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Maria SolàStructural MitoLab
IBMB-CSIC
Macromolecular Crystallography School MCS-2018
Production of protein samples for protein crystallization:
Expression, purification and stabilization
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
Bacterial strains
• BL21 DE3
• C41 - C43
• Origami-2
• Rosetta
• GroEs
• pLys S
• Rosetta-gami2
•Takara
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
In vitro protein expression (cell free systems): template (cdDNA, mRNA) + cell extract from cells, (insect, mammalian, bacteria) and contain RNA polym, ribosomes, tRNA and aa, cofactors and ATP, chaperones in the right proportions. More recently, includes glycosylation. However, yields are low and materials expensive (>1500 Eu/20 reactions)
Tags to improve protein solubility and/or allow affinity purification in E. coli
• 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
Domain 3
Domain 2
Domain 1
Limited proteolysis (Trypsin, trombin, Subtilisyn, Factor Xa):- T (on ice, RT, 37ºC)- sample concentration (1-10 mg/ml)- protease concentration
Cloning of subdomains after degradation: how to determine the N- and C-terminus of a fragment?
Based on the structure: - substitute flexible loops with short -GS(AS)nSG-- generate a chimera by fusing interacting units
M BPAP Chromatography fractions
M, markers; BF, before proteolysis, AP, after digestion
Crystallization of PhoB TF + s70 4thsd + DNA
PhoBE s70sd4
PhoBE
DNA
RNAPs70 sd4
How to get protein stable smaller fragments
Chimera
Blanco et al., EMBO J, 2011
Cloning techniques: ESPRIT – Darren Hart‘s Lab (IBS, Grenoble, France)Expression 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). - In each experiment, up to 30,000 individual clones are assayed in parallel for yield and solubility using a highly automated colony array format. Expressed polypeptides include an N-terminal His-tag and a C-terminal biothin acceptor peptide.
High Throughput
Constructs are made as a random library, they are biothinilated and printed on nitrocellulose membranes for soluble expression analysis by hybridisation of fluorescent antibodies.
Cloned gene5’ his tag3’ biothin peptide
Linearization + 5’ unidirectional truncation
(ideally, 1bp deletion)
Exo
Ligation
- Transformation- Printed on membranes- Protein expression, - Lysis in situ- Hybridization with fluo-streptavidine (18,432 colonies)
Herpes virus packaging terminase endonucleaseNadal et al., PNAS 2010
- Colony picking ofintense positive clones- 4ml express. cultures- Ni affinity purification- SDS analysis
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
Medium throughput: 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)
real-time PCR machine 96-well plate Buffers + additives
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
THANK YOU