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Recombinant protein expression and purification Lecture
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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.
What do you know about your protein? 5.
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Expressing your protein 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% 30% 8.
Getting your gene 9.
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Vectors, tags and solubility 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. Induction test 13. Purification 14. 15. Chromatography (GF)(HIC)(IEX)(AC)(RPC) 16. Chromatography 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, ionicstrength or polarity. 4. Affinity medium is re-equilibrated with binding buffer. 18. GST fusion protein purification Column:GSTrap 1mlLane 1: MW StdsSample:8 ml cytosolic extract fromE.coli Lane 2: E.coli expressing a GST fusion proteincytosolic extractBinding buffer (BB):PBS, pH 7.3Lane 3:GSTrapElution buffer(EB):50 mM Tris-HCl, pH 8.0 withelution 10 mM reduced glutathioneFlow rate:1ml/min[CV=column volume] Chromatographic sequence:4 CV BB 8ml sample 10 CV BB 5 CV EBSystem: KTAexplorer 10 FPLC (Amersham Pharmacia biotech) glutathione sepharose 19.
Purification additives? 20. Removal of the Tag
Units 21. Case Study-Human 1AcidGlycoprotein >humanalpha1AcidGlycoprotein IPL C ANLVPVPITNATLDQITGKWFYIASAFRNEEYNKSVQEIQATFFY FTPNKTEDTIFLREYQTRQDQ C IYNTTYLNVQRENGTISRYVGGQEHFAH LLILRDTKTYMLAFDVNDEKNWGLSVYADKPETTKEQLGEFYEALD C LRI PKSDVVYTDWKKDK C EPLEKQHEKE Schnfeld DLet al.,J. Mol. Biol. (2008) 384, 393405 22. Recombinant protein production and purification Human AGP (SWISS-Prot entry P02763) was produced inE. coli K12 strain MC4100skpusing theexpression vector pAGP1and the foldinghelper plasmid pTUM4essentially as previously described.cDNA for variant F1 cloned from human liverand encodes a fusion protein with theN-terminal OmpA signal peptide( effecting periplasmic secretion ) and theC-terminal Streptag IIof nine residues. Preparative protein production was performed at25 C in a fermenterfollowing a published protocol. Briefly, gene expression was induced at an optical cell density of OD550=20 by addinganhydrotetracyclineto a final concentration of 0.5 mg/L, and cells were harvested by centrifugation after 3 h. Theperiplasmic extractwas prepared by resuspending the cells in ice-cold 0.5 M sucrose, 15 mM ethylenediaminetetraacetic acid, 100 mM TrisHCl (pH 8.0), and 250 g/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 against150 mM NaCl, 1 mM ethylenediaminetetraacetic acid,100mMTrisHCl (pH 8.0), andaffinity-purifiedon a column with immobilized engineered streptavidin, followed bygelfiltrationon a Superdex 75 column (Amersham Pharmacia, Uppsala, Sweden) using 100 mM NaCl, 10 mM TrisHCl(pH 8.0) as running buffer. AGP eluted in a homogeneous peak corresponding to the monomeric protein, and its finalyield was ca 2 mg/L E. coli culture . Extract from methods-Schnfeld DLet al.,J. Mol. Biol. (2008) 384, 393405 23. Protein engineeringHuman 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 24. Thats All Folks!