Maria SolàStructural MitoLab
IBMB-CSIC
Macromolecular Crystallography School MCS-2017
Production of protein samples for protein crystallization:
Expression, purification and stabilization
CONTEXT OF A PROTEIN
LEVELS OF PROTEIN STRUCTURE
E.coli response regulator dimerisation domain of PhoB.
Crystallisation
Atomiccoordinates
RecombinantOverexpression
Organisms ortissues
Protein purification
DNAsequence
Steps to determine a protein crystal structure
Diffraction
Structure solution and refinement
The quality of the purified sample correlates with the crystal quality
The quality of the monocrystals is reflected in the diffraction pattern.
The ideal sample for crystallization
How it should be?
Large amounts:Extensive screening to find crystallization conditions: 5 to 25 mg/ml, 15 ul/plate minimum, 0.5 to 2 mg required (at least).
Quality:- High purity:
Superior to 95 % in SDS-PAGE
- Homogeneity:Chemical: no degradationStructural: no aggregation, one single conformation, low
polydispersity (monodispersity?).
Crystallisation
Atomiccoordinates
RecombinantOverexpression
Organisms ortissues
Protein purification
DNAsequence
Steps to determine a protein crystal structure
Diffraction
Structure solution and refinement
LacI Lac Promotor T7 RNA pol
Bacterial geneLac 0
-
T7 promotor GENE1 2
Expression plasmid
Lac 0
Cloning: E. coli DE3 system
Polymerase Chain Reaction (PCR) GENE1 2
Restr. sites:are not universal
Lac repressor
IPTG
XAllolactose
Isopropyl β-D-1-thiogalactopyranoside
20 reactions 353.00 $
High throughput: a universal restriction site or by recombination Ligation independent cloning, LICTopo system: blunt ends
Gateway
T4 Pol: pol and exo
Bacterial strains
Takara. Takara's pCold TF DNA Vector expresses Trigger Factor (TF) chaperone as a soluble tag. Trigger Factor is a prokaryotic ribosome-associated chaperone protein (48 kDa) which facilitates co-translational folding. Of E. coli origin, TF is highly expressed in E. coli expression systems.
• BL21 DE3
• C41 - C43
• Origami-2
• Rosetta
• GroEs
• pLys S
• Rosetta-gami2
What do the bacterial systems lack for heterologous protein expression?
Post-translational modifications
Eukaryotic expression systemsBaculovirus: subcloning into specific plasmids (E. Coli step), transfection into insect cells and amplification of the stock. With high titer stock infect insect cells and test expression
Mammalian cells (protocols for SeMet incorporation): cloning in specific vectors, small-scale transfection and expression tests. Positive clones, large –scale transfection, collect medium and purification. > Weeks
Leshmania tarentolae (Jena Biosc: LEXSY): Lizard parasiteFull eukaryotic folding machineryPost-translational protein modifications:- Homogeneous glycosilation
Eukaryotic expression systems
Yeast ~ E. coli costs, but reduced cloning strategies
Cell-freesystems
bacteriayeast
Cell culture
Animals
Plants
Difficulty in handling
Price
Tags to improve protein solubility and/or allow affinity purification
• His-protein
• Protein-His
• MBP-protein
• GST-protein
• GB1-protein
• Sumo protein
• NusA-protein
•Trx-protein
• LSL-protein
T7 promotor GENELac 0 FUSION
• His-protein
• Protein-His
• MBP-protein
• GST-protein
• GB1-protein
• Sumo protein
• NusA-protein
•Trx-protein
•Ztag-proteinc
Bacterial Strains:
BL21 DE3, C41 - C43, Origami-2, Rosetta
GroEs, pLys S, Rosetta-gami2
Eukaryotic system: Yeast, Baculovirus, Mammalian, Leishmania
Third parameter we can play with?
X
Combination of vectors, bacterial strains and automatic technology
Cloning techniques like ESPRIT – Darren HartExpression of soluble protein by random incremental truncation.
A diverse random library of DNA constructs is generated and screened to identify rare clones of interest (soluble expressers). Additionally, they have developed a "scanning" version to identify internal domains. In each experiment, 30,000 individual clones are assayed in parallel for yield and solubility using a highly automated colony array format.
High Throughput
The whole process is performed by atomatic means
Webpages that can serve as a guide
http://wolfson.huji.ac.il/purification/
Google Mario Lebendiker protein purification facility
https://www.embl.de/pepcore/pepcore_services/
Google embl protein purification
Don’t get lost!
Try simplest things first, as implemented in your own lab
pCri system
https://www.addgene.org/kits/pcrisystem
B (expression in the periplasm or extracellular )
A (expression in the cytoplasm) a
b
5’
3’
-> One single PCR product cloned into a collection of plasmids <-
Goulas T, Cuppari A, Garcia-Castellanos R, Snipas S, Glockshuber R, Arolas JL, Gomis-Rüth FX. The pCri System: a vector collection for recombinant protein expression and purification.PLoS One. 2014 Nov 11;9(11):e112643. doi: 10.1371/journal.pone.0112643. eCollection 2014.
pCri system
https://www.addgene.org/kits/pcrisystem29 vectors
NcoI - XhoI
pCri system
https://www.addgene.org/kits/pcrisystem
NdeI / NcoI - XhoI
CASE:
search for optimal construct –optimal fusion – optimal strain
Medium throughput:Strategy to get soluble helicase
Mitochondria sequence Motif I
Motif II Motif III
Motif VIMotifVMotif IV linker
linker Motif H 1 Motif H 1a
Motif H 4Motif H 3Motif H 2
Strategy to get soluble helicase:Design of constructs.
Step One: each construct is fused to different tags and expression is assayed.
Step two: find solubility:
X
Primase HelicaseLinker
Primase
Helicase
HelicaseLinker
Primase
Primase Linker
• His-protein
• Protein-His
• His-MBP-protein
• His-Ztag-protein
• His-GB1-protein
• His-Trx-protein
• His-GST-protein
• His-NusA-protein
• BL21 DE3
• BL21 DE3*
• C43
• Origami-2
• Rosetta
• GroEs
• pLys S
• Rosetta-gami2
8 Tags 8 strains
X
15 Constructs
* Phosphate 100mM pH 7.5 NaCl 700mM BME 5mM
* Bicine 100mM pH 9.5 NaCl 700mM BME 5mM
* Phosphate 100mM pH 7.5 NaCl 700mM glycerol 20%
* Citrate 100mM pH 6.0 NaCl 100mM BME 5mM
* Bicine 100mM pH 9.5 NaCl 1M BME 5mM
* MES 100mM pH 6.5 NaCl 700mM BME 5mM
* Citrate 100mM pH 6.5 NaCl 700mM BME 5mM
* Tris.HCl 100mM pH 8.5 NaCl 700mM BME 5mM
* Hepes 100mM pH 7.2 NaCl 300mM MgCl2 100mM BME 5mM
* Tris.HCl 100mM pH 8.5 MgCl2 200mM NaCl 200mM BME 5mM
* Phosphate 100mM pH 7.5 NaCl 300mM BME 1mM
* Phosphate 100mM pH 7.5 NaCl 500mM glycerol 10%
X
12 Buffers
Strategy to get soluble helicase
pCRI system
Step One: Clone each construct with a different tag and test the expression.
Constructs with positive expression
X
One construct with different tags
Eight E. coli strains
PrimaseMBP
PrimaseNusA
PrimaseGB1
PrimaseTrx
PrimaseZtag
PrimaseGSTSDS-PAGE for each construct-strain combination
MBP- Nterm domain
Type cell expression
BL21DE3 Yes
BL21DE3* No
Rosetta Yes
C43 Yes
Origami2 Yes
GroEs No
pLys No
Non
indu
ced
BL21
DE3
Indu
ced
BL21
DE3
Non
indu
ced
BL21
DE3
*
Indu
ced
BL21
DE3
*
Non
indu
ced
C43
Indu
ced
C43
Non
indu
ced
Orig
ami-2
Indu
ced
Orig
ami-2
Strategy to get soluble helicase
Constructs with positive expression
1. Phosphate 100mM pH 7.5 NaCl 700mM BME 5mM
2. Bicine 100mM pH 9.5 NaCl 700mM BME 5mM
3. Phosphate 100mM pH 7.5 NaCl 700mM glycerol 20%
4. Citrate 100mM pH 6.0 NaCl 100mM BME 5mM
5. Bicine 100mM pH 9.5 NaCl 1M BME 5mM
6. Citrate 100mM pH 6.5 NaCl 700mM BME 5mM
7. Tris.HCl 100mM pH 8.5 NaCl 700mM BME 5mM
8. Phosphate 100mM pH 7.5 NaCl 300mM BME 1mM
X
BL21DE3I M 1 2 3 4 5 6 7 8
I 1 M 2 3 4 5 6 7 8
I M 1 2 3 4 5 6 7 8 I 1 2 M 3 4 5 6 7 8
M I 1 2 3 4 5 6 7 8 GroES
Origami 2 Rosetta
BL21DE3*
MBP- Nterm domain
Type cell expression
BL21DE3 Yes
BL21DE3* No
Rosetta Yes
C43 Yes
Origami2 Yes
GroEs No
pLys No
Buffers
Step Two: analysis of the construct solubility.
Strategy to get soluble helicase
BL21DE3I M 1 2 3 4 5 6 7 8
I 1 M 2 3 4 5 6 7 8
I M 1 2 3 4 5 6 7 8 I 1 2 M 3 4 5 6 7 8
M I 1 2 3 4 5 6 7 8 GroES
Origami 2 Rosetta
BL21DE3*
Step three: Soluble constructs are analyzed for purification.
Ni2+-column binding analysis
Size exclusion analysis
Tag-cleavability analysis
Finger printing analysis
Strategy to get soluble helicase
Crystallisation
Atomiccoordinates
RecombinantOverexpression
Organisms ortissues
Protein purification
DNAsequence
Steps to determine a protein crystal structure
Diffraction
Structure solution and refinement
chromatography
Fractions (t)
OD
Aff Aff
Affinity Size exclusion
GF GF
THERMOFLUOR
Temperature denaturation of a protein gradually exposes more and more hydrophobic patches that would have been buried in the native fold. Thermofluor dye allows to monitor this as it binds to these patches and thereupon becomes much more fluorescent.
This assay can conveniently be performed in 96-well format using a real-time PCR machine. In this way, in can be used to screen the influence of buffer conditions, ligand binding or concentration on protein stability.
More accurate measurement of a melting temperature is obtained using differential scanning calorimetry or measuring the dependence of circular dichroism on temperature. However, the Thermofluor assay requires only small sample amounts and is much higher throughput. It is often used to establish suitable crystallisation conditions.
Thermofluor: fluorescence-based thermal stability assay
Sypro Orange (Molecular Probes)
1 FAD 2.52 NAD 53 Lysine 54 b-Octylglucoside 55 NADP 56 Proline 57 AMP–PNP 2.58 FeCl2 159 GDP 5
10 MgAc 1511 MnCl2 1512 Glucose 513 CuCl2 1514 ATP 515 CoCl2 1516 Mannose 517 Fructose 518 Maltose 5
19 CaAc 1520 NiCl2 1521 NADH 522 Haemin 2.523 UTP 524 DM 1025 DDM 1026 Cholic acid 1027 Chaps 10
28 Glycerol 10%29 Vanilic acid 1030 ZnCl2 1031 Glycine 1032 Phenylalanine 1033 4-Hydroxy benzoic acid 1034 Protoporphyrin 1035 Coproporphyrin 1036 Trimethanine 10
1 Sodium acetate 4.52 Sodium citrate 4.73 Sodium acetate 5.04 Potassium phosphate 5.05 Sodium phosphate 5.56 Sodium citrate 5.57 Mes 5.88 Potassium phosphate 6.0
9 Mes 6.210 Sodium phosphate 6.511 Sodium cacodylate 6.512 Mes 6.513 Potassium phosphate 7.014 Hepes 7.015 Ammonium acetate 7.316 Sodium phosphate 7.5
17 Tris 7.518 Imidazole 8.019 Hepes 8.020 Tris 8.021 Bicine 8.022 Tris 8.523 Bicine 9.0
Additives
List of the buffers and their pH values used in the screen
Tm is defined as the midpoint of temperature of the protein-unfoldingtransition.
Dynamic Light Scattering
Dynamic Light Scattering: Polydispersity
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