Maria SolàStructural MitoLab

IBMB-CSIC

Macromolecular Crystallography School MCS-2014

Production of protein samples for protein crystallization:

Expression, purification and stabilization

CONTEXT OF A PROTEIN

LEVELS OF PROTEIN STRUCTURE

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 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

Proteinpurification

DNAsequence

Steps to determine a protein crystal structure

Diffraction

Structure solutionand refinement

LacI Lac Promotor T7 RNA pol

Bacterial geneLac 0

-

T7 promotor GENE1 2

Expression plasmid

Lac 0

Cloning: E. coli DE3 system

IPTG

X

Isopropil-β-D-thiogalactpiranosyde, ~allolactose

Polymerase Chain Reaction (PCR) GENE1 2

Restr. sites:are not universal

20 reactions 353.00 $

High throughput: a universal restriction site or by 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. 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

http://www.embl-hamburg.de/~geerlof/webPP/vectordb/bact_vectors/vectordb.pdf

What do the bacterial systems lack 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

• 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 toidentify rare clones of interest (soluble expressers). Additionally, they 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 array format.

High Throughput

The whole process is performed by atomatic means

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: Polydispersity

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

LUNCHTIME!