Protein Purification & Crystallization. From cDNA to crystal structure. Design of the (best) constructs. BLAST-P your sequence (set PDB as database), find homologues; identify domains
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Protein Purification & Crystallization
Design of the (best) constructs • 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=on http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi • predict disordered regions http://bioinf.cs.ucl.ac.uk/disopred/ http://www.strubi.ox.ac.uk/RONN • predict transmembrane regions http://www.ch.embnet.org/software/TMPRED_form.html • predict the secondary structure http://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.html http://depts.washington.edu/ventures/UW_Technology/Express_Licenses/rosetta.php
What you should know before starting to express and purify your protein • 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.html http://www.cbs.dtu.dk/services/NetPhos/ • what is the molecular weight (MW)? http://us.expasy.org/tools/protparam.html http://us.expasy.org/tools/pi_tool.html • what is the isoelectric point (pI)? http://us.expasy.org/tools/protparam.html http://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?
III II I The three Phase Purification Strategy
Purification procedures • Precipitation techniques • Affinity chromatography • Ion-exchange chromatography • Hydrophobic interaction chromatography • Gel filtration
Affinity chromatography Makes use of specific binding interactions between molecules 1- Incubate crude sample with the immobilized ligand 3- Elute 2- Wash away non bound sample components from solid support
Affinity chromatography • 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
Ni-NTA columns - the specificity of the interaction between histidine residues and immobilized nickel ions
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 chromatography If pH mobile phase =7.2 Then charge of the proteins: (-) (-) (+) (+) Anion exchange column = + charged
+ + + Cl- + + + Cl- - + + + Cl- - - + + + Cl- + + + Cl- Na+ - Na+ - Na+ Na+ Na+ - - Na+ Na+ + + Na+ Cl- - Na+ Ion-Exchange chromatography - - + + Increased salt concentration
Hydrophobic Interaction Chromatography H H H H H = hydrophobic region H H H H H H H H H H H H H H H H At low salt concentration At high salt concentration H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H Elute undesired molecules with decreasing salt gradient Column with Resin Load with high salt buffer Elute target molecules with low salt buffer
Signs 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 columnprotein “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
SUMMARY 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 aBL21(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)
Crystallization of proteins • Vapor diffusion procedures: - hanging drop - sitting drop • Microbatch • Dialysis • Free interface diffusion (FID)
Vapor diffusion procedures Hanging drop Sitting Drop Cover slip (attached with grease) Drop (Protein/precipitant mix) Clear tape Well (precipitant)
Microbatch • crystallization (at microscale); droplet is covered by oil. • this prevents the evaporation of the very small drop.
Dialysis • sample placed in a dialysis cell or a dialysis button • sealed with a dialysis membrane
Free interface diffusion Protein and precipitant solutions are in contact (free interface)
Crystallization of proteins Curr. Opin. Struct. Biol. 14, 577–583 (2004)
Discussion between a protein purification expert and a crystallographer...