production of protein samples for protein crystallization · production of protein samples for...
TRANSCRIPT
Maria SolàStructural MitoLab
IBMB-CSIC
Macromolecular Crystallography School MCS2010
Production of protein samples for protein crystallization
- around 4000 known structures from X-ray crystallography and 2-D NMR studies- structure data base widely available for analysis, the Protein Data Bank- water soluable proteins are "globular," tight packed, water excluded frominterior, folded up. - bond lengths and bond angles don't vary much from equilibrium positions. - structures are stable and relatively rigid.
E.coli response regulatordimerisation domain of PhoB.
Crystallisation
Atomiccoordinates
RecombinantOverexpression
Organisms ortissues
Proteinpurification
DNAsequence
Steps to determine a protein crystal structure
Diffraction
Structure solutionand refinement
The quality of thepurified samplecorrelates withthe crystal quality
The quality of the monocrystals is reflected in the diffraction pattern.
The ideal sample for crystallization
How it should to be?
Large amounts:Extensive screening to find crystallization conditions: 5 to 25 mg/ml, 15 ul/plate minimum, 0.5 to 2 mg required (atleast).
Quality:- High purity:
Superior to 95 % in SDS-PAGE
- Homogeneity:Chemical: no degradationStructural: no aggregation, one single conformation
sample, monodispersity.
Crystallisation
Atomiccoordinates
RecombinantOverexpression
Organisms ortissues
Proteinpurification
DNAsequence
Steps to determine a protein crystal structure
Diffraction
Structure solutionand refinement
LacI Lac Promotor T7 RNA pol
Bacterial geneLac 0
-
? ?
T7 promotor GENE
Expression plasmid
Lac 0
Cloning: E. coli DE3 system
IPTG
X
Isopropil-b-D-thiogalactpiranosyde, allolactose
Traditional cloning: a multiple cloning site with restriction sites
Cloning: E. Coli DE3 system
EcoRI5'GAATTC 3'CTTAAG
T7 promotor
Expression plasmid
Lac 0
BamHI5'GGATCC 3'CCTAGG
Polymerase Chain Reaction (PCR)5´gaattcttat ccattcagcc attctgtatc ttctaaatgg 3´
GENE
Cloning: E. Coli DE3 system
T7 promotor GENE
Expression plasmid
Lac 0
Traditional cloning: a multiple cloning site with restriction sites
EcoRI5'GAATTC 3'CTTAAG
BamHI5'GGATCC 3'CCTAGG
20 reactions 353.00 $
High throughput: a universal restriction site or recombinationLigation independent cloning, LICTopo system: blunt ends
Gateway
Bacterial strains
Takara. Takara's pCold TF DNA Vector expresses TriggerFactor (TF) chaperone as a soluble tag. Trigger Factor is a prokaryotic ribosome-associated chaperone protein (48 kDa) which facilitates co-translational folding. E. coli origin, TF is highly expressed in E. coli expression systems.
• BL21 DE3
• C41 - C43
• Origami-2
• Rosetta
• GroEs
• pLys S
• Rosetta-gami2
http://www.embl-hamburg.de/~geerlof/webPP/vectordb/bact_vectors/vectordb.pdf
What is a bacterial system lacking for heterologous protein expression?
Post-translational modifications
Eukaryotic expression systemsBaculovirus: subcloning into specific plasmids (E. Coli step), transfection intoinsect cells and amplification of the stock. With high titer stock infect insect cellsand test expression
Mammalian cells (protocols for SeMet incorporation): cloning in specificvectors, 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
Cellculture
Animals
Plants
Difficulty in handling
Complexity of proteins
Price
Tags to improve protein solubility and/or allow affinitypurification
• His-protein
• Protein-His
• MBP-protein
• GST-protein
• GB1-protein
• Sumo protein
• NusA-protein
•Trx-protein
•Ztag-protein
T7 procmotor 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 Expression of soluble proteinby random incremental truncation.
A diverse random library of DNA constructs is generated and screened toidentify rare clones of interest (soluble expressers). Additionally, we have developed a "scanning" version to identify internaldomains. In each experiment, 30,000 individual clones are assayed in parallel for yield and solubility using a highly automated colony arrayformat.
High Throughput
The whole process is performed by atomatic means
CASE 1:
search for optimal construct –optimal fusion – optimal strain
Medium throughput:Strategy to get soluble helicase
Mitochondria sequence MotifI
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 OneStep One: each construct is fused to different tags and expression is assayed.
Step twoStep 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
Linker
Strategy to get soluble helicase
Step OneStep 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
NopLys
NoGroEs
YesOrigami2
YesC43
YesRosetta
NoBL21DE3*
YesBL21DE3
expressionType cell
MBP- Nterm domain
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 NaCl700mM 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 NaCl1M 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*
NopLys
NoGroEs
YesOrigami2
YesC43
YesRosetta
NoBL21DE3*
YesBL21DE3
expressionType cell
MBP- Nterm domain
Buffers
Step TwoStep Two: analysis of the construct solubility.
Strategy to get soluble helicase
BL21DE3
I 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 threeStep 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
Proteinpurification
DNAsequence
Steps to determine a protein crystal structure
Diffraction
Structure solutionand 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 thenative fold. Thermofluor dye allows to monitor this as it binds tothese patches and thereupon becomes much more fluorescent.
This assay can conveniently be performed in 96-well format usinga real-time PCR machine. In this way, in can be used to screenthe influence of buffer conditions, ligand binding or concentrationon protein stability.
More accurate measurement of a melting temperature is obtainedusing differential scanning calorimetry or measuring thedependence of circular dichroism on temperature. However, theThermofluor assay requires only small sample amounts and ismuch higher throughput. It is often used to establish suitablecrystallisation 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
Macromolecular Complexes Challenge: get a high amount of pure and
homogeneous sample.
If the complexes are not stable, the stoichiometry is lost.
The homogeneity is lost
Aff
GF
IDEALLY
- Fractions +
OD
columna de afinidad + GF
Sup6 1run 001beadbeater25L2ndround001:10_UV1_280nm Sup6 1run 001beadbeater25L2ndround001:10_UV2_260nm Sup6 1run 001beadbeater25L2ndround001:10_FractionsSup6 1run 001beadbeater25L2ndround001:10_Inject
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Ve
OD260 > OD280
> PROTOCOLO DE PURIFICACIÓN?
Selected fractions loaded into a SDS-PAGE
Sup6 1run RPDNasedigestA8910:10_UV1_280nm Sup6 1run RPDNasedigestA8910:10_UV2_260nm Sup6 1run RPDNasedigestA8910:10_FractionsSup6 1run RPDNasedigestA8910:10_Inject
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Eliminación de ácidos nucleicos: Digestión con DNAsa
Agunas fracciones de la elución de la GF se incuban con DNAsa, y se carga el complejo en GF otra vez para ver el perfil
Sup6 1runRPRNase2uLA10first001:10_UV1_280nm Sup6 1runRPRNase2uLA10first001:10_UV2_260nm Sup6 1runRPRNase2uLA10first001:10_FractionsSup6 1runRPRNase2uLA10first001:10_Inject
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Eliminación de ácidos nucleicos: Digestión con RNAsa
Agunas fracciones de la elución de la GF se incuban con RNAsa, y se carga el complejo en GF otra vez para ver el perfil
?
Sup6 1runRPRNase2uLA10first001:10_UV1_280nm Sup6 1runRPRNase2uLA10first001:10_UV2_260nm Sup6 1runRPRNase2uLA10first001:10_FractionsSup6 1runRPRNase2uLA10first001:10_Inject
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OD280> OD260
Eliminación de ácidos nucleicos: Digestión con RNAsa
Heparin1mL001RPafterTEV001:10_UV1_280nm Heparin1mL001RPafterTEV001:10_UV2_260nm Heparin1mL001RPafterTEV001:10_ConcHeparin1mL001RPafterTEV001:10_Fractions Heparin1mL001RPafterTEV001:10_Inject
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Columna de Heparina
El perfil no es claro y el gel muestra que el complejo sigue apareciendo en todas las fracciones
Eliminación de ácidos nucleicos: Columna de heparina
ResourceQ1mL001RPafterTEV001:10_UV1_280nm ResourceQ1mL001RPafterTEV001:10_UV2_260nm ResourceQ1mL001RPafterTEV001:10_ConcResourceQ1mL001RPafterTEV001:10_Fractions ResourceQ1mL001RPafterTEV001:10_Inject
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Columna ResourceQ
Claramente aparecen tres picos.
Sup6 1run RPafterResQfractB11001:10_UV1_280nm Sup6 1run RPafterResQfractB11001:10_UV2_260nm Sup6 1run RPafterResQfractB11001:10_FractionsSup6 1run RPafterResQfractB11001:10_Inject Sup6 1run RPafterResQfractB11001:10_UV1_280nm@01,BASEM
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7.11
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9.17 9.649.89
10.09
10.9713.50
GF Pico 2 tras ResourceQ
El pico sale antes: Posible agregación.
Muestra heterogénea.
ResourceQ1mL001RPafterTEV001:10_UV1_280nm ResourceQ1mL001RPafterTEV001:10_UV2_260nm ResourceQ1mL001RPafterTEV001:10_Conc ResourceQ1mL001RPafterTEV001:10_Fractions ResourceQ1mL001RPafterTEV001:10_Inject
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Sup6 1run 001lidafter1500mMwashResQ001:10_UV1_280nm Sup6 1run 001lidafter1500mMwashResQ001:10_UV2_260nmSup6 1run 001lidafter1500mMwashResQ001:10_Fractions Sup6 1run 001lidafter1500mMwashResQ001:10_Inject
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GF Pico 1 tras ResourceQ
ResourceQ1mL001RPafterTEV001:10_UV1_280nm ResourceQ1mL001RPafterTEV001:10_UV2_260nm ResourceQ1mL001RPafterTEV001:10_Conc ResourceQ1mL001RPafterTEV001:10_Fractions ResourceQ1mL001RPafterTEV001:10_Inject
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Cual sería el protocolo?
Rational design of a protocol for a transcription factor
SD 75 26 60 mtTFA001:10_UV1_280nm SD 75 26 60 mtTFA001:10_UV2_260nm SD 75 26 60 mtTFA001:10_Fractions
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Purification protocol:pH far from pILow to high amounts of NaCl
Protein-DNA complexwith different
oligonucleotides
DNA
22DN
A 22
DNA
ohDN
A 21
DNA oligos_________________________________________________
cry1
cry2 overhang 29pb No crystals
cry2 oh2
cry1/27/5’ 27pB No crystals
cry2/27/5’ oh
cry/27/3’ 27pB No crystals
cry/27/3’ oh
cry/26
cry/26 oh 26pb No crystals, spherullites
cry/26 oh2
cry/22 22pb No crystals
cry/22 oh 5’
cry22 oh 5’ 22pb Crystals at frist condition(*) 4Acry23 oh 5’
cry21 21pb No crystalscry21 oh 5’
cry21 21pb No crystalscry21
cryhalf1 15pb Crystals, at several conditions 6-20Acryhalf1
cryhalf2 16pb Crystal at one condition. No up-scalingcryhalf2
cry22 22pb Crystals at first condition(*). 3.2Acry22