The document discusses recombinant protein expression and engineering. It describes:
1) Cloning or synthesizing the gene of interest, making an expression construct, transfecting cells, purifying the recombinant protein.
2) Factors to consider like the protein's origin (prokaryotic/eukaryotic), required post-translational modifications, and available expression systems.
3) A case study expressing recombinant human alpha-1-acid glycoprotein in E. coli, including vector construction, periplasmic extraction, affinity purification, and yield.
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!
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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%
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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
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.
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
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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
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
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