title: Purification and characterization of the fusion protein trypsin-streptavidin expressed in Escherichia coli.
abstract: Expression of fusion protein trypsin-streptavidin (TRYPSA) in Escherichia coli was evaluated and the protein purified. Protein expression was induced by 1 mM isopropylthio-beta-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 degrees 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.
authors.affiliation: Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh, North Carolina 27695, USA.
authors.forename: F
authors.initials: F
authors.lastname: Zhao
authors.forename: D A
authors.initials: DA
authors.lastname: Clare
authors.forename: G L
authors.initials: GL
authors.lastname: Catignani
authors.forename: H E
authors.initials: HE
authors.lastname: Swaisgood
mesh_terms: D015153:Blotting, Western; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D019809:Streptavidin / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry*
chemical_list: D011993:Recombinant Fusion Proteins; D019809:Streptavidin; D014357:Trypsin