在大肠杆菌中表达的水稻（Oryza sativa L.）两种抗坏血酸过氧化物酶的纯化与特性分析

Purification and characterization of two ascorbate peroxidases of rice (Oryza sativa L.) expressed in Escherichia coli.

作者信息

Lu Zhenqiang, Takano Tetsuo, Liu Shenkui

机构信息

Alkali Soil Natural Environmental Science Center (ASNESC), Stress Molecular Biology Laboratory, Northeast Forestry University, 150040, Harbin, P.R. China.

出版信息

Biotechnol Lett. 2005 Jan;27(1):63-7. doi: 10.1007/s10529-004-6587-0.

DOI:10.1007/s10529-004-6587-0

PMID:15685422

Abstract

To clarify the diversity and function of isozymes of ascorbate peroxidase (APX) in plants, a method of producing large quantities of these proteins is needed. Here, we describe an Escherichia coli expression system for the rapid and economic expression of two rice APX genes, APXa and APXb (GeneBank accession Nos. D45423 and AB053297, respectively). The two genes were cloned into the pGEX-6p-3 vector to allow expression of APX as a glutathione-S-transferase (GST) fusion protein. The GST-APXa and GST-APXb fusion proteins were purified by affinity chromatography using a glutathione-Sepharose 4B column, with final yields of 40 and 73 mg g(-1) dry cells, respectively. Specific activities were 15 and 20 mM ascorbate min(-1) mg(-1) protein, respectively. The K(m) values for ascorbate were 4 and 1 mM, respectively, and those for H(2)O(2) were 0.3 and 0.7 mM, respectively indicating that the two rice isoenzymes have different properties.

摘要

为阐明植物中抗坏血酸过氧化物酶（APX）同工酶的多样性和功能，需要一种大量生产这些蛋白质的方法。在此，我们描述了一种用于快速且经济地表达两个水稻APX基因APXa和APXb（分别为基因库登录号D45423和AB053297）的大肠杆菌表达系统。这两个基因被克隆到pGEX - 6p - 3载体中，以使APX作为谷胱甘肽 - S - 转移酶（GST）融合蛋白表达。GST - APXa和GST - APXb融合蛋白通过使用谷胱甘肽 - 琼脂糖4B柱的亲和层析进行纯化，最终产量分别为每克干细胞40毫克和73毫克。比活性分别为15和20毫摩尔抗坏血酸每分钟每毫克蛋白质。抗坏血酸的K(m)值分别为4毫摩尔和1毫摩尔，H(2)O(2)的K(m)值分别为0.3毫摩尔和0.7毫摩尔，这表明这两种水稻同工酶具有不同的特性。