production of protein samples for protein crystallization · production of protein samples for...

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


Organisms ortissues

Proteinpurification

DNAsequence


Diffraction


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


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.


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


Crystallisation

Atomiccoordinates


Organisms ortissues

Proteinpurification

DNAsequence


Diffraction


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

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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0.0 5.0 10.0 15.0 20.0 25.0 mlF2 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 B12 B11 B10 B9 B8 B7 B6 B5 B4 B3 Waste Waste

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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0.0 5.0 10.0 15.0 20.0 25.0 mlF2 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 B12 B11 B10 B9 B8 B7 B6 B5 B4 B3 B2 B1 C1 C2 C3 C4 C5

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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0.0 5.0 10.0 15.0 20.0 25.0 mlF2 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 B12 B11 B10 B9 B8 B7 B6 Waste Waste

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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0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 mlF2 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 B12 B11 B10 B9

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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0.0 5.0 10.0 15.0 20.0 25.0 mlA1 A2 A3 A4 A5 A6 A7 A8 F2 A9 A10 A11 A12 B12 B11 B10 B9 B8 B7 B6 B5 B4 B3 B2 B1 C1 C2 C3 C4 C5 C6

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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0.0 5.0 10.0 15.0 20.0 mlA1 A2 A3 A4 A5 A6 A7 A8 Waste A9 A10 A11 A12 B12 B11 B10 B9 B8 B7 B6 B5 B4 B3 B2 B1 C1

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

8.21

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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120.0 130.0 140.0 150.0 160.0 170.0 180.0 190.0 ml1 2 3 4 5 6 7 8 9 1011 Waste 12 13 14 15 16 17 Waste

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

production of protein samples for protein crystallization · production of protein samples for...

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