1 / 29

Protein Purification & Crystallization

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

bardia
Download Presentation

Protein Purification & Crystallization

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Protein Purification & Crystallization

  2. From cDNA to crystal structure

  3. 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

  4. 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?

  5. III II I The three Phase Purification Strategy

  6. Purification procedures • Precipitation techniques • Affinity chromatography • Ion-exchange chromatography • Hydrophobic interaction chromatography • Gel filtration

  7. Precipitation techniques

  8. 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

  9. 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

  10. Ni-NTA columns - the specificity of the interaction between histidine residues and immobilized nickel ions

  11. Affinity Separation. An example

  12. 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

  13. - - + + + + + + + + + - + + + + - - - - + + + - + + + + Ion-Exchange chromatography If pH mobile phase =7.2 Then charge of the proteins: (-) (-) (+) (+) Anion exchange column = + charged

  14. + + + Cl- + + + Cl- - + + + Cl- - - + + + Cl- + + + Cl- Na+ - Na+ - Na+ Na+ Na+ - - Na+ Na+ + + Na+ Cl- - Na+ Ion-Exchange chromatography - - + + Increased salt concentration

  15. Typical IEX gradient elution

  16. 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

  17. Typical HIC gradient elution

  18. Size-exclusion chromatography

  19. Gel filtration elution

  20. 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 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

  21. Storage of purified proteins

  22. 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)

  23. Crystallization of proteins • Vapor diffusion procedures: - hanging drop - sitting drop • Microbatch • Dialysis • Free interface diffusion (FID)

  24. Vapor diffusion procedures Hanging drop Sitting Drop Cover slip (attached with grease) Drop (Protein/precipitant mix) Clear tape Well (precipitant)

  25. Microbatch • crystallization (at microscale); droplet is covered by oil. • this prevents the evaporation of the very small drop.

  26. Dialysis • sample placed in a dialysis cell or a dialysis button • sealed with a dialysis membrane

  27. Free interface diffusion Protein and precipitant solutions are in contact (free interface)

  28. Crystallization of proteins Curr. Opin. Struct. Biol. 14, 577–583 (2004)

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

More Related