title: [Expression in Escherichia coli, purification and enzymatic properties of chicken aminopeptidase H].
abstract: Aminopeptidase H (APH) is an universally distributed aminoendopeptidase in the tissue of many organism. However, it is hard to investigate its mechanism underlying the catalysis and the function in cell. In this paper, the full DNA sequence of this enzyme was cloned from chicken liver, then subcloned to the vector pET22 b(+). The recombined vector was transformed into E. coli Rosetta(DE3), and the APH gene was expressed by the induction of IPTG. It was found the recombinant protein exhibited same mo lecular weight as authentic APH on SDS-PAGE analysis; the expression level increased with induction time and approached maximum of 94.7 mg/L till 6 hours, which contained 16.7% of the total protein. Moreover, this recombinant protein showed similar prop erties of subunit composition, thermal stability and optimum pH with native APH, based on the enzymatic assay, purification and analysis of enzymological properties. Therefore, it is confirmed that APH was expressed in this prokaryote system with a high-level of 1636 u/L aminopeptidase activity. These results would help to elucidate the catalysis mechanism and biological function of APH by providing enough material.
authors.affiliation: Institute of Biotechnology in Fuzhou University, Fuzhou 350001, China.
authors.forename: Qingan
authors.initials: Q
authors.lastname: Lai
authors.forename: Shutao
authors.initials: S
authors.lastname: Liu
authors.forename: Wanhua
authors.initials: W
authors.lastname: Lu
authors.forename: Li
authors.initials: L
authors.lastname: Chen
authors.forename: Toshihide
authors.initials: T
authors.lastname: Nishimura
authors.forename: Pingfan
authors.initials: P
authors.lastname: Rao
mesh_terms: D000626:Aminopeptidases / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D002645:Chickens; D010450:Endopeptidases / Q000096:biosynthesis* / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D011994:Recombinant Proteins; D010450:Endopeptidases; D000626:Aminopeptidases; C030596:hydrolase H