title: Novel affinity purification of monomeric sarcosine oxidase expressed in Escherichia coli.
abstract: An efficient affinity-purification protocol for Bacillus monomeric sarcosine oxidase (SOX) expressed in Escherichia coli BL21 (DE3) was developed. 4-Aminopyrrole-2-carboxylic acid was chosen as the affinity ligand, which was coupled with Sepharose CL 4B via spacers composed of epichlorohydrin and ethylenediamine. With the affinity medium, the purification process consisted of only one affinity chromatography step to capture monomeric SOX. The purified SOX was 94 and 96% pure when analyzed on an HPLC Vydac C8 column and reducing SDS-PAGE. Meanwhile, the recoveries of typical SOX activity and protein were 90.8 and 37.5%, respectively, which were higher than other reported traditional protocols. Reducing SDS-PAGE analysis revealed that the enzyme was a single polypeptide with the mass of ~46 kDa. The desorption constant Kd and theoretical maximum absorption Qmax were 35 μg/mL and 52.7 mg/g, respectively, in absorption analysis. All results indicated that the method would be of great potential for purifying monomeric SOX on an industrial scale.
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, P. R. China.
authors.forename: Yanjun
authors.initials: Y
authors.lastname: Tong
authors.forename: Yu
authors.initials: Y
authors.lastname: Xin
authors.forename: Hailin
authors.initials: H
authors.lastname: Yang
authors.forename: Ling
authors.initials: L
authors.lastname: Zhang
authors.forename: Xiumei
authors.initials: X
authors.lastname: Tao
authors.forename: Hui
authors.initials: H
authors.lastname: Xu
authors.forename: Wu
authors.initials: W
authors.lastname: Wang
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D050879:Sarcosine Oxidase / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D050879:Sarcosine Oxidase
keywords: 4-Aminopyrrole-2-carboxylic acid; Affinity purification; Bacillus sp; Sarcosine oxidase