一种重组豌豆细胞质果糖-1,6-二磷酸酶的纯化与特性分析

Purification and characterization of a recombinant pea cytoplasmic fructose-1,6-bisphosphatase.

作者信息

Jang Hye-Kyung, Lee Sang-Won, Lee Youn-Hyung, Hahn Tae-Ryong

机构信息

Plant Metabolism Research Center (PMRC) and Graduate School of Biotechnology, Kyung Hee University, Suwon 449-701, Republic of Korea.

出版信息

Protein Expr Purif. 2003 Mar;28(1):42-8. doi: 10.1016/s1046-5928(02)00654-x.

DOI:10.1016/s1046-5928(02)00654-x

PMID:12651105

Abstract

Full-length cDNA encoding pea cytoplasmic fructose-1,6-bisphosphatase (cyFBPase) was cloned from a pea cDNA library. The cloned cDNA was introduced into the Escherichia coli expression vector pET-15b. The recombinant cyFBPase was expressed in E. coli BL21 (DE3) cells in a soluble form and purified to homogeneity by Ni(+)-NTA affinity chromatography. The identity of the recombinant cyFBPase was confirmed by SDS-PAGE and immunoblot analysis using a polyclonal anti-His tag antibody. The recombinant cyFBPase was active at neutral pH ranges (6.6-9.0) and thermostable as other cyFBPases. The activation energy (E(a)) and Arrhenius frequency factor were 17.4 kcal/mol and 2.6 x 10(12)/s, respectively. The K(M) and V(max) values of the recombinant enzyme were calculated as 10.47 microM and 109 micromol/min, respectively. In case of removal of histidine tag, the K(M) value was calculated as 5.03 microM. The recombinant enzyme was non-competitively and competitively inhibited by AMP and fructose-2,6-bisphosphate, respectively.

摘要

从豌豆cDNA文库中克隆出编码豌豆细胞质果糖-1,6-二磷酸酶（cyFBPase）的全长cDNA。将克隆的cDNA导入大肠杆菌表达载体pET-15b。重组cyFBPase在大肠杆菌BL21(DE3)细胞中以可溶形式表达，并通过Ni(+)-NTA亲和层析纯化至同质。使用多克隆抗His标签抗体通过SDS-PAGE和免疫印迹分析确认了重组cyFBPase的身份。重组cyFBPase在中性pH范围（6.6-9.0）具有活性，并且与其他cyFBPase一样具有热稳定性。活化能（E(a)）和阿仑尼乌斯频率因子分别为17.4千卡/摩尔和2.6×10(12)/秒。重组酶的K(M)和V(max)值分别计算为10.47微摩尔和109微摩尔/分钟。去除组氨酸标签后，K(M)值计算为5.03微摩尔。重组酶分别被AMP和果糖-2,6-二磷酸非竞争性和竞争性抑制。