Purification and characterization of the fusion protein trypsin-streptavidin expressed in Escherichia coli |
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Authors: | Zhao F Clare D A Catignani G L Swaisgood H E |
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Institution: | (1) Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, North Carolina, 27695 |
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Abstract: | Expression of fusion protein trypsin-streptavidin (TRYPSA)4 in Escherichia coli was evaluated and the protein purified. Protein expression was induced by 1 mM isopropylthio- -D-galactoside (IPTG), and the enzyme activity was measured by the hydrolysis rate of p-toluenesulfonyl-l-arginine methyl ester (TAME). Expression of the fusion protein in the cell-free extract decreased with increased induction time; correspondingly, that in the inclusion bodies increased. The total expression in Luria–Bertani broth (LB) and Terrific Broth (TB) media reached the highest levels in 2 hr at 30°C. The optimum expression level was 35 and 48 U/L in LB and TB, respectively. Expression of the fusion protein was verified by Western Blot analysis using streptavidin antiserum, and the fusion protein was purified using a benzamidine Sepharose 6B affinity column at room temperature. The molecular size of the soluble purified fusion protein was determined by size-exclusion chromatography using Superose 12 FPLC. A molecular weight of 39–40 kDa was obtained, indicating that the soluble protein exists as a monomer; thus, the presence of the trypsin domain must prevent the streptavidin domain from tetramer formation. |
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Keywords: | Affinity column fusion protein protein expression protein purification trypsin streptavidin |
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