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JCSG Center for Innovative Membrane Protein Technologies
Cell-Free Expression
• Used in industry for soluble proteins Swartz, J Microbiol Biotechnol, 33 (7), 476-485, 2006
• Becoming popular for membrane proteins Klammt, C, Schwarz, D, Löhr, F, Schneider, B, Dötsch, V, Bernhard, F, Cell-free expression as an emerging technique for the large scale production of integral membrane protein, FEBS J, 273, 18, 2006
• Expression possible in the presence of certain detergents and/or lipids
• Works very well with small membrane proteins (~20kD), including those that oligomerize.
• Progress towards expression of larger membrane proteins
JCSG Center for Innovative Membrane Protein Technologies
JCSG Center for Innovative Membrane Protein Technologies
Cell-Free Expression
• CF Protein expression from DNA plasmids and/or even PCR fragments
• CF Reaction Composition– Lysate– Amino acids and nucleotides– Buffer– ATP and secondary energy source– DNA template
• E. coli and wheat germ are popular• Reticulocyte, insect cell, PURE• Several styles of operation
JCSG Center for Innovative Membrane Protein Technologies
JCSG Center for Innovative Membrane Protein Technologies
Cell-Free Expression of Membrane Proteins
1. Grow and lyse cells2. Prepare extract (centrifugation)3. Add detergent/lipids, salts, substrates4. Add template5. Incubate (~ 6 hours)
Klammt et al. (2006) FEBS J Vol 273 (18) 4141-4153
JCSG Center for Innovative Membrane Protein Technologies
JCIMPT Membrane Protein Cell Free Expression
• Our project is a collaboration with Invitrogen, Inc.- Based on one of the most active bacterial based lysate currently on the market - cDNA clone collection and cloning technology available under this collaboration- Relatively high interest to convert this into commerical product
• Several advantages of the CF system over the in vivo approach- No toxicity issues- High level of control (eliminate “biology”) and reproducibility- Selective labeling (Se, 15N, 13C, deuteration)- Relatively high yields possible (0.6-1.2 mg purified protein per ml reaction typical)- Capable of rapid screening and scaling is linear
• Initial focus on bacterial lysate- Bacterial based lysate is relatively cheap and quick to produce- System takes advantage of bacterial transcriptional/translation efficiency- Introduction of mammalian lipids and also insertion machinery in vitro- Possibility of incorporating membrane protein directly into micelle or other surfactants- No post-translation modifications that introduce heterogeneity- Several sources of lysate are possible and to be developed
JCSG Center for Innovative Membrane Protein Technologies
• Increased yield of certain membrane proteins (< 20 kD molecular weight)(some up to ~1.5 mg/ml of lysate) (Tier 1)
• Successful to overexpress a much larger range of bacterial membrane proteins of ~40-50 kD molecular weight range. (Tier 2)
• We have increased the efficiency and robustness of our CF system toaccomodate the expression of certain mammalian membrane proteins(~50 kD molecular weight). (Tier 3)
• Tested function of these membrane proteins by ligand binding/microcalorimetry
• Investigated possible alternative lysate sources and the incorporation ofadditional lipids/membranes such as those derived from thykoloid or fromother sources.
• Working with Invitrogen to commercialize for membrane proteins
JCIMPT Cell-Free Membrane Protein Expression Where are we now?
JCSG Center for Innovative Membrane Protein Technologies
JCSG Center for Innovative Membrane Protein Technologies
YiiP(33 kD)
EmrD(42 kD)
MscL(14 kD)
EmrE(11 kD)
(1-1.2µg/µl/ml)
(0.6-0.8µg/µl/ml)
Cell Free Expression of Bacterial Membrane Proteins
(43 kD)(40 kD)
JCSG Center for Innovative Membrane Protein Technologies
EmrE - Small Multidrug Transporter
• First test of our CF system for membrane protein x-ray structure determination
• Bacterial, Small Multidrug Resistance (SMR)• 110 Amino Acid, ~11 kD wild-type• 4 transmembrane helices• Functional as dimer• Critical and conserved residue Glu14 required
for substrate binding/transport• Hetero-dimers like EbrAB are well studied• Controversy on the packing of the dimer
JCSG Center for Innovative Membrane Protein Technologies
JCSG Center for Innovative Membrane Protein Technologies
EmrE SeMet Labeling
Wild-type 13986 DLabeled 14181 D
Sulfur 32.06 DSelenium 78.96 D
4 Met labeled with Se
MNPYIYLGGAILAEVIGTTLMKFSEGFTRLWPSVGTIICYCASFWLLAQTLAYIPTGIAYAIWSGVGIVLISLLSWGFFGQRLDLPAIIGMMLICAGVLIINLLSRSTPH 91 and 92
21
JCSG Center for Innovative Membrane Protein Technologies
CF and in vivo EmrEfunctionally similar
Tryptophan Fluorescence quenching Fluorescence anisotropy Competitive Bindingusing Fluorescence anisotropy
JCSG Center for Innovative Membrane Protein Technologies
EmrE – New SeMet Data
JCSG Center for Innovative Membrane Protein Technologies
EmrE - Small Drug Transporter
C2 Crystal Form P21 Crystal Form
JCSG Center for Innovative Membrane Protein Technologies
EmrE - Small Drug Transporter
JCSG Center for Innovative Membrane Protein Technologies
JCSG Center for Innovative Membrane Protein Technologies
EmrE x-ray structure with EM densityUbarretxena-Belandia et al, (2003)
EMBO J 22:6175-6181.
JCSG Center for Innovative Membrane Protein Technologies
CF Expression of Larger (~50 kD) Membrane Proteins
• Expressed as full-length protein• Stays solubilized if expressed with
– Brij series (35, 58, 78)– Digitonin– Liposomes
• Detergent exchange possible. Some targets may aggregate.
• Proved to be functional after reconstituted into liposome
JCSG Center for Innovative Membrane Protein Technologies
JCSG Center for Innovative Membrane Protein Technologies
Human/mammalian targets for cell-free expression
Hybrid transporter/channel. Only known high affinity Cu uptake system in mammals. Anti-cancer drug (cisplatin) transporter. Trimer.
321CTR1 - Copper transport
Solute carrier family 2. Responsible for insulin-regulated glucose disposal.
1255GLUT4 - insulin-regulated glucose transporter
One of a few lysosomal membrane proteins. Transports nucleosides, drugs, steroids, etc. Possible target for development of novel chemotherapeutic agents.
428LAPTM4A - Lysosomal-associated protein transmembrane 4 alpha
Controls organ formation (in the heart) and electrical synchronization (in the brain). Hexamer.
426/32/43CX26/32/43 - Connexin(Gap junction) protein family
SignificanceNo. of TMMW (KDa)Protein
JCSG Center for Innovative Membrane Protein Technologies
JCSG Center for Innovative Membrane Protein Technologies
In vitro expression and purification of human membrane proteins using E. coli extract
• Proteins were extracted with Fos-Choline 14 and purified using Ni-NTA column• Yield: 0.5 – 1 mg purified protein/mL of cell-free reaction.
JCSG Center for Innovative Membrane Protein Technologies
Large-scale expression and purification of GLUT4 using CF without detergent (Mode A)
A. GF peak #1B. GF peak #2
Monodispersed population of CF-expressed GLUT4
Gel filtration profile of Ni-NTA purified GLUT4in Fos-14
Void
Void
M: MarkerP: PelletS: Supernatant
A B
JCSG Center for Innovative Membrane Protein Technologies
Large-scale CF expression, purification and detergent exchangeof GLUT4 with Brij-35 as solubilization agent (Mode B)
1. Gel filtration peak of GLUT4/Brij-35
Purification of GLUT4 using Brij-35 Detergent exchange of Brij-35-purified GLUT4
1. OG2. UDM3. DDM*4. Cymal 55. Cymal 66. CHAPS
Void
Superdex 200 16/60
JCSG Center for Innovative Membrane Protein Technologies
0
20
40
60
80
100
120
140
160
180
0 2 4 6 8 10 12 14 16 18 20
3H-SubstrateUV280Conduct
Cell-Free Expression of Mammalian Receptors
ETAROGR1Void
Monomer
Superdex 200 16/60OGR1
Superdex 200 10/30ETAR
~0.2-0.3 mg/ml reaction
JCSG Center for Innovative Membrane Protein Technologies
Biology and Relevanceof MsbA and Pgp
• ~128kD homodimer
• Shown to bind and hydrolyze ATP
• Transports lipopolysaccharide (LPS), an integral part of the outer bacterial membrane, from the inner leaflet to the outer leaflet of the inner membrane
• MsbA is the only essential ABC transporter identified in gram negative bacteria
• MsbA is a structural and functional homologue of Human P-glycoprotein (Pgp; hMDR1), a clinically important transporter
• Pgp is a multidrug transporter responsible for chemotherapy resistance
JCSG Center for Innovative Membrane Protein Technologies
NH3
-
+
CO2
TMD
ABC
JCSG Center for Innovative Membrane Protein Technologies
MsbA Structural Data• Density at 3.7Å• AMPPNP Density at 3.7Å• Hg and Vanadate at 4.5 Å
JCSG Center for Innovative Membrane Protein Technologies
X-ray Structures of MsbA
Inward Facing Inward Facing Outward FacingAMPPNP and ADP-Vi
JCSG Center for Innovative Membrane Protein Technologies
Conformational flexibility of MsbA based on structures
JCSG Center for Innovative Membrane Protein Technologies
Acknowledgements
Srinivas ChittaboinaRitu RoyQinghai Zhang
InvitrogenToni KudlickiJulia Fletcher
Yen-Ju ChenSamantha LieuOwen PornillosAndy ChenAlex Ma
Tuan NguyenAndrey Kariakin
Paul SzewczykXiao He
Andrew WardChristopher L. ReyesJodie YuChristopher Roth
Steve Aller
SSRLALSAPS
NIHNASADOD
Stevens LabYeager LabKuhn LabFinn Lab
Ina UrbatschLab (TTUHSC)
Department of Molecular BiologySkaggs Institute for Chemical BiologyThe Scripps Research Institute