Recombinant protein expression and purification Lecture

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Recombinant protein expression and purification Lecture

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Recombinant protein expression and purification Lecture

  1. 1. RECOMBINANT PROTEIN EXPRESSION AND PROTEIN ENGINEERING
  2. 2. <ul><li>Why recombinant proteins? </li></ul><ul><ul><li>  To identify the polypeptide coded by a DNA-sequence </li></ul></ul><ul><ul><li>To analyze the biological activity </li></ul></ul><ul><ul><li>To study the structure-function relationships, interactions </li></ul></ul><ul><ul><li>To study the 3D-structure </li></ul></ul><ul><ul><li>To do protein engineering and design </li></ul></ul><ul><ul><li>To raise specific antibodies </li></ul></ul><ul><ul><li>To develop a target-specific drug </li></ul></ul><ul><ul><li>To produce therapeutic proteins </li></ul></ul><ul><ul><li>To produce vaccines </li></ul></ul><ul><ul><li>To produce biotechnological enzymes </li></ul></ul>
  3. 3. How to make recombinant proteins         clone OR synthesize the gene         make an expression construct         transfect and grow cells         purify the recombinant protein         problem: Recombinant protein production-not a trivial task!
  4. 4. <ul><ul><li>A little bio-informatics can be very helpful </li></ul></ul><ul><ul><li>Blast for obtaining all of the related sequences </li></ul></ul><ul><ul><li>ClustalW (or some other alignment tool) can be used to determine the boundaries of your domains </li></ul></ul><ul><ul><li>Don’t assume you know what is correct; that is, there is often some ambiguity, so don’t rely on a single construct </li></ul></ul><ul><ul><li>Use other people’s experience: if you want to produce a Tyrosine kinase, don’t bother with E. coli as an expression host </li></ul></ul><ul><ul><li>What are the possible post-translational modifications that may be required for folding/ function (glycosylation, etc.)? </li></ul></ul><ul><ul><li>What are the co-factors that may be required for folding/ function? </li></ul></ul><ul><ul><li>Cysteines? Do you have disulfides or free cysteines on the surface? Methionines? Are you using SeMet to phase? </li></ul></ul>What do you know about your protein?
  5. 5. <ul><li>Selection of protein production host depends on: </li></ul><ul><li>produced protein- prokaryotic/eukaryotic; cytoplasmic/ </li></ul><ul><li>secreted; organelle specific </li></ul><ul><li>amounts needed- analytical, functional studies (ng); antibody </li></ul><ul><li>production (  g-mg); structural biology (mg); commercial (g-kg) </li></ul><ul><li>demands on authenticity- active/inactive=native/denatured; </li></ul><ul><li>modified/not modified (glycosylation etc.); molecular weight; sequence;  expression=  authenticity </li></ul><ul><li>$ money $ </li></ul><ul><li>protein production </li></ul><ul><li>systems available in the lab- see above  </li></ul>
  6. 6. <ul><ul><li>Which host? For human proteins, mammalian cells may be the best, but they are also the slowest and most expensive to use. </li></ul></ul><ul><ul><li>Insect cells (baculovirus) have many of the advantages of mammalian cell culture (post-translational modifications, eukaryotic cell system, etc.) but they are faster and cheaper to use. </li></ul></ul><ul><ul><li>Yeast is an option for some things, but the differences can bite (particularly in glycosylation pattern and extraction). </li></ul></ul><ul><ul><li>E. coli is the cheapest, easiest and fastest by far, but it just doesn’t work for some proteins. </li></ul></ul><ul><ul><li>Do you need to add co-factors to the media? Do you need a chaperone or second protein for correct folding? </li></ul></ul>Expressing your protein
  7. 7. Characteristics of different expression systems Characteristic E.coli Yeast Mammalian Insect Proteolytic cleavage ? ? Y Y Glycosylation N ? Y ? Sectretion ? Y Y Y Folding ? ? Y Y Phosphorylation N ? Y ? Acetylation N Y Y ? Amidation N Y Y Y % yield >50% 1% <1% >30%
  8. 8. <ul><ul><li>Clone by phone- There are plenty of gene synthesis shops around the world that will make your gene for you. The advantage to this is that they can bias the codon usage to the expression host of choice </li></ul></ul><ul><ul><li>Or PCR your heart away. This will often break your heart ;-) </li></ul></ul><ul><ul><li>Taq? Vent? Pfu/Pfx? </li></ul></ul><ul><ul><li>Length and melting temperature of your primers </li></ul></ul><ul><ul><ul><li>Order from plasmid banks: </li></ul></ul></ul><ul><ul><ul><li>The Harvard plasmid repository: </li></ul></ul></ul><ul><ul><ul><li>http://plasmid.med.harvard.edu/PLASMID/ </li></ul></ul></ul><ul><ul><ul><li>The Addgene plasmid bank: </li></ul></ul></ul><ul><ul><ul><li>http://www.addgene.org/pgvec1 </li></ul></ul></ul><ul><ul><ul><li>Least reliable-human generosity: </li></ul></ul></ul><ul><ul><ul><li>Ask a scientist who has published a report using the gene of interest. </li></ul></ul></ul>Getting your gene
  9. 9. <ul><li>Expression vector structure </li></ul><ul><li>Promoter-transcription initiation </li></ul><ul><li>Stop codon </li></ul><ul><li>Localization signals </li></ul><ul><li>Codon usage </li></ul><ul><li>polyA signal </li></ul><ul><li>Fusion partners </li></ul><ul><li>Stable/inducible </li></ul><ul><li>expression </li></ul><ul><li>Control of copy number </li></ul><ul><li>Episomal/integrating </li></ul><ul><li>Resistance marker </li></ul>
  10. 10. <ul><ul><li>The highest level of expression is not always the best High expression can also give you the most insoluble material </li></ul></ul><ul><ul><li>Sometimes reducing the temperature and the expression level can give you more overall soluble material </li></ul></ul><ul><ul><li>Tags and protease sites can effect your protein solubility/ stability Most tags will make your protein more soluble, but it may not stay soluble if you pull the tag off. </li></ul></ul><ul><ul><li>Some proteases will take off more than you expect! </li></ul></ul><ul><ul><li>Some proteolytic sites can effect your protein (Kurz et al., Protein Expression and Purification, v. 50, p.68-73, 2006: Incorporating a TEV cleavage site reduces the solubility of nine recombinant mouse proteins) </li></ul></ul>Vectors, tags and solubility
  11. 11. Fusion protein vectors Affinity tag Residues Sequence Matrix Poly-His Usually 6 HHHHHH Ni Poly-Arg Usually 5 RRRRR Cation-exchange Glutatione S-transferase 211 Protein Glutathione Maltose-binding protein 396 Protein Cross-linked amylose Streptavidin binding protein 38 Peptide Streptavidin Calmodulin-binding protein 26 Peptide Calmodulin Chitin-binding protein 51 Protein domain Chitin c-myc 11 EQKLISEEDL Anti-body HA(Hemaglutanin) 9 YPYDVPDYA Anti-body Flag/x3 Flag 8/24 DYKDDDK/ DYKDDDK x3 Anti-body T7 11 MASMTGGQQMG Anti-body
  12. 12. Induction test
  13. 13. Purification
  14. 15. Chromatography (GF) (HIC) (IEX) (AC) (RPC)
  15. 16. Chromatography
  16. 17. Affinity purification 1. Affinity medium is equlibrated with binding buffer. 2. Sample application under conditions that favor binding to the complementary structure on the medium. Target protein binds specifically, but reversibly, unbound material eluted. 3. Target protein recovery: *Specifically-competitive ligand *Non-specifically-pH, ionic strength or polarity. 4. Affinity medium is re-equilibrated with binding buffer.
  17. 18. GST fusion protein purification Column: GSTrap 1ml Lane 1: MW Stds Sample: 8 ml cytosolic extract from E.coli Lane 2: E.coli expressing a GST fusion protein cytosolic extract Binding buffer (BB): PBS, pH 7.3 Lane 3: GSTrap Elution buffer (EB): 50 mM Tris-HCl, pH 8.0 with elution 10 mM reduced glutathione Flow rate: 1ml/min [CV=column volume] Chromatographic sequence: 4 CV BB  8ml sample  10 CV BB  5 CV EB System: ÄKTAexplorer 10 FPLC (Amersham Pharmacia biotech) glutathione sepharose 
  18. 19. <ul><ul><li>Cofactors and additives to stabilze the protein </li></ul></ul><ul><ul><li>To β-ME or not to β-ME: that is the question: Whether 'tis nobler in solution to suffer The slings and arrows of outrageous reduction, Or to take arms against a sea of oxidation, And by opposing end them? </li></ul></ul><ul><ul><li>(or DTT? or TCEP?) </li></ul></ul><ul><ul><li>Glycerol? </li></ul></ul><ul><ul><li>Detergents? (beware of Bugbuster, B-Per, etc.) </li></ul></ul><ul><ul><li>Metal ions? </li></ul></ul><ul><ul><li>High salt? (i.e. > 200mM) </li></ul></ul><ul><ul><li>Phosphate buffer? </li></ul></ul><ul><ul><li>ATP/ADP? GTP/GDP? NADP/NADPH? or other cofactors... </li></ul></ul>Purification additives?
  19. 20. Removal of the Tag <ul><li>Factors to consider with choosing a protease </li></ul><ul><li>Cost </li></ul><ul><li>Specificity </li></ul><ul><li>Vector availability </li></ul><ul><li>Removal of the protease </li></ul><ul><li>Incubation time and efficiency </li></ul>Units
  20. 21. Case Study-Human α 1AcidGlycoprotein >humanalpha1AcidGlycoprotein IPL C ANLVPVPITNATLDQITGKWFYIASAFRNEEYNKSVQEIQATFFY FTPNKTEDTIFLREYQTRQDQ C IYNTTYLNVQRENGTISRYVGGQEHFAH LLILRDTKTYMLAFDVNDEKNWGLSVYADKPETTKEQLGEFYEALD C LRI PKSDVVYTDWKKDK C EPLEKQHEKE Schönfeld DL et al., J. Mol. Biol. (2008) 384, 393–405
  21. 22. Recombinant protein production and purification Human AGP (SWISS-Prot entry P02763) was produced in E. coli K12 strain MC4100Δskp using the expression vector pAGP1 and the folding helper plasmid pTUM4 essentially as previously described. cDNA for variant F1 cloned from human liver and encodes a fusion protein with the N-terminal OmpA signal peptide ( effecting periplasmic secretion ) and the C-terminal Streptag II of nine residues. Preparative protein production was performed at 25 °C in a fermenter following a published protocol. Briefly, gene expression was induced at an optical cell density of OD550=20 by adding anhydrotetracycline to a final concentration of 0.5 mg/L, and cells were harvested by centrifugation after 3 h. The periplasmic extract was prepared by resuspending the cells in ice-cold 0.5 M sucrose, 15 mM ethylenediaminetetraacetic acid, 100 mM Tris–HCl (pH 8.0), and 250 μg/mL lysozyme, and by incubating them on ice for 30 min. The resulting spheroplasts were sedimented by centrifugation, and the supernatant containing the recombinant protein was recovered. The protein extract was dialyzed against 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 100mMTris–HCl (pH 8.0), and affinity-purified on a column with immobilized engineered streptavidin, followed by gel filtration on a Superdex 75 column (Amersham Pharmacia, Uppsala, Sweden) using 100 mM NaCl, 10 mM Tris–HCl (pH 8.0) as running buffer. AGP eluted in a homogeneous peak corresponding to the monomeric protein, and its final yield was ca 2 mg/L E. coli culture . Extract from methods-Schönfeld DL et al., J. Mol. Biol. (2008) 384, 393–405
  22. 23. Protein engineering Human α 1AcidGlycoprotein >humanalpha1AcidGlycoprotein IPL C ANLVPVPITNATLDQITGKWFYIASAFRNEEYNKSVQEIQATFFY FTPNKTEDTIFLREYQTRQDQ C IYNTTYLNVQRENGTISRYVGGQEHFAH LLILRDTKTYMLAFDVNDEKNWGLSVYADKPETTKEQLGEFYEALD C LRI PKSDVVYTDWKKDK C EPLEKQHEKE >humanalpha1AcidGlycoprotein IPL D ANLVPVPITNATLDQITGKWFYIASAFRNEEYNKSVQEIQATFFY FTPNKTEDTIFLREYQTRQDQ K IYNTTYLNVQRENGTISRYVGGQEHFAH LLILRDTKTYMLAFDVNDEKNWGLSVYADKPETTKEQLGEFYEALD K LRI PKSDVVYTDWKKDK D EPLEKQHEKE S-S bridges Salt bridges
  23. 24. That’s All Folks!

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