粘质沙雷氏菌寡肽酶 B。I. 野生型和重组酶变体的一级结构测定、分离和纯化。

Oligopeptidase B from Serratia proteamaculans. I. Determination of primary structure, isolation, and purification of wild-type and recombinant enzyme variants.

机构信息

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia.

出版信息

Biochemistry (Mosc). 2009 Oct;74(10):1164-72. doi: 10.1134/s0006297909100137.

PMID:19916930

Abstract

A novel trypsin-like protease (PSP) from the psychrotolerant gram-negative microorganism Serratia proteamaculans was purified by ion-exchange chromatography on Q-Sepharose and affinity chromatography on immobilized basic pancreatic trypsin inhibitor (BPTI-Sepharose). PSP formed a tight complex with GroEL chaperonin. A method for dissociating the GroEL-PSP complex was developed. Electrophoretically homogeneous PSP had molecular mass of 78 kDa; the N-terminal amino acid sequence 1-10 was determined, and mass-spectral analysis of PSP tryptic peptides was carried out. The enzyme was found to be the previously unknown oligopeptidase B (OpdB). The S. proteamaculans 94 OpdB gene was sequenced and the producer strain Escherichia coli BL-21(DE3) pOpdB No. 22 was constructed. The yield of expressed His(6)-PSP was 1.5 mg/g biomass.

摘要

一种新型的胰蛋白酶样蛋白酶（PSP）从耐冷革兰氏阴性微生物产硷假单胞菌中被分离出来，通过 Q-Sepharose 离子交换层析和固定化碱性胰蛋白酶抑制剂（BPTI-Sepharose）亲和层析进行纯化。PSP 与 GroEL 伴侣蛋白形成紧密复合物。开发了一种分离 GroEL-PSP 复合物的方法。电泳纯的 PSP 分子量为 78 kDa；确定了 N 端氨基酸序列 1-10，并对 PSP 胰蛋白酶肽进行了质谱分析。该酶被发现是以前未知的寡肽酶 B（OpdB）。对产硷假单胞菌 94 号 OpdB 基因进行测序，并构建了表达菌株大肠杆菌 BL-21(DE3) pOpdB No. 22。表达的 His(6)-PSP 的产量为 1.5 mg/g 生物质。