From cDNA to crystal structureFrom cDNA to crystal structure

Design the constructs

Cloning

Expression of the proteins

Solubility/ stability tests

Expression optimization

Large scale expression

Purification

Crystallization screening

Crystal optimization

X-ray data collection

Phase determination

Structure determination

• BLAST-P your sequence (set PDB as database), find homologues; identify domains

http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins&PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=onhttp://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi

• predict disordered regionshttp://bioinf.cs.ucl.ac.uk/disopred/http://www.strubi.ox.ac.uk/RONN

• predict transmembrane regionshttp://www.ch.embnet.org/software/TMPRED_form.html

• predict the secondary structurehttp://bioinf.cs.ucl.ac.uk/psipred

• predict the 3D-structure http://bioinf.cs.ucl.ac.uk/psipred/http://www.bioinfo.rpi.edu/~bystrc/hmmstr/about.htmlhttp://depts.washington.edu/ventures/UW_Technology/Express_Licenses/rosetta.php

Design of the (best) constructsDesign of the (best) constructs

• from what organism does it originate?

• is your protein extracellular, intracellular or is it a membrane protein? If intracellular – where is it localized in the cell?

http://www.cbs.dtu.dk/services/TargetP/http://ihg2.helmholtz-muenchen.de/ihg/mitoprot.html

• what kind of posttranslational modification(s) does it contain?http://www.cbs.dtu.dk/services/NetNGlyc/http://mendel.imp.ac.at/sat/PrePS/index.htmlhttp://www.cbs.dtu.dk/services/NetPhos/

• what is the molecular weight (MW)?http://us.expasy.org/tools/protparam.htmlhttp://us.expasy.org/tools/pi_tool.html

• what is the isoelectric point (pI)?http://us.expasy.org/tools/protparam.htmlhttp://us.expasy.org/tools/pi_tool.html

• what is the extinction coefficient?http://us.expasy.org/tools/protparam.html

• does your protein display a measurable activity? How your protein can be assayed?

What you should know before starting to What you should know before starting to express and purify your proteinexpress and purify your protein

The three Phase Purification StrategyThe three Phase Purification Strategy

I

II

III

Purification proceduresPurification procedures

• Precipitation techniques

• Affinity chromatography

• Ion-exchange chromatography

• Hydrophobic interaction chromatography

• Gel filtration

Precipitation techniquesPrecipitation techniques

Affinity chromatographyAffinity chromatography

Makes use of specific binding interactions between molecules

1- Incubate crude sample with the immobilized ligand

2- Wash away non bound sample components from solid

support

3- Elute

• Commonly used affinity partners:– Ni2+ binds to poly-histidines (example 6xHis)– specific antibodies (anti-Flag tag)– glutathione binds to GST– Protein A or G binds antibodies

• Possible elution strategies:– pH modification– ionic strength modification– competitor ligand or analog

Affinity chromatographyAffinity chromatography

- the specificity of the interaction between histidine residues and immobilized nickel ions

Ni-NTA columnsNi-NTA columns

Affinity Separation. An exampleAffinity Separation. An example

Ion exchange chromatograpy (IEC)Ion exchange chromatograpy (IEC)

• Protein charge varies according to surrounding pH:

• When pH above pI: binding to ANION exchanger

(pH > pI Anion exchange)

• When pH below pI: binding to CATION exchanger

(pH < pI Cation exchange)

• Elution with salt concentration gradient (stepwise or continuous)

• Separation of proteins with different charge properties at different pH values

Ion-Exchange chromatographyIon-Exchange chromatography

If pH mobile phase =7.2

Then charge of the proteins: (-) (-) (+) (+)

-- + +

+

+

+

+

+

+-

--

--

-+

+

+

+

+

+

+

+

+

+

+

+

Anion exchange column = + charged

Increased salt concentration

Ion-Exchange chromatographyIon-Exchange chromatography

-- + +

+

+

+

+

+

--

Na+Na+

Na+

Na+Na+

Na+

+

+

+

+

+

+ Cl-

Cl-Cl-

Cl-

Cl-

Cl-

+

+

+

+

+

+-

-

-

--

-

Na+

Na+Na+

Typical IEX gradient elutionTypical IEX gradient elution

At low salt concentration

At high salt concentration

H

H H

H

H = hydrophobic region

HH

HH

HH

HH

HH

HH

HH

HH

Column with Resin

Hydrophobic Interaction ChromatographyHydrophobic Interaction Chromatography

HH

HH

HH

HH

HH

HH

HH

HH

Load with high salt buffer

Elute undesired molecules with decreasing salt

gradient

HH

HH

HH

HH

HH

HH

HH

HH

Elute target molecules with low salt buffer

HH

HH

HH

HH

HH

HH

HH

HH

Typical HIC gradient elutionTypical HIC gradient elution

Size-exclusion chromatographySize-exclusion chromatography

Gel filtration elutionGel filtration elution

Signs of unstable/ insoluble proteinSigns of unstable/ insoluble protein• cell-free lysate does not contain the protein

• protein is soluble but cannot be eluted from the affinity column (or, cleaved on the column protein “disappears”)

• protein precipitates during concentration

• multiple or asymmetric gel-filtration profile

• multidispersity in DLS

• multiple bands in native electrophoresis

Potential remedies:• glycerol• change the buffer• change the pH of the buffer• add a ligand• add some mild (non-ionic) detergent• clone/ express another construct

Storage of purified proteinsStorage of purified proteins

SUMMARY OF CONSENSUS PROTOCOLSUMMARY OF CONSENSUS PROTOCOL• Obtain the cDNA by amplifying genomic DNA (prokaryotic genes, or eukaryotic genes with no

introns) or sequence-verified cDNAs (eukaryotes) or by total gene synthesis.

• Use ligation-independent cloning (LIC) into an E. coli expression vector. Use T7 RNA polymerase–driven expression and an N-terminal oligohistidine tag (include a cleavage site for a protease to enable removal of the tag).

• Express the protein in a BL21(DE3) strain, with induction at low temperature (15–25 °C) in rich medium and with good aeration.

• Solubilize and purify the protein in a well-buffered solution containing an ionic strength equivalent to 300–500 mM NaCl.

• Use immobilized metal affinity chromatography (IMAC) as the initial purification step.

• If additional purification is required, use size-exclusion chromatography (gel filtration). If necessary, use ion exchange chromatography as a final ‘polishing’ step.

• The affinity tag may be removed. Use a recombinant, hexahistidine-tagged protease and reapply the sample to IMAC column to remove the protease.

From Nat. Method, 5, 138-146 (2008)

• Vapor diffusion procedures:

- hanging drop

- sitting drop• Microbatch• Dialysis• Free interface diffusion (FID)

Crystallization of proteinsCrystallization of proteins

Vapor diffusion proceduresVapor diffusion procedures

Hanging drop Sitting Drop

Cover slip(attached with grease)

Clear tape

Drop(Protein/precipitant mix)

Well(precipitant)

MicrobatchMicrobatch

• crystallization (at microscale); droplet is covered by oil.

• this prevents the

evaporation of the

very small drop.

DialysisDialysis

• sample placed in a

dialysis cell or a dialysis button• sealed with a dialysis membrane

Free interface diffusionFree interface diffusion

Protein and precipitant solutions are in contact (free interface)

Curr. Opin. Struct. Biol. 14, 577–583 (2004)

Crystallization of proteinsCrystallization of proteins

Discussion between a protein purification Discussion between a protein purification expert and a crystallographer...expert and a crystallographer...