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

THANK YOU