figure 5.1.1. Preparation and the Capture, Intermediate purification and Polishing strategy.
Figure is taken from Amersham Pharmacia Biotech Protein Purification Handbook.
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5.2. Expression of recombinant human FLT3 receptor domain 1 and domain 1 to 2 in eukaryotic expression system
The extracellular domain of FLT3 receptor showed small similarity with other class III RTKs by the presence of CXXXGXPXPXXXWXXC signature in the immunoglobuline-like domain 2 (IG2) and immunoglobuline-like domain 5 (IG5). In addition, there was homology observed between IG1 and IG4 as well as between IG2 and IG5 suggesting that an internal duplication may have taken place in the ancestor of the FLT3 gene (Figure 5.2.1) (Rosnet et al., 1991).
Figure 5.2.1. Immunoglobuline-like domains (IG) FLT3 receptor comparison. Sequence identities in some residues were identified, particularly the residues presence in the class III RTKs signature sequence (*). Figure is adapted from Rosnet, Marchetto et al. 1991.
Based on the sequence similarity between IG1-IG4 and IG2-IG5, it is interesting to investigate the structure of domain 1 and domain 1 to 2 and to study their interactions with the ligand.
5.2.1 Cloning of the extracellular domains of rhFLT3 receptor
There were two extracellular regions of human FLT3 receptor cloned into the pcDNA4/TO vector. They were Ig-like domain 1 (pcDNA4/TO-hFLT3d1) and the Ig-like domain 1 to 2 (pcDNA4/TO-hFLT3d12) (Figure 5.2.2). Both of the recombinant proteins are fused with C-terminal His6
tag and N-terminal Mu secretion signal by using AgeI and KpnI restriction sites of the pcDNA4/TO vector. DNA sequencing of both constructs confirmed their correctness.
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Figure 5.2.2. pcDNA4/TO vector of human FLT3 receptor domain 1 (left) and domain 1 to 2 (right).
5.2.2. Expression test of human FLT3 receptor domain 1 and domain 1 to 2 in HEK293T cells
Five days after induction with tetracycline and sodium butyrate, the three ml medium was collected for Western Blot analysis. Western Blot is a technique to detect protein specifically and it has high sensitivity. In this technique, the proteins are separated according to their molecular weight using gel electrophoresis. The proteins were subsequently transferred to a nitrocellulose membrane where they were probed by specific antibodies against the protein of interest. In this experiment we used anti-His antibody against the C-terminal His
The pcDNA4/TO-hFLT3d1 and pcDNA4/TO-hFLT3d12 were used for transient expression. Small scale expression test for both cell lines were conducted to check the expression level of the recombinant proteins. The cells were grown in six-wells plates under 0% fetal calf serum (FCS), with or without kifunensine conditions.
6
The predicted molecular weight (MW) of recombinant human FLT3 receptor domain 1 (rhFLT3d1) was 16.2 kDa and recombinant human FLT3 receptor domain 1 to 2 (rhFLT3d12) was 26.5 kDa. However, from Figure 5.2.3 we see that the apparent molecular weight of rhFLT3d1 is more and less between 20 and 25 kDa and rhFLT3d12 is more and less at 37 kDa. The difference between the predicted and the apparent MW is possibly due to glycosylations. In the absence of kifunensine, rhFLT3d1 shows three bands close to each other, but one is dominant. This suggested the presence of three glycosylation forms where one was the most dominant. However, the expression of rhFLT3d12 was lower in the presence of kifunensine, although it showed equal homogeneity with and without kifunensine.
tag.
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Figure 5.2.1. Western Blot of rhFLT3 domain 1 and domain1 to 2 where rhFLT3 domain 1 to 5, 1 to 4 and 1 to 3 were used as positive controles. d5 to d3 are rhFLT3 domain 1 to 5, domain 1 to 4 and domain 1 to 3 respectively. d2+K; rhFLT3 domain 1 to 2 treated with kifunensine, d1+K; rhFLT3 domain 1 treated with kifunensine, d2-K; rhFLT3 domain 1 to 2 not treated with kifunensine, d1+K; rhFLT3 domain 1 treated with kifunensine.
We observed that the expression level was high for rhFLT3d1 treated with kifunensine (d1+K) as well as rhFLT2d1 not treated with kifunensine (d1-K). d1+K was seen as multiple bands while d1-K showed three bands closed to each other. As kifunensine inhibits α-mannosidase I, the sugar trees of d1+K are more homogenous than d1-K. On the other hand, d1-K showed less homogeneity where three rhFLT3d1 glycoforms were observed. One rhFLT3d1 glycoform was more dominant than the others.
5.2.2. Large scale production of recombinant human FLT3 receptor domain 1 in HEK293S cells and purifications
The HEK293S-GnTI-/-
rhFLT3d1 stable cells were grown for large scale expression in 175 cm² giant falcons. Five days after induction with tetracycline and sodium butyrate, the medium was harvested and filtered through a 0.22 µm filter. To capture the recombinant proteins, IMAC was carried out by using a Talon column. After a wash step, the recombinant proteins were eluted with 200 mM imidazole. The elution fraction was concentrated to one ml and then injected to Superdex 200 gel filtration column. The purification profile shows a convincing result where rhFLT3d1 is separated from contaminants (Figure 5.2.4). rhFLT3d1 came out at the expected volume, which is after 75 ml with an elution rate of 1 ml/min while the void volume is between 40 ml and 50 ml.
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Figure 5.2.4. Superdex 200 purification chromatogram of rhFLT3d1. Peak 1 was the void volume, peak 2 to 4 were contaminants, peak 5 was rhFLT3d1 eluted at the expected volume, which was after 75 ml with elution rate of 1 ml/min.
One fraction of peak 2 to 4 and three fractions of peak 5 were tested on SDS-PAGE (15% acrylamide). From the result of SDS-PAGE (Figure 5.2.5) we are convinced that peak 2 to 4 were indeed contaminanting proteins and peak 5 was rhFLT3d1. However, we found four bands of rhFLT3d1 on the gel which were closed to each other, while in Western Blot we found three bands. This results suggested that there might be four different glycoforms of rhFLT3d1. In addition, there were two bands found on 75 and 50 kDa and also a smear of protein until 25 kDa where rhFLT3d1 reside. We suspected that the two bands and the smear were also rhFLT3d1. All of the fractions under peak 5 were collected and concentrated until it reached concentration of 8.3 mg/ml. This was enough for crystallization which required protein concentration at least 5 mg/ml.
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Figure 5.2.5. SDS-PAGE gel of fractions from peak 2, 3, 4 and 5. Fraction 4, 16 and 23 are contaminants, fraction 31, 35 and 38 are rhFLT3d1 (predicted MW of 16.2 KDa).
5.2.3. Large scale production of recombinant human FLT3 receptor domain 1 to 2 in HEK293T cells and purifications
Identical to what we performed for rhFLT3 domain 1, rhFLT3d12- GnTI-/-
The amount of rhFLT3d12 was very low. The low expression level might be caused by the instability of the recombinant protein which make it difficult to produce it. Structural studies have shown that there are interactions between domain 2 and domain 3 (personal communication with K. Verstraete). Domain 2 might be unable to fold properly without these interactions. Due to lack of time, we put further experiments of rhFLT3d12 on hold.
stable cells were also grown for large scale in 175 cm² giant falcons. Five days after induction with tetracycline and sodium butyrate, the medium is harvested and filtered through a 0.22 µm filter. To capture the recombinant proteins, affinity chromatography was carried out by using a 25 ml talon column. After a wash step, the recombinant proteins are eluted with 200 mM imidazole. The elution peak was concentrated to one ml and then injected to Superdex 75 gel filtration column. Unfortunately, the purification profile didn’t show a convincing result (Figure 5.2.6).
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Figure 5.2.6. Superdex 75 purification chromatogram of rhFLT3d12. The rhFLT3d12 peak was much smaller than the void volume.
Fraction 3, 5, 7, 13, 14, 16, 17 and the pool before SD 75 purification were tested on SDS-PAGE (15% acrylamide). The result shown in Figure 5.2.7 showed that Pool contained of contaminating proteins and rhFLT3d12. In fraction 3, 5 and 7, contaminating proteins and rhFLT3d12 were present while fraction 13 to 17 showed only rhFLT3d12.
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Figure 5.2.7. SDS-PAGE gel of void volume peak and rhFLT3d12 peak. Pool consisted of contaminating proteins and rhFLT3d12; fraction 3, 5 and 7, contained contaminating proteins and rhFLT3d12; fraction 13 to 17 contained rhFLT3d12.
5.3. Complex formation study of human recombinant FLT3 ligand and human recombinant FLT3 receptor domain 1
rhFLT3d1 was mixed with equimolar amount of recombinant human FLT3 ligand (rhFL) and incubated for five minutes at room temperature. The sample was injected in analytical gel filtration column. The sample eluted at the same time as rhFLT3d1 and rhFL alone (Figure 5.3.1) suggesting that there was no binding formed. Binding of rhFLT3d1 and rhFL would have resulted in a shift of the peak.
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Figure 5.3.1. Elution peaks of rhFLT3d1, rhFL and rhFLT3d1+rhFL. In blue is rhFLT3d1 alone, pink is rhFL alone and brown is rhFLT+rhFL. rhFLT3d1+rhFL eluted at the same time as rhFLT3d1 and rhFL alone.
5.4. Complex formation study of human recombinant FLT3 ligand and human recombinant FLT3 receptor domain 1 to 2
rhFLT3d12 was added into an excess of rhFL in equimolar amount and incubated for five minutes in room temperature. The sample was injected in analytical gel filtration column. The sample eluted at the same time as rhFLT3d12 and rhFL alone (Figure 5.4.1) suggesting that there was no binding formed.
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Figure 5.4.1. Elution peaks of rhFLT3d12, rhFL and rhFLT3d12+rhFL. rhFLT3d12+rhFL eluted at the same time as rhFLT3d12 and rhFL alone. In black is rhFLT3d12 alone, pink is rhFL alone and brown is rhFLT+rhFL.
5.5. Conclusion
The expression level of rhFLT3d1 was high and the recombinant protein is heterogenous. Unfortunately the expression level rhFLT3d12 was low which might be due to instability of the recombinant protein. Structural studies have shown that there are interactions between domain 2 and domain 3 (personal communication with K. Verstraete). Domain 2 might be unable to fold properly without these interactions.
From complex formation studies we concluded that neither rhFLT3d1 or rhFLT3d12 bind to rhFL. The role of domain 1 and domain 2 is still unclear for FLT3 receptor. However, rhFLT3d123 complex formation studies showed ligand binding. This information is obtained by personal communication with K.Verstraete.
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Chapter 6
Crystallization of recombinant human FLT3 receptor domain 1
6.1. Introduction
X-ray crystallography is a form of very high resolution microscopy and it is nowadays the most favored technique for threedimensional structure determination of proteins and biological macromolecules. With this technique researchers are able to study proteins interactions, conformational changes, etc. In microscopy, the amount of detail or also known as the resolution, is limited by the wavelength of the electromagnetic radiation. X-rays are used to obtain a molecular structure because its electromagnetic radiation wavelength is around 1 Å, which is close to the bond length distance atoms in a protein.
X-ray scattering from a single molecule is very weak and cannot be detected above the noise level which include scattering of water and air. On the other hand, a crystal arranges large numbers of molecules in the same orientation whereby the scattered waves constructively interfere and raise the signal to a measureable level above the noise level.
Diffraction of a crystal gives rise to constructive and destructive effects in the diffraction pattern which appears on the detector as a series of discrete spots known as reflections. Each of detected reflection contains information on all atoms in the molecule and conversely, each atom contributes to the reflection intensity. As to combine a diffraction pattern of every spot, the amplitude and phase are required for each reflection. Unfortunately only the amplitude can be determined experimentally and the relative phase angles for the different diffracted spots are missing. The latter is known as the phase problem. There are several methods to solve this phase problem, such as isomorphous replacement, single anomalous dispersion (SAD) and multiple anomalous dispersion (MAD).
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6.2. Recombinant human FLT3d1 crystallization
rhFLT3d1 that we obtained from SD 200 purification (paragraph 5.2.3) was concentrated to 8.3 mg/ml and screened for crystallization by using PEG ion screen 1 and 2, Crystal Screen 1 and 2 from Hampton Research readily available for direct use. From a total of 200 conditions, there was only one condition that gave rise to forming of crystal. This condition contains 0.01 M Cobalt (II) chloride hexahydrate (CoCl2), 0.1 M MES monohydrate pH 6.5 and 1.8 M ammonium sulfate ((NH4)2SO4). More crystals were produce by conducting crystallization using variations of CoCl2 and (NH4)2SO4 concentrations. The size of the crystals is more likely to be dependent on (NH4)2SO4 concentrations than on CoCl2
concentrations (Figure 6.2.1).
Figure 6.2.1. rhFLT3d1 crystals. Cyrstals are formed in the presence of (NH4)2SO4 and CoCl2. From left to right was increasing (NH4)2SO4 concentrations and difference in crystal size was (NH4)2SO4
Crystallization trials were further conducted using Co
concentration dependent.
2+, Ni2+, Zn2+, Ca2+ and Mg2+ at 10 mM, 100 mM MES monohydrate pH 6.5 and 1.8 M (NH4)2SO4. We observed that there was no crystal formed by using Zn2+, Ca2+ and Mg2+ which suggested Co2+ and Ni2+ are important in the packaging into crystals. This is possibly because of the His6 tag that was still present in rhFLT3d1 meaning that Co2+ and Ni2+ could have bind to the tag and helped the packaging of rhFLT3d1 into crystals. Ni2+, Co2+ and Zn2+ are similar (in the same row in table of Mendelejev) and they all bind to His6 tag. However, we observed that precipitants were formed in the presence of Zn2+. Apparently it behaves differently in the protein than Co2+ and Ni2+. As for Ca2+ and Mg2+, they are more similar to each other (in the same column in table of Mendelejev) than to Co2+,Ni2+, or Zn2+
Then we tested the influence of Co
.
2+ concentration to the crystal packaging at 1 mM, 10 mM, 50 mM and 200 mM. Here we observed that there were only crystals formed at 10 mM and 50 mM (result not shown). The size of rhFLT3 crystals varies with (NH4)2SO4 concentrations. The higher (NH4)2SO4 concentrations, the faster supersaturation was reached and thereby more nuclei were formed, resulting in numerous small crystals.
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6.3. Crystal tests
After we obtained rhFLT3d1 crystals, we tested the crystal in three different ways to verify that those are indeed the recombinant hFLT3d1 protein crystals and not salt crystals before performing X-rays diffraction. These following tests were performed on the crystal: Izit Crystal Dye, SDS-PAGE Silver-staining and crystal crushing. Izit Crystal Dye is a simple test provided by Hampton Research to see directly the difference between salt crystals and protein crystals. The crystal dye is blue and protein crystals will be also blue by penetration of solvent channels while salt crystals will not absorb the dye and stay colorless.
The Izit Crystal Dye test was performed by adding 0.2 µl of the dye into the 1+1 drop. The absorption took place directly and all the crystals became blue (Figure 6.3.1). This gave a direct indication that our crystals were protein crystals.
Figure 6.3.1. Crystals proteins. Izit Crystal Dye gave rise to blue crystals protein.
SDS-PAGE (15% acrylamide) silver staining test was performed also to investigate which protein is present in the crystals. The crystals were washed with the precipitant solvent several times before they were loaded into the SDS-PAGE. Soluble rhFLT3d1 was used as positive controls. As shown in Figure 6.3.2, the crystals exhibited all the bands as the positive controls which indicated that the crystals were rhFLT3d1 protein. These crystals broke easily which indicated they were protein crystals as salt crystals don’t.
Western Blot was also performed where we used anti-His antibody for specific probing of C-terminal His6 tag of rhFLT3d1 protein. This test was needed to investigate if all the bands present in the SDS-PAGE silver staining were rhFLT3d1 protein. Three crystal samples (1 µl, 1.5 µl and 2 µl) were loaded into SDS-PAGE (15% acrylamide) and after electrophoresis the proteins were subsequently transferred to a nitrocellulose membrane where they were probed by anti-His antibody. This test turned out to be positive as well which indicated that the crystals were solely rhFLT3d1 and also confirmed the fourth rhFLT3d1 glycoform hypothesis (Figure 6.3.2). Although rhFLT3d1 was heterogenous, packing of rhFLT3d1 in crystal was
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not prevented by its heterogenous glycosylation. Note that all four glycosylation forms were present in the crystal.
Figure 6.3.1. SDS-PAGE silver staining (left) and Western Blot (right) of rhFLT3d1 crystals.
6.4. Recombinant human FLT3 receptor domain 1 crystals diffraction
To cryoprotect the crystals, rhFLT3d1 crystal was brought into cryoprotectant solutions first. All cryoprotectant solutions had the same composition as the mother liquor, supplemented with 20%-25% glycerol, 25% sucrose, 25% sorbitol, 3 M (NH4)2SO4
rhFLT3d1 crystals diffracted to a resolution of 4.7 Å (Figure 6.4.1). We can perceive that the diffraction was anisotropic meaning that the crystals tend to diffract asymmetrically to certain direction and there was a twinning in the crystal. The predicted space group was face centered with two-fold symmetrical axis (F 2 2 2). Although our crystals have good shape and size, the resolution was still low. Heterogeneity and twinning of rhFLT3d1 could be the reason of the resolution problem.
. Paraton was also used as cryoprotectant directly. Diffraction of the crystal was performed using PXIII beamline at Paul Scherrer Institute in Switzerland.
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Figure 6.4.1. rhFLT3d1 diffraction spots.
6.5. Conclusion
rhFLT3d1 was heterogenous and rhFLT3d1 crystals were formed in a condition containing Co2+ or Ni2+, MES pH 6.5 and (NH4)2SO4
. rhFLT3 crystals were diffracted at 4.7 Å resolution, the predicted space group was F 2 2 2 and the scattering was anisotropic. There was a possibility of twinning in the crystals. Deglycosylation of rhFLT3d1 and polishing purification using cation exchange can be performed in the future to obtain better crystals.
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Chapter 7
Overexpression of recombinant human FLT3 ligand construct in E. coli, its refolding and purification 7.1. Introduction
As already mentioned in the paragraph 5.1, E. coli is still the most commonly used and the cheapest strategy for the expression of proteins in large amount even though it lacks the N-glycosylation machinery. Forming of inclusion bodies (IB) is a problem that often occurs when a eukaryotic protein is being overexpressed in the cytoplasm of a prokaryotic system. This phenomena arises because of hydrophobe aggregation of badly folded intermediates. A refolding protocol must be performed to obtain the functional native protein. But an advantage of this situation is that the proteins can be expressed with a high yield and is very homogenous.
The Rosetta-gami (D3) E. coli expression system was chosen for the large amount production of human FLT3 ligand (rhFL) using pET15b vector. This vector is specially developed for overexpression in prokaryotic systems. The N-terminal of the recombinant protein is fused with a cleaveable N-terminal hexahistidine (His6) tag to facilitate purification, followed by a thrombine cleaving site. The His6 tag allows purification of the recombinant protein using immobilized metal affinity chromatography (IMAC).
rhFL IB were isolated and purified under denaturing conditions using Ni Sepharose column. The denatured rhFL IB were refolded in a drop-wise manner and then the refolded rhFL was captured using Ni Sepharose column. After His6 tag removal, rhFL was purified using gel filtration column Superdex 75. The gel filtration separates proteins based on their molecular weight. Polishing purification was performed afterwards using ion exchange chromatography column. Capturing and purifications were performed in real time by following the 280 nm absorption.
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7.2. Overexpression of recombinant human FLT3 ligand, its refolding and purification
Two rhFL expression-plasmid (Figure 7.2.1) were available to be used for experiments. Plasmid pET15b-rhFL contains the full-length of human FLT3 ligand (rhFL) cDNA encoding residues 1-134 of the rhFL ectodomain, thrombin cleaving site and a cleaveable N-terminal His6
tag. pET15b-rhFL-AVI plasmid contains all the components of pET15b-rhFL with addition of an AVI tag. The overexpression of the contructs was under control of inducible T7 promotor.
Figure 7.2.1. rhFL expression-plasmid. Indicated clockwise; human FLT3 ligand cDNA encoding residues 1-134 cloned between NdeI and BamHI restriction sites, LacI coding region, ampilicine resitance gene.
As noted earlier, rhFL formed IB that needed to refolded to obtain functional and native form of the protein. In this experiment rapid dilution refolding was performed following the method written by Verstraete et al. 2009. In this method, the IB were added drop wise into refolding buffer. The refolding buffer contains high concentration of L-arginine which prevents hydrophobic aggregation and a redox couple that catalyses the correct forming of disulfide bridges (Verstraete et al., 2009).
Four hour after induction, the cells were lysed by sonication and this was followed by centrifugation to separate cell debris and IB. The IB were then washed with buffer containing Triton X-100 to remove hydrophobically associated proteins. Following this, IB were solubilized in 6 M GuHCl buffer and centrifuged to remove unsolubilized material. The supernatant was then loaded onto Ni-Sepharose column and rhFL was eluted using a stepwise pH gradient. The refolding of rhFL occured overnight at 4°C by drop-wise addition of denatured rhFL to a refolding buffer which contained redox couple GSH:GSSG, 1 M L-arginine. The redox couple promote disulfide exchange of the cysteines while L-arginine prevented hydrophobic aggregation. Note that protease inhibitor was not necessary because in the previous refolding experiments protein degradation was not observed. L-arginine was removed afterwards by dialysis because concentration higher than 1 M are not compatible
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with IMAC (GE Handbook). The refolded rhFL was then loaded on to Ni-Sepharose column and the proteins were eluted with 500 mM imidazole. After the proteins were desalted by phosphate-buffered saline (PBS), the concentrated proteins were digested overnight by using biotinylated thrombin to remove N-terminal His6
Three variants of rhFL were loaded into SDS-PAGE, followed by electrophoresis:
-tag. Thrombine was removed using streptavidin-agarose beads. rhFL was then purified further using SD 75 and MonoS with buffer containing 20 mM MES, 1 M NaCl pH 6 (MES buffer). Note that in this experiment, rhFL with AVI tag (rhFL-AVI) was first biotinylated before thrombin digest and further purifications.
• rhFL3; rhFL without AVI tag and His6
• rhFL-AVI-B; rhFL with AVI tag and without His
tag (digested with thrombin), expected molecular weight 15.856 kDa.
6
• rhFL-AVI-B-His
tag (biotinylated and digested with thrombin), expected molecular weight 17.781 kDa
6; rhFL with AVI tag and with His6
From the result shown in Figure 7.2.2, the difference in molecular weight of the three rhFL variants were detectable. At rhFL3 and rhFL-AVI-B lanes there was only one band detected each time meaning that thrombin digest occurred completely.
tag (biotinylated but not digested with thrombin), expected molecular weight 19.663 kDa
Figure 7.2.2. SDS-PAGE of rhFL variants. rhFL3, rhFL without AVI tag and His6 tag (15.856 kDa); rhFL-AVI-B, biotinylated rhFL with AVI tag and without His6 tag (17.781 kDa); rhFL-AVI-His6, biotinylated rhFL with AVI tag and His6
tag (19.663 kDa).
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7.3. Conclusion
Three rhFL variants were successfully made using pET15b vector and Rosetta-Gami as host strain. The thrombin digest to eliminate His6
tag occurred completely, resulting in rhFL3 and biotinylated rhFL-AVI
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Chapter 8
Interaction studies of recombinant human FLT3 ligand with recombinant human FLT3 domain 1 to 3, domain 1 to 4 and domain 1 to 5 receptor constructs
8.1. Isothermal Titration Calorimetry
Isothermal Titration Calorimetry (ITC) is a thermodynamic technique to measure the heat released or absorbed during a biomolecular binding event. Measurement of the heat enables accurate determination of affinity constants (KA
In our experiments, a solution of rhFL was titrated into recombinant human FLT3 receptor (rhFLT3) solution. We used rhFLT3 domain 1 to 3 (rhFLT3d123), rhFLT3 domain 1 to 4 (rhFLT3d1234) and rhFLT3 domain 1 to 5 (rhFLT3d12345) for interaction studies. HEK293S-GnTI
), reaction stoichiometry (N), enthalpie (ΔH) and entropy (ΔS). Hereby a complete thermodynamic profile of the molecular interaction is available in a single experiment.
-/-
• HEK293S-GnTI
stable cell lines for these rhFLT3 variants were already available to be used.
-/-
• HEK293S-GnTI-rhFLT3d123
-/-
• HEK293S-GnTI-rhFLT3d1234
-/-
Note that each of the recombinant proteins produced by these stable cell lines are fused a C-terminal His
-rhFLT3d12345
6 tag to facilitate the purification using IMAC. The interaction between the ligand and its receptor were tested in NaPO4,
MOPS, HEPES and Tris buffers. The choice of using these buffers was based on their different deprotonation enthalpies (Table 8.1.1).
51
Table 8.1.1. Deprotonation enthalpy (∆H) of NaPO4,
Buffer
MOPS, HEPES and Tris buffers
Deprotonation ∆H (kcal/mole)
NaPO 0,7 4
MOPS 4,9
HEPES 5,16
Tris 11,13
Heat released upon interaction of rhFL3 with rhFLT3 variants was monitored over time. Excess amount of rhFL in the syringe were titrated into the ITC cell containing rhFLT3. The quantity of heat absorbed or released was equally proportional to the amount of binding. When the system reached saturation, the heat signal decreased until only heat of dilution was observed. A binding curve can be obtained from a plot of the total integrated heat from each injection against the ratio of rhFL and rhFLT3 in the cell. The binding curve was analyzed with one set binding sites model to determine KA (1/KD), N, ΔH and ΔS. KD
The results of ITC experiments and the calculated Gibbs free energy (ΔG), -TΔS are given in Table 8.1.2. The enthalpy of complex formation in different buffers could correlate with the deprotonation ΔH of the buffers due to deprotonation or protonation events. This was however not the case in our experiments although the pH of the buffers were brought correctly at 37°C. The binding reaction was high exothermic and the negative entropy was penalty. Binding of ligand to rhFLT3 must have caused movement stringency of rhFLT3 where it lost some degree of freedom.
is the dissociation constant.
The N values of rhFLT3d1234 and rhFLT3d12345 were in the same range and they didn’t deviate a lot from the predicted N (0,5). However, this was not the case for rhFLT3d123. Previous binding studies (personal communication with K. Verstraete) showed a fraction of inactive rhFLT3d123 that didn’t bind to the ligand. The deviation of rhFLT3 N values could be due to the presence of this inactive fraction.
The affinity of rhFLT3d123 and rhFLT3d1234 were in the same range while the affinity of rhFLT3d12345 was significantly higher. This result indicated that domain 5 stabilizes the complex, possibly by mediating receptor-receptor interaction. However this contact couldn’t be defined as salt bridge interactions as one might expect a gain in enthalpy and loss in entropy by salt bridge formation. The finding that rhFLT3d123 and rhFLT3d1234 have similar binding affinity indicates that domain 4 does not mediate receptor-receptor interactions in contrast to what is seen for other members of the family e.g. KIT and PDGFR (Yang et al., 2008; Liu et al., 2007). The homotypic interactions of domain 4 in PDGFR are mediated by one conserved salt bridge which is not present in FLT3 receptor, and play an important role in their activation (Yang et al., 2008).
52
Table 8.1.1. Thermodynamics parameters. Dissociation constant (KD
), reaction stoichiometry (N), enthalpie (ΔH), calculated Gibbs free energy (ΔG) and entropy (ΔS).
rhFLT3d123
N K ΔH D ΔS ΔG -TΔS Buffer (nM) (cal/mol) (cal/mol) (cal/mol) (cal/mol) MOPS 0,11 12,7 -71111 -193,30 -11188,09 59922,91 HEPES 0,22 49,8 -35290 -80,46 -10347,26 24942,74
rhFLT3d1234
N K ΔH D ΔS ΔG -TΔS Buffer (nM) (cal/mol) (cal/mol) (cal/mol) (cal/mol) HEPES 0,45 40,1 -55920 -146,58 -10480,57 45439,43
Tris 0,43 31 -60030 -159,33 -10638,96 49391,04 NaPO 0,42 4 55,5 -69030 -189,51 -10280,58 58749,42
rhFLT3d12345
N K ΔH D ΔS ΔG -TΔS Buffer (nM) (cal/mol) (cal/mol) (cal/mol) (cal/mol) HEPES 0,42 8,7 -51749 -130,09 -11420,86 40328,14
Tris 0,45 6,9 -65690 -174,60 -11563,50 54126,50 NaPO 0,45 4 8,3 -59760 -155,84 -11449,82 48310,18
The ΔG, ΔH and –TΔS values including their error means are represented in graph (Figure 8.1.1).
53
Figure 8.1.1. rhFLT3d123, rhFLT3d1234 and rhFLT3d12345 ΔG, ΔH and –TΔS values, with their error means.
8.2. Surface Plasmon Resonance
Surface Plasmon Resonance (SPR) is a technique to measure biomolecular interactions which allows the verification of a binding event, the determination of binding affinity and the actual dissociation and association rates in a label free environment. An advantage of SPR is that the method can be used to study interactions which are enthapily neutral and also only small amounts of proteins are needed to obtain measurable signal.
In our experiments we immobilized the biotinylated rhFL containing AVI tag (rhFL-AVI-B) to the sensor surface. We choose not to immobilize the receptors to favor receptor-receptor interaction for complex formation. The rhFLT3 receptor variants were free in solution and passed over the surface. The association between rhFL and rhFLT3 was measured in respons units (RU).
For the experiments we also used the three rhFLT3 variants as in ITC and for each variant there were six samples prepared with different concentrations. The concentrations from high to low: 1 µM, 0.5 µM, 250 nM, 0.125 µM, 0.0625 µM and 0.03125 µM. The dilution series were made with buffer containing 10 mM HEPES, 150 mM NaCl pH 7.4 (HEPES buffer)
54
because Superdex 200 purifications of rhFLT3 variants were performed with this buffer. rhFL-AVI-B were first dialyzed from MES buffer into HEPES buffer.
We performed the steady-state analysis to analyze the interactions between the rhFLT3 receptor variants and the ligand.
The association and the dissociation were measured in RUs and given in a sensorgram (Figure 8.2.1).
55
Figure 8.2.1. rhFLT3d123, rhFLT3d1234 and rhFLT3d12345 steady state analysis sensorgram.
56
The measured RUs were then plotted as a function of rhFLT3 concentrations as shown in Figure 8.2.2. and also the KD Table 8.2.1’s were calculated ( ).
Figure 8.2.2. Respons units (RUs) plotted in function of rhFLT3 concentration ([rhFLT3]) in µM. rhFLT3d123, recombinant human FLT3 domain 1 to 3; rhFLT3d1234, recombinant human FLT3 domain 1 to 4; rhFLT3d12345, recombinant human FLT3 domain 1 to 5.
The value of the KD’s suggested that the affinity of rhFLT3d1234 and rhFLT3d12345 were in the same range, while the affinity of rhFLT3d123 was much lower. This is in contradiction to the KD’s attained from ITC experiments. However, taking the inactive rhFLT3d123 fraction into account, we suggested that if the active rhFLT3d123 was five times higher then what we had then its KD
would have decreased by factor five meaning that the affinity would be higher. In addition, we observed that the dissociation occurred slower as the domain became larger (Figure 8.2.1.) which corresponds to our ITC results that domain 5 stabilizes the complex.
Table 8.2.1. KD
s of rhFLT3 variants
rhFLT3d123 rhFLT3d1234 rhFLT3d12345KD (nM) 960.6 187.2 112.6
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8.3. Conclusion
Results of ITC experiment showed that the affinity of rhFLT3d123 and rhFLT3d1234 were in the same range while rhFLT3d12345 was higher. Based on this result, we concluded that domain 5 stabilizes the complex, possibly by mediating receptor-receptor interaction while domain 4 does not mediate receptor-receptor interactions in contrast to what is seen for KIT and PDGFR. The latter corresponds to the available structural information of FLT3 receptor where contacts between domain 4 are not observed (personal communication with K. Verstraete). On the other hand, results of SPR experiment showed that the affinity of rhFLT3d1234 and rhFLT312345 were in the same range while rhFLT3d123 was much lower. This is possibly due to the presence of inactive fraction of rhFLT3d123. This inactive fraction might also cause the deviated N at the ITC experiment. From the steady state analysis we observed that rhFLT3d12345 showed slow dissociation which confirmed that domain 5 stabilizes the receptor-receptor binding.
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Chapter 9 Selenium-Methionine incorporated recombinant human FLT3 receptor and recombinant human FLT3 ligand
9.1. Introduction
Nowadays it is possible to label proteins with Selenium-Methionine (SeMet) in a variety of expression systems. The SeMet incorporation can be performed in many expression systems that are being used in research such as yeast, insect and mammalian cells. This labeling can be used to produce protein samples to overcome the Phase problem in X-ray crystallography, such as being done by the group of Aricescu, Hon et al. in 2006.
A disadvantage of SeMet labeling is that the amount of the protein produced is lower than without labeling owing to the toxicity of SeMet. However, reported by Aricescu and co-workers they were able to phase the structures of two secreted proteins transiently expressed in mammalian cells with 60% SeMet incorporation. Based on this result it is worth trying to label recombinant human FLT3 receptor and recombinant FLT3 ligand with SeMet and try to crystallize the labeled proteins.
9.2. Overexpression of SeMet incorporated recombinant human FLT3 ligand and its purification
To overexpress SeMet incorporated recombinant human FLT3 ligand (rhFL-His6-SeMet), the B834 (DE3) pRARE E. coli strain was used. This strain is methionine auxotroph, protease deficient, chloramphenicol resistent and contains the pRARE plasmid which allows expression of genes encoding tRNAs for rare argentine codons AGA, AGG, and CGA, glycine codon GGA, isoleucine codon AUA, leucine codon CUA and proline codon CCC. A construct containing the full-length of rhFL cDNA encoding residues 1-134 of the rhFL ectodomain, thrombin cleaving site and N-terminal His6 tag, was available for direct use. The cells were cultured in medium containing SeMet after electroporation and grew normally in the presence of toxic selenium. The induction was then carried out by adding 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG).
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rhFL-His6-SeMet also formed inclusion bodies whereby the correct refolding procedure is performed to obtain functional and native form of the protein as described in paragraph 7.2. Small fraction of rhFL-His6-SeMet was separated before thrombin digest and it was loaded into SDS-PAGE gel(15% acrylamide) where electrophoresis followed. The rest of rhFL-His6-SeMet was concentrated and digested overnight using biotinylated thrombin to remove the His6 tag. rhFL-SeMet was loaded into anion exchange column (MonoS) and the eluted protein was loaded into SDS-PAGE where electrophoresis followed. Results of both electrophoresis showed rhFL-His6-SeMet with predicted molecular weight 17.738 kDa and rhFL-SeMet with predicted molecular weight 15.856 kDa (Figure 9.2.1
).
Figure 9.2.1. SDS-PAGE of recombinant human FLT3 ligand with SeMet. On the left side is recombinant human FLT3 with SeMetbefore thrombine digest (rhFL-His6-SeMet
9.3. Overexpression of SeMet incorporated recombinant human FLT3 domain 1 to 4 and its purification
) with predicted molecular weight 17.738 kDa and on the right side is after thrombine digest (rhFL-SeMet) with predicted molecular weight 15.856 kDa.
Two HEK293S-GnTI-/--rhFLT3d14 stable cell lines, colony 10 and colony 14 were used to overexpress recombinant human FLT3d1234 containing C-terminal His6
After the induction, the cells were observed every day until day five to see if they stay alive in the presence of toxic selenium, which they did. The medium was then harvested and filtered through 0.22 µm filter. To capture the recombinant proteins, IMAC was carried out by using a
tag (rhFLT3d1234). The cells were grown in normal medium containing methionine for large scale in 175 cm² giant falcons. Before induction with tetracycline and sodium butyrate, the medium was change into minimal medium without methionine and SeMet was added into the medium. In this way the cells could only use the added SeMet to synthesized recombinant proteins.
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25 ml talon column. After a wash step, the recombinant proteins are eluted with 300 mM imidazole. The elution fraction was concentrated to one ml and then injected to SD200 gel filtration column.
The amount of SeMet incorporated recombinant human FLT3d1234 (hFLT3d1234-SeMet) was very low (Figure 9.3.1). rhFLT3d1234-SeMet came out between 70 ml and 80 ml at elution rate of 1 ml/min which was similar to rhFLT3d1234 (result not shown). The concentration of rhFLT3d1234-SeMet was 5 mg/ml which is sufficient for crystallization.
Figure 9.3.1. Superdex 200 purification chromatogram of hFLT3d1234-SeMet. hFLT3d1234-SeMet came out after 70 ml at elution rate of 1 ml/min with low amount.
Two fractions of the peak were tested on SDS-PAGE to confirm rhFLT3d1234-SeMet. The bands reside at more and less expected height (47.756 kDa) which indicated the present of rhFLT3d1234-SeMet (Figure 9.3.2).
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Figure 9.3.2. SDS-PAGE of rhFLT3d1234-SeMet and rhFL-His6
-SeMet. rhFLT3d1234-SeMet reside at 47.756 kDa.
9.4. Complex formation of SeMet incorporated recombinant human FLT3d1234 and SeMet incorporated recombinant human FL and its purification.
To make rhFLT4d1234-SeMet and rhFL-SeMet protein complex, the receptor was added to an excess of ligand and incubated at room temperature for five minutes. The sample was then injected to SD 200 column and eluted with 20 mM MES, 150 mM NaCl pH 6.5 buffer. The peak came out between 62.5 ml and 70 ml at elution rate of 1 ml/min (Figure 9.4.1). This peak shift suggested that there was a complex formed between the receptor and the ligand. The peak which came after predominant peak was the excess of rhFL-SeMet protein (Figure 9.4.1).
One fraction of the predominant peak was tested on SDS-PAGE to confirm the complex formation which gave positive result (result not shown). The complex were then used for crystalization trials.
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Figure 9.4.1. Superdex 200 purification chromatogram of rhFLT3d1234-SeMet and rhFL-His6
-SeMet complex. There was a shift in the predominat peak which gave indication of rhFLT4d1234-SeMet and rhFL-SeMet complex forming. Excess of rhFL-SeMet came out as second and smaller peak.
9.5. Conclusion
Incorporation of SeMet in rhFLT3d1234 and rhFL were performed successfully although the yield of rhFLT4d1234-SeMet was low. rhFLT3d1234-rhFL complex is on crystallization trials.
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Section IV Materials and methods
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Chapter 10
Cloning of recombinant human FLT3 domain 1 and domain 1 to 2, small and large scale expression, purification, crystallization trials and complex formation study
10.1. Cloning of recombinant human FLT3 domain 1 and domain 1 to 2
10.1.1. Cloning to pCR2.1-TOPO vector
rhFLT3 cDNAs encoding the extracellular domain 1 and domain 1 to 2 of the receptor were synthetised. Polymerase chain reaction (PCR) was performed using M13 reversed primers, M13 (-20) forward primers and Easy-A polymerase (Invitrogen, Carlsbad, CA, USA) which has proofreading capacity and generates 3’-adenine sticky ends. The content of each reaction mix is given Table 10.1.1.
Table 10.1.1. PCR reaction mix.
Volume (µl) Forward primer (5 µmol/µl) 15 Reverse primers (5 µmol/µl) 15
DNA template (10 ng/µl) 3 dNTP’s 3
10X Easy-A buffer (containing Mg) 15 Easy-A polymerase 1,5
Distilled water 97,5
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PCR was carried out following a program describe in Table 10.1.2. The annealing was performed at 50, 55 and 60°C to investigate which temperature gives the best results.
Table 10.1.2. PCR program.
Step Temperature (°C) Time (min) Hot start 95 2 Initiation 95 1 Annealing 50-55-60 1 Extension 75 2 Repeating Initiation to Extention 29 times
Final extention 72 10
The PCR products were separated on a 1% agarose gel. The DNA bands at the expected height were purified from agarose gel extraction kit by the QIAquick protocol (Qiagen, Venlo, Netherlands). The purified DNA was cloned into pCR2.1-TOPO vector (Figure 10.1.1
) (Invitrogen, Carlsbad, CA, USA) using TOPO cloning technology and then transformed into TOP10 electrocompetent cells (Invitrogen, Carlsbad, CA, USA).
Figure 10.1.1.
Using Birnboim-Doly protocol for plasmid extraction, one colony was selected that had incorporated gene of interest. The plasmid was then prepared using Qiagen Plasmid Midi kit
pCR2.1-TOPO vector map (Invitrogen, Carlsbad, CA, USA). Indicated clockwise from +1: Plac, promoter; Multi Cloning Site (MCS), lacZα coding region, f1ori, kanamycine resistance gene, ampicilin resistance gene, pUC ori.
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(Qiagen, Venlo, Netherlands). The rhFLT3d1 and rhFLT3d12 fragments were cut from pCR2.1-TOPO vector using AgeI and KpnI restriction enzymes. The fragments were tested on a 1% agarose gel. The DNA bands reside at the expected height were purified from agarose gel following the QIAquick Gel Extraction Kit protocol (Qiagen, Venlo, Netherlands).
10.1.2. Cloning to pcDNA4-TO vector
To generate a stable HEK293S-GnT-/-
Figure 10.1.2 cell line, the rhFLT3d1 and rhFLT3d12 fragments
obtained from pCR2.1/TO vector cloning were ligated in pcDNA4-TO vector ( ) which was linearized using AgeI and KpnI restriction enzymes (Invitrogen, Carlsbad, CA, USA), designated as pcDNA4/TO-hFLT3d1 and pcDNA4/TO-hFLT3d12. By using AgeI an KpnI restriction enzymes, the recombinant proteins were fused with C-terminal His6 tag and N-terminal Mu secretion signal. The ligation was performed using T4 DNA ligase (Promega, Madison, USA) and the
pcDNA4-TO vector consist of a Tetracyline Regulated Expression System (T-Rex, Invitrogen, Carlsbad, CA, USA) which allows inducible expression, applicable for transient as well as stable expression. The pcDNA4-TO vector contains the following key elements:
pcDNA4/TO-hFLT3d1 and pcDNA4/TO-hFLT3d12 were prepared by QIAquick Spin (Qiagen, Venlo, Netherlands).
• A cytomegalovirus (CMV) promoter followed by additional control elements of the bacterial tetracycline resistance operon (2X TetO2) to repress the effective transcription of one of the strongest mammalian promoter sequences. Tetracycline repressor protein (TR) binds to the operator and 2X TetO2 thus blocking transcription initiation. When tetracycline (Tet) is added into the medium, it binds TR and causing conformational change in TR. This conformational change in turn will released TR from 2X TetO2 whereby transcription is unrepressed starting from CMV promoter and a high expression of the desired protein takes place.
• Multi cloning site (MCS) for easy cloning of construct of interest. • Zeocine resistance gene for effective selection to create a stable mammalian cell.
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Figure 10.1.2. pcDNA4/TO vector map (Invitrogen, Carlsbad, CA, USA). Indicated clockwise: Multi Cloning Site (MCS), BGH polyadenylation sequence; f1 ori, SV40 ori, EM-7 promoter, zeocin resistance gene, SV40 early polyadenylation sequence, pUC ori, ampicilin resistance gene, cytomegalovirus (CMV) promoter, TATA box and Tetracycline operator (2X TetO2) sequence.
pcDNA4/TO-hFLT3d1 and pcDNA4/TO-hFLT3d12 were transformed into TOP10 electrocompetent cells (Invitrogen, Carlsbad, CA, USA). Following Birnboim-Doly protocol for plasmid extraction, one colony was selected that had incorporated gene of interest. The plasmid was then prepared using Qiagen Plasmid Midi kit
(Qiagen, Venlo, Netherlands). The correctness of pcDNA4/TO-hFLT3d1 and pcDNA4/TO-hFLT3d12 were confirmed by DNA sequencing using BGHREV, CMV-FORWARD primers for both constructs (COGENICS, Beckman Coulter GENOMICS, UK).
10.2. Small scale expression of recombinant human FLT3 domain 1 and domain 1 to 2
Small scale expression of rhFLT3d1 and rhFLT3d12 was performed in HEK293T cells, according to the protocol described by Aricescu et.al 2006. This protocol starts with an expression test in six-well plate using 0% FCS DMEM, with or without kifunensine in order to check if the recombinant protein is formed, to have an idea about the expression level and about the influence of kifunensine on glycoprotein. Kifunensine is an alkaloid produced by actinomycete Kitasatosporia kifunensine and it is a potent α-mannosidase I (MI) inhibitor (Figure 10.2.1) (Chang et al., 2007). Adding kifunensine to the expression system will increase homogeneity of the recombinant proteins.
If the expression level is high, transient expression can be used for large scale production. On the other hand stable cell line must be generated in case of low expression level.
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Figure 10.2.1. Small section of glycosylation pathway. MI is α-mannosidase I. represents N-Acetlyglucosamine, represents mannose. Picture is modified from Chang, Crispin et al. 2007.
HEK293T cells were grown in six-well plates until the cells were 90% confluent. Dulbecco’s Modified Eagle’s Medium (DMEM) (DMEM high glucose, Sigma) was used as growth medium with addition of 10% fetal calf serum (FCS), 1% L-glutamine, 0.2% penicillin and streptomycin. For transient expression per well, a DMEM mixture was made of plasmid DNA, 25 kDa branched polyethyleneimine (PEI) in order to attain PEI/DNA ratio of 1.5 and two ml DMEM. The growth medium was removed and replaced with DMEM mixture.
Five days after induction with Tet and sodium butyrate, the expression medium was collected. The medium was centrifugated at high speed for one minute to precipitate contaminants. 30 µl of the medium was mixed with 20 µl leammli buffer and incubated for 10 minutes at room temperature. 20 µl of the samples was loaded into SDS-PAGE gel (15% acrylamide) where electrophoresis followed. Subsequently Western Blot was performed where the proteins were transferred to a nitrocellulose membrane at which they were probed by specific antibodies against the protein of interest. In this experiment we used anti-His antibody against the C-terminal His6
tag. From this experiment we observed that the expression was low and therefore stable cell line must be generated.
10.3. Large scale expression and purification of recombinant human FLT3 domain 1 and domain 1 to 2
Large scale expression of rhFLTd1 and rhFLT3d12 were performed in HEK293S-GnTI-/- cells. These cells are deficient in N-Acetylglucosamine transferase I (GnTI) gene (GnTI-/-
). Adding kifunensine to this expression system will increase sugar homogeneity of the recombinant proteins (Figure 10.3.1).
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Figure 10.3.1. Small section of glycosylation pathway. MI is α-mannosidase I, GnTI is N-Acetylglucosamine transferase I. Symbol repesents N-Acetlyglucosamine, represents mannose. Picture is modified from Chang, Crispin et al. 2007.
rhFLT3d1 and rhFLT3d12 HEK293S-GnTI-/- stable cell lines were obtained under zeozine selection. They were grown until 90% confluent in 175 cm² giant falcons (Greiner Bio-One) by using DMEM as growth medium. We used giant falcons because our HEK293S-GnTI-/-
Five days after induction the medium was collected and centrifuged at 11.000 g, 4°C for 30 min to remove the cells and contaminants. The medium was then filtered through 0.22 µm filter (Corning) and loaded onto Talon Superflow column (Clontech Laboratories, Inc), connected to an ÅKTA Explorer system (GE Healthcare). Column was first equilibrated with binding buffer (BB) containing 50 mM NaPO
stable cells were unable to stay attach in the roller bottle. This medium contained 10% FCS, 1% L-arginine, 0.2% penicillin and streptomycin. The medium was then removed and replaced with DMEM medium containing 0% FCS while the rest of the components were still present. The induction was performed by adding 4.5 ml/l Tet (1 M stock solution) and 4.5 ml/l sodium butyrate (400 µg/ml stock solution).
4 and 300 mM NaCl pH 7.2. Recombinant proteins were eluted using elution buffer (EB) containing 50 mM NaPO4
, 300 mM NaCl pH 7.2 and 200 mM imidazole. Protease inhibitor mix (Complete, Roche) was added into the protein solution. The protein solution was concentrated using 5.000 kDa cut-off concentrator until one ml. Subsequently the concentrated protein solution was injected onto onto a Prep-Grade HiLoad 16/60 Superdex 200 or Superdex 75 column (GE Healthcare, Buckinghamshire, UK), connected to an ÅKTA Explorer system (GE Healthcare). Note that the column was equilibrated with HEPES buffer containing 20 mM HEPES and 150 mM NaCl pH 7.4. The eluted recombinant proteins were then fractionated. The total amount of recombinant receptors produced were estimated based on the absorbance of the sample at 280 nm and calculated absorption 1% of 0.721 and 0.775 g/l respectively.
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10.4. Crystallization (trials) of recombinant human FLT3 domain 1
Sitting drop vapor diffusion method was used for crystallization. A 24-well plate (Hampton Research) was utilized for crystallization trials where 500 µl of reagent was added into liquid reservoir and then one µl of protein solution was mixed with one µl of reagent in the vapor equilibration. Initially the droplet of protein solution contain half of the precipitant concentration as in the reservoir which is insufficient for crystallization. As water vaporizes from the drop and transfers to the liquid reservoir, the precipitant concentration increases to a level optimal for crystallization. Since the system is in equilibrium, these optimum conditions are maintained until the crystallization is complete. A figure of a well in sitting drop plate is given in Figure 10.4.1.
Figure 10.4.1. Sitting drop-well. Precipitant concentration in the drop [ppt]drop is half of precipitant concentration in the reservoir [ppt]reservoir
PEG Ion Screen 1 and 2 and Crystal Screen 1 and 2 were used as reagent (Hampton Research). After crystals were formed in 0.01 M CoCl
. Water will be extracted from the drop over time to reach concentration equilibrium. Picture is taken from Hampton Research.
2, 0.1 M MES pH 6.5 and 1.8 M (NH4)2SO4
The crystals were washed six times using the same reagent as they were formed and subsequently loaded into SDS-PAGE gel (15% acrylamide). The washing steps were necessary to remove any impurities, if such existed. After electrophoresis, the gel was colored following the silver staining protocol. The crystals were also tested using Western Blot where they probed specifically using anti-His antibody to confirm that the higher bands were also rhFLT3d1.
, Izit Crystal Dye (Hampton Research) test was performed to investigate if the crystals were protein crystal by adding 0.2 µl dye into the drop.
To cyroprotect the crystals, cryoprotectors were prepared which have the same composition as the mother liquor, supplemented with:
• 20-25 % glycerol • 20% sucrose
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• 20% sorbitol • 3 M (NH4)2SO
Paraton is a cryoprotectant and can be used directly. 4
10.5. Complex formation studies
The complex formation studies between rhFLT3d1, rhFLT3d12 with rhFLT3 ligand were performed with the aim to crystallize the complex. All samples were centrifuged at 25.000 g, 4°C for five minutes and then injected separately into analytical Superdex 200 column at elution rate of 40µl/min. The column was equilibrated by buffer containing 20 mM HEPES and 150 mM NaCl pH 7.4. An equimolar amount of rhFLT3d1 and rhFLT3d12 was mix with rhFLT3 ligand and incubated at room temperature for five minutes. The mixture was injected into analytical Superdex 200 column at rate of 40 µl/min.
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Chapter 11
Recombinant human FLT3 ligand refolding and purifications
11.1. Expression of recombinant human FLT3 ligand in E. coli
A cDNA clone containing human FLT3 ligand (full-length) cDNA (rhFL) and FLT3 ligand (full-length) cDNA with AVI tag (rhFL-AVI) were already cloned in the pET-15b vector, designated as pET15b-rhFL and pET15b-rhFL-AVI respectively, available to be used for expression experiments for both ligand variations.
Both vectors were used to transform Rosetta-Gami (DE3) strain (Novagen) which carries the chloramphenicol and pRARE plasmid for selection. Expression cultures were grown at 37 °C in Luria-Bertani (LB) medium, containing carbenicilin (100 µg/ml) and chlorampenicol (34 µg/ml). Expression of rhFL was induced when the culture reached OD600
= 0.6 by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Hereafter, the culture was grown for an additional 4 h. The bacteria were then harvested by centrifugation at 6.000 g, 4 °C for 20 min.
11.2. Isolation of rhFL inclusion bodies and its purifications under denaturing conditions
The bacterial pellet was resuspended in lysis buffer containing 50 mM Tris pH 8.0, 100 mM NaCl, 1% Triton X-100, 1 mM EDTA and 0.2 M PMSF. The cells were lysed by sonication on a 250-D Branson sonifier with total run time of 4 min, 30 s pulses at 30% output interspersed with 30 s of down time. Cell debris and inclusion bodies (IB) were then isolated by centrifugation at 20.000 g, 4 °C for 30 min. The IB were washed twice following this protocol:
• Pellets containing the IB were solubilized in lysis buffer and sonicated for 30 s at 30% output.
• The suspention was incubated on a ‘head-overhead’ shaker for 30 min followed by centrifugation at 20.000 g, 4 °C for 30 min.
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• Finally, the washed IB were solubilized in guanidium hydrochloride (GuHCl) buffer containing 6M Guanidium Chloride, 100 mM NaPO4
Insoluble material was discarded by centrifugation at 100.000 g, 4 °C for 30 min. The supernatant was loaded onto a Ni-Sepharose 6 FF (GE Healthcare) column. This column was equilibrated first by GuHCl buffer pH 8.0. The column was washed afterwards with five column volumes (CV) GuHCl buffer at pH 6.3 and pH 5.8. Denatured rhFL was eluted with five CV GuHCl buffer at pH 4.5 and neutralized with Tris pH 8.0. The total amount of rhFL produced was estimated based on the absorbance of the sample at 280 nm and calculated extinction coefficients of 19855 /M cm and 25355 /M cm for rhFL and rhFL-AVI respectively.
, 10 mM β-mercaptoethanol pH 8.0 by gentle rotation in a ‘head-overhead’ shaker for 2 h at room temperature.
11.3. In vitro refolding of rhFL (and rhFL-AVI) and its purification
The denatured rhFL was added drop-wise to refolding buffer containing 100 mM Tris pH 8.5, 1 M L-arginine, 5 mM GSH, 0.5 GSSG and 0.2 mM PMSF overnight at 4°C under rapid mixing, to a final concentration of 0.1 mg/ml. The refolding mixture was then dialyzed against 20 mM Tris pH 8.0. Centrifugation followed subsequently to remove aggregates and the clarified refolding mixture was loaded onto a Ni Sepharose 6 FF column. The column was first equilibrated with buffer containing 50 mM NaPO4 and 300 mM NaCl pH 8.0. Refolded rhFL was then eluted with equilibration buffer containing 500 mM imidazole. The eluted rhFL was desalted into 10 mM NaPO4, 150 mM NaCl, pH 7.0 phosphate-buffered saline (PBS). Afterwards, the protein solution was incubated overnight at room temperature with one unit of biotinylated thrombine (Novagen) per milligram rhFL to remove the N-terminal His6
After His
tag. SDS-PAGE electrophoresis was performed to ensure proteolysis cleavage occurred completely. The biotinylated thrombine was removed using streptavidin-agarose (Novagen). Note that, rhFL-AVI was first biotinylated before thrombin digest and further purifications.
6
tag removal, rhFL was injected onto a Prep-Grade HiLoad 16/60 Superdex 75 column (GE Healthcare, Buckinghamshire, UK) which was connected to an ÅKTA Purifier system (GE Healthcare). This column was equilibrated with PBS. The fractions corresponding to rhFL were pooled, concentrated and then diluted into cation-exchange running buffer containing 20 mM MES pH 6.0 to obtain 25 mM NaCl final concentration. The diluted rhFL was loaded onto a MonoS HR 5/5 column (GE Healthcare) connected to ÅKTA Purifier system. Elution of rhFL occurred using a linear gradient from 1 to 1 M NaCl.
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Chapter 12
Interaction studies of recombinant human FLT3 ligand with recombinant human receptors
12.1. Isothermal Titration Columetry
12.1.1. Introduction
Isothermal Titration Calorimetry (ITC) is a thermodynamic technique to measures the heat released or absorbed during a biomolecular binding event. Measurement of the heat enables accurate determination of affinity constants (KA
), reaction stoichiometry (N), enthalpie (ΔH) and entropy (ΔS). Hereby a complete thermodynamic profile of the molecular interaction is available in a single experiment. A figure of ITC cells and syringe is given in Figure 12.1.1.
Figure 12.1.1. Diagram of ITC cells and syringe. Figure is taken from MicroCal Technology.
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12.1.2. Sample preparations and interaction studies
rhFLT3d123, rhFLT3d1234 and rhFLT3d12345 HEK293S stable cell lines were available for direct use. The recombinant protein expression and purification occurred as already explained in paragraph 10.3 for the receptor. The total amount of rhFLT3d123, rhFLT3d1234 and rhFLT3d12345 produced were estimated based on the absorbance of the sample at 280 nm and calculated extinction coefficient of 37650, 50850 and 76205 /M cm respectively. The receptors and rhFL (without AVI tag) were dialyzed separately against MOPS, HEPES, Tris, NaPO4
All samples were centrifugated at 25000 g, 4 °C for five minutes to remove possible aggregates and partikels and then degassed before brought into ITC device (MicroCal Technology, GE Healthcare). The ITC device was first brought to 37 °C and the working buffer was injected to reference cell. Approximately 2200 µl of recombinant receptors (1 µM) were then injected into the Sample Cell (Figure 12.1.1). An excess of rhFLT3 (10 µM) of approximately 800 µl were brought into the syringe. The injection of rhFL3 into the receptor took place as follows:
buffers. These buffers were chosen because their pKa’s at 37 °C were 7.02, 7.31, 7.72 and 7.20 respectively, in the range of physiological pH.
• Time between subsequent injections: 300 s • Volume injected: 10 µl • Amount of injections: 20
Heat released upon interaction of rhFL3 with rhFLT3 receptors was monitored overtime. As rhFL in the syringe were titrated into the ITC cell, the quantity of heat absorbed or released was equally proportional to the amount of binding. When all receptors were saturated, the heat signal decreased until only heat of dilution was observed. A binding curve can be obtained from a plot of the heat from each injection against the ratio of rhFL and rhFLT3 in the cell. An example of ITC binding curve is given in Figure 12.1.2. The binding curve was analyzed with one set binding sites model to determine KA (1/KD) where KD
is dissociation constant, N, ΔH and ΔS.
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76
Figure 12.1.2. rhFLT3d12345+rhFL3 ITC binding curve. N, reaction stoeichiometry; K, affinity constant; ΔH, reaction enthalpy; ΔS, reaction entropy.
12.2. Surface Plasmon Resonance
12.2.1. Introduction
Surface Plasmon Resonance (SPR) is a physical process occurring when plane-polarized light hits a metal film under total internal reflection conditions. Physics courses have taught us that when a light beam coming from a dense medium hits a half circular prism, it is bended towards the plane of interface as it going out to less dense medium. In SPR a gold layer is inserted between the dense and less dense medium (e.g. glass and buffer). Light energy is lost to the gold electrons which oscillate to form surface plasmons. Resonance occurs when the momentum of the incoming photons is equal to the momentum of the plasmons. In short, at a certain angle there is a minimum intensity of light. SPR measures changes in the resonance angle in real time. The change in the SPR is directly correlated to the amount of protein near the surface of the chip. The principles of Biacore SPR technology we used are depicted in Figure 12.2.1
.
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77
Figure 12.2.1.
Biacore SPR technology principles.
12.2.2. Immobilization of rhFL and interaction studies
The Sensor Chip SA (GE Healthcare) contains four flow channels (Fch). The active Fch one (Fch 1) and two (Fch 2) were equilibrated with running buffer, HEPES buffer (10 mM HEPES, 150 mM NaCl pH 7.4) in our case, until the baseline was stable. Fch 1 was used as reference. The biotinylated rhFL with AVI tag (rhFL-AVI-B) dialyzed against HEPES buffer. Note that all samples were centrifuged at 25000 g, 4 °C for five minutes to remove possible aggregates and particles. In order to immobilize rhFL-AVI-B the on Fc 2, protein sample was 106X diluted and injected for 120 s to the sensor surface. The association and the dissociation were measured in RUs. The final immobilization RU was obtained by substracting the RU of Fch 2 with Fch 1.
For the steady state analysis, there were six 1 ml rhFLT3 dilutions prepared; 1 µM, 0.5 µM, 250 nM, 0.125 µM, 0.0625 µM and 0.03125 µM. The dilution series was prepared in running buffer. Each sample was injected for 3000 s to the sensor surface. The association and dissociation occurred were measured in RU. The RU values of rhFLT3d123, rhFLT3d1234 and rhFLT3d12345 binding with ligand were plotted as function of rhFLT3 concentration using GraphPad Prism software and also the KD
’s were calculated.
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Chapter 13
Large scale expression of Selenium-Methionine incorporated recombinant human FLT3 receptors and recombinant human FLT3 and purifications
13.1. Large scale expression of Selenium-Methionine incorporated recombinant human FLT3 receptors
The rhFLT3d1234 HEK293S stable cell line was already available. With the aim of solving the Phase problem by SAD or MAD, we used rhFLT3d1234. Previous studies have shown that rhFLT3d1234 and rhFLT3 ligand complex can be diffracted at resolution 4.2 Å (personal communication with K. Verstraete). The cell culturing and induction were performed in the same way as described in paragraph 10.3. However, when the cells were 90% confluent, the DMEM medium was removed and the cells were wash using PBS in order to remove methionine (Met) traces. Subsequently DMEM medium without Met and 30 mg/L Selenium-methionine (SeMet) was added. Purifications were performed as described in paragraph 10.3.
13.2. Large scale expression of Selenium-Methionine incorporated recombinant human FLT3 ligand
B834 (DE3) pRARE E.coli strain was used to over express SeMet incorporated rhFL-His6-SeMet. This strain is methionine auxotroph, protease deficient, chloramphenicol resistent and contains the pRARE plasmid which allows expression of genes encoding tRNAs for eukaryotic arginine codons AGA, AGG, and CGA, glycine codon GGA, isoleucine codon AUA, leucine codon CUA and proline codon CCC. A construct containing the full-length of rhFL rcDNA encoding residues 1-134 of the rhFL ectodomain, thrombin cleaving site and N-terminal His6 tag, was available for electrocompetent cells transformation.
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Expression cultures were grown at 37°C in SelenoMet Base Medium plus Nutrient Mix (AthenaES) minimal medium. By using chemically defined minimal medium without methionine, the bacteria will incorporate the SeMet when they produce recombinant proteins. SeMet (30 mg/l), carbecilin (100 µg/ml) and chlorampenicol (34 µg/ml) were added into the minimal medium. Expression of rhFL was induced when the culture reached OD600
The isolation of IB and its purification under denaturing conditions were performed as described in paragraph 11.2. Afterwards, in vitro refolding and further purification were carried out as described in paragraph 11.3.
= 0.6 by adding IPTG to a final concentration of 1 mM. Hereafter, the culture was grown for an additional 4 h. The bacteria were then harvested by centrifugation at 6.000 g, 4 °C for 20 min.
13.3. Complex formation
SeMet incorporated receptor and ligand complex were prepared for crystallization trials. An equimolar amount of rhFLT3d1234-SeMet was mixed with rhFL-SeMet and incubated at room temperature for five minutes. The mixture was then centrifuged at 25000 g, 4°C for five minutes and injected into analytical Superdex 200 column at rate of 40 µl/min.
General conclusion
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Section V
General conclusion
General conclusion
81
General conclusion
The threedimensional structure of the FLT3 receptor and the architecture of the FLT3 ligand-receptor complex are still unknown today. This master thessi was conducted in a framework that aim to elucidate the X-ray structure of the FLT3 complex and characterize the contribution of the various Ig-like domains of the receptor by methods as analytical gel filtration, SRP and ITC.
In the laboratory it was previously shown that FLT3 Ig-like domains confine the high affinity ligand binding site, as was shown for the two other RTK members, FMS and KIT. The aim of this master thesis was to produce rhFLT3d1 and rhFLT3d12 and investigate their role and binding. Additionally, a high resolution X-ray structure of such a minimal FLT3 receptor could provide essential phase information to solve the existing 4.2 Å dataset for the (FLT3d14)2
It was found that both rhFLT3d1 and rhFLT3d12 can be produced as secreted proteins by an inducible stable HEK293 GnT
-FL complex.
-/- cell line. It was shown that neither rhFLT3d1 and rhFLT3d12 shows a high affinity complex with the ligand. So, it was shown that domain 3 is necessary to for the binding site, which was later confirmed by X-ray studies (personal communication with K. Verstraete). The atypical Ig-like domain1 of FLT3 can be crystallized in the presence of Co2+ and Ni2+
ITC and SPR data or the interaction of rhFL and various constructs of it receptor led to the conclusion that domain 5 stabilizes the complex possibly by mediating homotypic interactions, and that domain 4 does not form such contacts in contradiction with the other RTK members. This is in agreement with the finding that salt bridge that mediates these domain 4 receptor contacts is absent in FLT3 receptor and with the emerging structure of FLT3d1234-rhFL complex.
as salt. The crystals diffracted maximally to a resolution of 4.7 Å. Moreover it was found that the crystals are twinned and diffract anisotropically. Lowering the heterogeneity due to N-linked glycosylation by prior treatment with EndoH or by high resolution ion exchange chromatography to resolve different glycosylation forms could improve crystal quality in the future.
List of references
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Section VI
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Samenvatting
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Section VII
Samenvatting
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Samenvatting Inleiding FMS-like tyrosine kinase 3 ligand FMS-like tyrosine kinase 3 ligand (FLT3 ligand) cytokine wordt veel vermeld in de literaturen. Blijkbaar is FLT3 ligand een groeifactor die in staat is om de proliferatie van hematopoëtische voorlopercellen van zowel lymfoïde en myeloïde oorsprong te stimuleren. FLT3 ligand is ook een belangrijke groeifactor voor dendritische cellen (DCs) en Natural Killer cells (NKCs) homeostasis in vivo.
FLT3 ligand werd ontdekt door twee verschillende onderzoeksgroepen (Lyman et al., 1993, Hannum et al., 1994) in een korte tijdspanne. Hoewel ze verschillende methoden hanteerden, gebruikten zowel Lyman als Hannum de FLT3 receptor om ligand FLT3 te isoleren. Ze isoleerden transmembraan alsook oplosbare FLT3 ligand, waar beide vormen zijn biologisch actief. Door comparative sequencing werd vastgesteld dat het FLT3 ligand ORF bij de muis alsook bij de mens structureel gerelateerd is aan KL en aan M-CSF, waarvan reeds bekend was dat deze de α-helix cytokine fold aanhalen en een disulfide brug netwerk hebben.
Een structuuractiviteitsstudie werd uitgevoerd door Graddis en zijn medewerkers waarbij ze willekeurig gemuteerd FLT3 ligand hebben gescreend door gebruik te maken van een FLT3 receptor-Fc fusieeiwit. FLT3 ligand werd als een homodimeer voorspeld, waarbij L27P alsook A64T mutaties de monomer-dimer equilibrium naar de monomer verschuiven en hun biologische activiteit verminderen. Er zijn drie “hot spots” van hoge aminozuur substitutie frequenties die zich groeperen in een klein surface patch van de tertiaire structuur, namelijk op posities 8-15,81-87 en 116-124. Door substitutie op posities 8,84,118 en 122 verbetert de binding en de biologische activiteit. Maar de H8R substitutie in plaats van H8Y leidt tot een verminderde FLT3 ligand activiteit. De structuur van FLT3 ligand werd volledig opgelost door Savvides et al. in 2000 door gebruik te maken van een multiple isomorfe replacement (MIR) bij een resolutie van 3.2 Ǻ, wat daarna verfijnd werd tot een resolutie van 2.2 Ǻ. Ze bevestigden dat FLT3 ligand inderdaad een homodimer is dat de short chain helical cytokine fold aanneemt.
FMS-like tyrosine kinase 3 receptor Verschillende polypeptiden die een belangrijke rol spelen in cel proliferatie, differentiatie, migratie en metabolisme voeren hun acties uit door zich te binden aan celoppervlakte receptoren met tyrosine kinase activiteit en ze daardoor te activeren. Deze celoppervlakte receptoren zijn bekend om receptor tyrosine kinases (RTKs) te zijn. Alle RTKs zijn glycoproteinen die uit geglycosyleerde extracellulaire domeinen, een hydrofobe
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transmembrane helix en cystoplasmic kinase domeinen bestaan. De receptoren zijn geoligomeriseerd bij ligand-binding en gevolgd door conformationele verandering van het extracellulaire domein. Deze laatste stabiliseert de interacties tussen de aangrenzende cytoplasmische domeinen en leidt tot activering van de kinase functie.
Wanneer de RTKs actief zijn, catalyseren ze de fosforilering van exogene subtraten alsook tyrosine residu’s binnenin hun eigen polypeptide keten, ook bekend als autofosforilering, Hierbij, één receptor molecule fosforileert de andere receptor in de dimeer. Autofosforilatie komt hoofdzakelijk voor bij twee verschillende klassen van tyrosine residu’s, namelijk op het beschermde tyrosine residu in de activation loop van het kinase domein, of op een tyrosine residu buiten de kinase domein dat dient als docking site for downstream signaaltransductie moleculen die Scr homologie 2 (SH2) domeinen bevatten.
Transmembraan (TM) domein van RTKs spelen een actieve rol in signalisering. TM domein draagt bij tot de stabiliteit van de full-length receptor dimeren en behouden een signaling-competent dimerische receptor conformatie. Het juxtamembrane domein is een korte regio dat het transmembrane domein en het cytoplasmische domein verbindt. De sequenties van deze regio zijn divergent tussen receptor subklassen maar geconserveerd tussen de leden van dezelfde subklasse en het is betrokken bij de modulatie van receptor functies. Klasse III, IV en V RTKs beschikken over de kinase insert domain, waardoor de kinase domein in twee wordt verdeeld. Deze insertie is tot 100 aminozuren waar de meeste hoofdzakelijk hydrofiele aminozuren zijn.
De onderzoeksgroep van Matthews heeft in 1991 degeneratieve oligonucleotiden gebruikt, in een PCR-gebaseede strategie om een novel receptor fragment te isoleren van hoog opgezuiverde muisfoetale leverstamcellen. Dit deel wordt dan gebruikt om de full-length receptor kloon te isoleren dat fetal liver kinase 2 (FLK-2) werd genaamd. De extracellulair domain protein sequenties van FLK-2, KIT en FMS werden vergelijkt. Het resultaat toonde aan dat 30% van de homologie gebaseerd is op consensus aminozuren en conservatieve aminozuur substitutie. Negen op de tien geconserveerde cysteine residu’s van de receptors familie zijn identiek geconserveerd in FLK-2. Echter, hebben ze ook aangetoond dat de extracellulaire domeinen additionele 12 cysteine residu’s bevatten die niet gezien zijn in KIT noch in FMS. Dit gaat in feite over hetzelfde eiwit als FLT3 receptor.
FMS-like receptor tyrosine kinase 3 (FLT3 receptor) werd ontdekt en gekarakteriseerd in de muis door de groep van Rosnet in 1991. Zij hebben dit geïdentificeerd door gebruik te maken van low stringency DNA hybridization. Bij een zoektocht naar nieuwe RTKs, werden verschillende cDNAs met homologie naar FMS geisoleerd. De receptoren die ze encodeerden FMS-like RTK werden genaamd. Biochemische karakterisering toonde aan dat het FLT3 eiwit inderdaad een eiwit met N-glycosylatie is. De twee kinase domeinen zijn verantwoordelijk voor de receptor tyrosine fosforilering en deze activiteit kan worden afgeschaft door een mutatie in de geconserveerde sequentie. Op ligand binding gebeurt autofosforilatie van de receptor op de cytoplasmische region. Een isoform van de FLT3 receptor in de muis is geïdentificeerd waarin de vijfde immunoglobuline-achtige regio in het extracellulair domein ontbreekt ten gevolge van alternatieve splicing. Deze isoform is aanwezig op een lager niveau dan de wild type receptor, maar het is nog steeds in staat om
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ligand te binden en wordt gefosforyleerd bij binding, wat aanduidt dat het vijfde lg domein van muis FLT3 niet nodig is voor ligand binding en ook niet voor fosforilatie.
Humane cDNA van een pre-B-cell line is gekloond en dan gekarakteriseerd door de groep van Rosnet, Schiff et all. in 1993. Hun werk toonde aan dat de cDNA sequentie van het FLT3 receptor eiwit gelijk is aan dat van muis FLT3 receptor cDNA. Gebaseerd op de primaire aminozuur sequentie is voorspeld dat het extracellulaire region van FLT3 ook gevouwen is in vijf lg-like domeinen, wat een typisch kenmerk is van typische klasse III RTKs. Het FLT3 eiwit is vastgesteld als 140 kDa band en 160 kDa band dankzij de N glycosylatie. De niet geglycosyleerd en oplosbare FLT3 receptor is het opgemerkt als 130 kDa band.
In contrast met zijn ligand, is de expressie van FLT3 op een meer beperkte aantal cellen. FLT3 expressie op CD34+
In de kwaadaardige menselijke hematopoietische cellen, voornamelijk in de acute myelogenous leukemia (AML) patient cellen, is vastgesteld dat de FLT3 transcript abnormale langere PCR producten bevat in aanvulling op de germline producten in de JM regio. Het extracellulaire en het transmembrane domein transcript lengte vertonen geen lengte alteratie. De JM regio bevat een tyrosine (Y)-rijke stretch 589YFYVDFREYEY599, welk geassocieerd wordt met signaal transductie. In het aberrante FLT3 transcript, sommige Y waren gedupliceerd, waar Y589/Y591/Y597/Y599 het meeste voorkomt is, gevolgd door Y597/Y599. Het JM domain mutaties induceren een extracellulaire conformationele verandering die dimerisatie begunstigen. Het is ontdekt dat punt mutaties van residuen in de activation loop ook constitutieve geactiveerde vormen van FLT3 receptor voortbrengen in sommige van de AML gevallen. Deze punt mutatie komt onafhankelijk voor van de FLT3/ITD en is niet aanzienlijk verbonden aan de oncogenesis.
stem-progenitor van zowel murine als humane hematopoietische cellen tonen aan dat ze waarschijnlijk betrokken zijn bij de groei en/of differentiatie van deze cellen.
Resultaten en discussie Het extracellulaire domein van FLT3 receptor vertonen kleine homologie met andere klasse III RTKs door de aanwezigheid van CXXXGXPXPXXXWXXC signature in het IG2 en IG5. Daarnaast, vertonen IG1 homologie met IG4 en IG2 met IG5 wat betekent dat een interne duplicatie zou hebben plaatsgevonden in de voorouder van de Flt3 gen. Op basis van de homologie tussen IG1-IG4 en IG2-IG5, is het interessant om de structuur van domein 1 en domein 1 tot 2 te onderzoeken en hun interacties met het ligand te bestuderen.
Er waren twee extracellulaire regio's van de humane FLT3 receptor in de pcDNA4/TO vector gekloneerd, namelijk de Ig-achtige domein 1 (pcDNA4/TO-hFLT3d1) en de Ig-achtige domein 1 tot 2 (pcDNA4/TO-hFLT3d12). De recombinante eiwitten zullen worden gefusioneerd C-terminale His6 tag als N-terminale Mu secretie-signaal door gebruik te maken
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van AgeI en KpnI restriction sites van de pcDNA4/TO vector. De pcDNA4/TO-hFLT3d1 en pcDNA4/TO-hFLT3d12 werden gebruikt voor zowel de transiente expressie als stabiele cellijn. Kleinschalige expressie test voor beide cellijnen werden uitgevoerd om na te gaan of de recombinante eiwitten worden gevormd en wat is het expressie niveau. De cellen werden gegroied in zes-well platen onder 0% FCS, met of zonder kifunensine. The apparent moleculair gewicht (MW) van rhFLT3d1 lag min of meer tussen de 20 en 25 kDa en rhFLT3d12 was min of meer 37 kDa, Er is een verschil in het apparent and verwachte MW, te wijten aan de glycosylaties. In aanwezigheid van kifunensine, de suiker bomen van rhFLT3d1 zijn meer homogene. Zonder kifunensine vertoonde FLT3d1 minder homogeniteit waar drie rhFLT3d1 glycovormen worden waargenomen. De HEK293S-GnTI-/- rhFLT3d1 en rhLT3d12 stabiele cellen werden gegroeid voor grootschalige expressie in 175 cm² reuze falcons en de recombinante eiwitten werden opgezuiverd. De opbrengst van rhFLT3d1 eiwit was hoog en kristallisatie trials werd uitgevoerd. De kristal vorming van rhFLT3d1 werd echter niet verhinderd door de heterogene glycosylaties. rhFLT3d1 kristallen werden gevormd in 10 mM CoCl2, 100 mM MES pH 6,5 en 1,8 M (NH4) 2SO4. rhFLT3d1 kristallen werden gediffracteerd op 4,7 Å resolutie. De diffractie was anisotrope en een twinnning werd waargenomen. De voorspelde ruimtegroep was face centered met tweevoudige symmetrische as (F222)
De expressie van rhFLT3d12 was zeer laag die door instabiliteit van het recombinante eiwit veroorzaakt kan worden. Structurele studies hebben aangetoond dat er interactie is tussen domein 2 en domein 3 (persoonlijke communicatie met K. Verstraete). Zonder deze interacties zou domein 2 vermoedelijk niet goed kunnen vouwen. Van complexe formatie studies namen we het besluit dat rhFLT3d1 en rhFLT3d12 geen interactie aangaan met rhFL.
Onze ITC experimenten toonden aan dat de affiniteit van rhFLT3d123 en rhFLT3d1234 waren in dezelfde orde, terwijl rhFLT3d12345 hoger was. Hiermee namen wij het besluit dat domein 5 het complex stabiliseert, vermoedelijk door receptor-receptor interacties en dat domein 4 niet betrokken is in receptor-receptor interacties zoals wordt gezien bij het KIT en PDGFR. De resultaten van SPR steady sate analyse experimenten toonde aan dat de affiniteit van rhFLT3d1234 en rhFLT312345 waren in dezelfde orde, terwijl rhFLT3d123 veel lager was. Dit is mogelijk te wijten aan de aanwezigheid van niet-actieve fractie van rhFLT3d123. rhFLT3d12345 vertoonde trage dissociatie welk bevestigd dat domein 5 de receptor-receptor binding stabiliseert.
Met het oog om de Phase problem op te lossen in rhFLT3d1234 structurele bepaling, SeMet rhFLT3d1234 en rhFL worden gelabeleld met SeMet. De opbrengst van rhFLT4d1234-SeMet was laag maar het was voldoende voor kristallisatie. Complex vorming van rhFLT3d1234-SeMet en rhFL-Semet werd daarna uitgevoerd voor kristallisatie trials.
Section VIII
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Section VIII
Addendum
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Isolation of plasmid-DNA by the method of Birnboim (after Birnboim and Doly, 1979)
1. Pick a single colony from a plate and inoculate 3 ml of LB-medium (containing the right antibiotics (by using a tip).
2. Incubate the culture overnight at 370
3. Take 1 ml and transfer it in a microcentrifuge tube. C with vigorous shaking.
4. Centrifuge for 1 min, 14000 rpm and take off the supernatans. 5. Re-suspend the bacterial pellet in 100 l of buffer P1.
P1 (stored at 4 degrees): 25 mM Tris-HCl pH 8.0 10 mM EDTA 100 g/mL RNase A
6. Add 100 l of lysis buffer P2. P2 (stored at room temp.): 200 mM NaOH 1% SDS Invert and incubate at room temp. for 5 minutes.
7. Neutralize the reaction by adding 100 l of buffer P3. P3 (stored at 4 degrees): 60 mM 5M KAc pH 5.5 11.5 ml Glacial acetic acid 28.5 ml water
8. Phenol extraction: add 100 l phenol/chloroform/isoamylalcohol (ratio 25:24:1). Vortex for 2 seconds and centrifuge 5 min, 14000 rpm.
9. Transfer the aqueous phase (+- 300 l) into a new 1.5 ml microcentrifuge tube. 10. Add 200 l 100% isopropanol, invert and centrifuge 15 min, 14000 rpm, 4 degrees. 11. Take off the isopropanol, add 100 l 70% ethanol and wash the DNA-pellet.
Centrifuge for 10 min, 14000 rpm, 4 degrees. 12. Take off the ethanol and let the pellet dry by air at room temp.
Restriction analysis of the Birmboim-DNA
1. Calculate the contents of master mix: - NEB buffer (10X): Check the NEB-website, Double Digest Finder, for the
properties of each enzyme and the suggested buffers. - BSA 10X (optional): to reduce star activity. - Restriction enzyme(s): 0.25 – 0.5 l per DNA-pellet. - Add fresh water to the final volume (20 l for each tube). - Always prepare a bit more than needed.
2. Add 20 l to each tube and mix by pipetting up and down a few times. 3. Incubate at 37C for 2 to 4 hours. 4. Add 5 l of loading buffer (6X) for agarose gel electrophoresis. 5. Load 12 – 14 l and start the gel (140V).
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QIAGEN Plasmid Midi Kit Protocol
1. Resuspend bacterial pellet in 4 ml Buffer P1. 2. Add4 ml Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4-6 times,
and incubate at room temperature (15-25°C) for 5 min. 3. Add 4 ml of chilled Buffer P3, mix immediately and thoroughly by vigorously
inverting 4-6 times, and incubate on ice for 15 min. 4. Centrifuge at ≥20.000g for 20 min at 4 °C. Remove supernatant containing plasmid
DNA promptly. 5. Equilibrate a QIAGEN-tip 100 by applying 4 ml Buffer QBT, and allow the column to
empty by gravity flow. 6. Apply the supernatant from step 4 to the QIAGEN-tip and allow it to enter the resin by
gravity flow. 7. Wash the QIAGEN-tip with 2 x 10 ml Buffer QC. 8. Elute DNA with 5 ml Buffer QF. 9. Precipitate DNA by adding 3.5 ml room temperature isopropanol to the eluted DNA.
Mix and centrifugate immediately at ≥15.000 g for 20 min at 4°C. Carefully decant the supernatant.
10. Wash DNA pellet with 2 ml of room temperature 70% ethanol and centrifugate immediately at ≥15.000 g for 10 min. Carefully dec ant the supernatant without disturbing the pellet.
11. Air-dry the pellet for 5-10 min and redissolve the DNA in 100 µl distilled water.
QIAquick Gel Extraction Kit Protocol 1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. 2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of
gel (100 mg ~ 100 μl). 3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help
dissolve gel, mix by vortexing the tube every 2–3 min during the incubation. 4. After the gel slice has dissolved completely, check that the color of the mixture is
yellow (similar to Buffer QG without dissolved agarose). 5. Add 1 gel volume of isopropanol to the sample and mix. 6. Place a QIAquick spin column in a provided 2 ml collection tube. 7. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min. 8. Discard flow-through and place QIAquick column back in the same collection tube. 9. Recommended: Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1
min. 10. To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min. 11. Discard the flow-through and centrifuge the QIAquick column for an additional 1 min
at 17,900 x g (13,000 rpm). 12. Place QIAquick column into a clean 1.5 ml microcentrifuge tube. 13. To elute DNA, add 50 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5)
to the center of the QIAquick membrane and centrifuge the column for 1 min.
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QIAquick PCR Purification Kit Protocol
1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix. It is not necessary to remove mineral oil or kerosene.
2. If pH indicator I has beein added to Buffer PB, check that the color of the mixture is
yellow. If the color of the mixture is orange or violet, add 10 μl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.
3. Place a QIAquick spin column in a provided 2 ml collection tube. 4. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s. 5. Discard flow-through. Place the QIAquick column back into the same tube. 6. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s. 7. Discard flow-through and place the QIAquick column back in the same tube. 8. Centrifuge the column for an additional 1 min.n Protocol 9. Place QIAquick column in a clean 1.5 ml microcentrifuge tube. 10. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to
the center of the QIAquick membrane and centrifuge the column for 1 min. Western Blot protocol Preparation of buffers:
• Protein Transfer Buffer (PTB): 25 mM Tris pH 8.3 3.03 g/l 192 mM Glycine 14.4 g/l 20% Methanol 200 ml/l
• Phosphate Buffer Saline (PBS): 8 g/l NaCl 0.2 g/l KCl 1.44 g/l NaH2PO4
0.24 g/l K
2HPO• Blocking Buffer (BB): 50 ml PBS + 2.5 g nonfat dry milk
4
• PBST: PBS + 0.1% Tween 20 • BBT: BB + 0.1% Tween 20
1. SDS-PAGE (15% acrylamide) electrophoresis. 2. Electro-blotting with ice overnight at 35 V. 3. Cut the ladder and put in distilled water. 4. Wash membrane several times with distilled water. 5. Blocking with BB for 2 h. 6. Wash with PBST: 3x20 s, 1x5 min, 1x10 min. 7. Antibody incubation (5 µl antibody in 5 ml BBT). 8. Wash with PBST: 3x20 s, 5x5 s. 9. Detection: 1 ml Supersignal (Thermo Scientific)
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SDS-PAGE silver staining protocol
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