title: Expression of a kallikrein-like protease from the snake venom: engineering of autocatalytic site in the fusion protein to facilitate protein refolding.
abstract: In order to circumvent the difficulty encountered in the expression and purification of the recombinant products in E. coli system, we have developed a novel and facile method of removing the polyhistidine tag from target proteins after heterologous gene expression. The expression of a serine protease (Tm-5) from Taiwan habu (Trimeresurus mucrosquamatus) is taken as an exemplar to illustrate the basic rationales and protocols involved. In place of an enterokinase recognition site, a polyhistidine tag linked to an autocatalyzed site based on cleavage specificity of the serine protease flanking on the 5'-end of Tm-5 clone sequence was engineered before protein expression in E. coli system. Renaturation of the fusion protein after expression revealed that the recombinant protease had refolded successfully from the inclusion bodies. Upon autocleavage of the expressed protease, the polyhistidine tag with additional amino acid residues appended to the N-terminus of the coding sequence is found to be removed accordingly. The protein expressed and purified by this new strategy possesses a molecular weight of approximately 28,000 in accord with the expected value for this venom protease. Further characterization of the recombinant protein employing a variety of techniques which include immunoblot analysis, RP-HPLC, ESI-MS, and N-terminal amino acid sequencing all shows indistinguishable properties to those of the isolated native protease. Most noteworthy is that the recombinant Tm-5 protease also exhibits amidase activity against N-benzoyl-Pro-Phe-Arg-p-nitroanilide, a unique and strict substrate for native Tm proteases reported previously.
authors.affiliation: Institute of Biochemical Sciences, College of Science, National Taiwan University, Taiwan.
authors.forename: C C
authors.initials: CC
authors.lastname: Hung
authors.forename: S H
authors.initials: SH
authors.lastname: Chiou
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000201:enzymology* / Q000235:genetics; D004765:Enteropeptidase / Q000378:metabolism; D004926:Escherichia coli; D015870:Gene Expression; D002479:Inclusion Bodies; D007610:Kallikreins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity
chemical_list: D003435:Crotalid Venoms; D011993:Recombinant Fusion Proteins; C029748:Trimeresurus venoms; D007610:Kallikreins; D004765:Enteropeptidase; D000581:Amidohydrolases; C019665:amidase