title: Affinity purification of a cholesterol oxidase expressed in Escherichia coli.
abstract: A cholesterol oxidase (COD) gene from Brevibacterium sp. (DQ345780) was expressed in Escherichia coli BL21 (DE3), an affinity protocol was developed for the preparation, and industrial application of this method was of great potential. Riboflavin was chosen as the affinity ligand, and it was coupled with Sepharose 4B through some spacers. With the affinity medium, the purification process consisted of only one affinity chromatography step to capture the target protein. The purified cholesterol oxidase was 99.5% pure analyzed on HPLC Vydac C4 column, and 98% with SDS-PAGE analysis. The yield of the expressed enzyme was 9.8% of crude extracted proteins; the recovery of typical cholesterol oxidase activity was 90.1%, higher than that of other reported traditional protocols. Reducing SDS-PAGE analysis showed that the enzyme was a single polypeptide with the mass of ∼50 kDa. The desorption constant K(d) and the theoretical maximum absorption Q(max) on the affinity medium were 1.0 μg/g medium and 74.5 mg/g medium in absorption analysis. K(m) and V(max) of cholesterol oxidase activity for the purified enzyme were 25.5 μM and 16.4 μmol/(min mg), respectively.
authors.affiliation: School of Biotechnology, Jiangnan University, Key Laboratory of Industry Biotechnology, Ministry of Education, Wuxi, Jiangsu, PR China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Xin
authors.forename: Hailin
authors.initials: H
authors.lastname: Yang
authors.forename: Xiaole
authors.initials: X
authors.lastname: Xia
authors.forename: Ling
authors.initials: L
authors.lastname: Zhang
authors.forename: Chen
authors.initials: C
authors.lastname: Cheng
authors.forename: Guocui
authors.initials: G
authors.lastname: Mou
authors.forename: Jiebing
authors.initials: J
authors.lastname: Shi
authors.forename: Yunfei
authors.initials: Y
authors.lastname: Han
authors.forename: Wu
authors.initials: W
authors.lastname: Wang
mesh_terms: D001951:Brevibacterium / Q000201:enzymology; D002789:Cholesterol Oxidase / Q000737:chemistry / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D011994:Recombinant Proteins; D002789:Cholesterol Oxidase