Optimisation of expression and purification of the recombinant Yo1066 (Rib2) protein from Saccharomyces cerevisiae

被引：16

作者：

Urban, A ^{[1
]}

Ansmant, I ^{[1
]}

Motorin, Y ^{[1
]}

机构：

[1] UHP Nancy 1, Fac Sci, CNRS, UMR 7567,Lab Maturat ARN & Enzymol Mol, F-54506 Vandoeuvre Les Nancy, France

来源：

JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES | 2003年 / 786卷 / 1-2期

关键词：

expression; purification; Saccharomyces cerevisiae; recombinant Yo1066 protein;

D O I：

10.1016/S1570-0232(02)00742-0

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Yeast protein Yo1066 (encoded by YOL066 ORF, also known as Rib2) possesses two distinct sequence domains: C-terminal deaminase domain and N-terminal part related to RNA:pseudouridine (psi)-synthases. The deaminase domain is implicated in the riboflavine biosynthesis, while the exact function of the RNA:Psi-synthase domain remains obscure. Here we report the optimisation of growth conditions and purification scheme for recombinant His(6)-tagged Yo1066 expressed in E. coli BL21(DE3) using pET28 plasmid. Production of soluble Yo1066 protein is best at low temperature (18degreesC) and IPTG concentration (50 KM) and Yo1066 purification was achieved using metal-affinity and ion-exchange chromatography. This optimised protocol yields about 10 mg of highly purified recombinant Yo1066 from 31 of E. coli culture. (C) 2002 Elsevier Science B.V. All rights reserved.

引用

页码：187 / 195

页数：9

共 37 条

[1] Identification of the Saccharomyces cerevisiae RNA:pseudouridine synthase responsible for formation of Ψ2819 in 21S mitochondrial ribosomal RNA [J].