人超氧化物歧化酶-过氧化氢酶融合基因的构建、表达及其产物的初步纯化和性质研究

Construction of superoxide dismutase-catalase fusion gene and its expression in Escherichia coli

尝试应用基因工程技术制备一种兼具人超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性的双功能融合蛋白CAT-PTD-SOD,其中的PTD是序列为RKKRRQRRR的短肽.首先通过重叠PCR构筑CAT-PTD-SOD融合基因,然后把该基因转化进入大肠杆菌表达菌株Rosetta 2(DE3)菌株.通过SDS-PAGE、CAT和SOD活性分析以及分离纯化组分的分析确认该重组菌株能够表达CAT-PTD-SOD,其表达量可达总蛋白的8.9%,SDS-PAGE显示其分子量约为85 kD.可溶性实验发现该融合蛋白大部分以兼具SOD和CAT活性的可溶形式存在.抗H2O2能力实验表明CAT-PTD-SOD具有很好的抗H2O2能力,在0.033 mol.L-1、甚至0.067 mol.L-1的H2O2溶液中,其SOD活性20 min内无明显下降.

In this study,gene engineering technology was used to prepare a double function fusion protein CAT-PTD-SOD to integrate the activity of superoxide dismutase（SOD） and catalase（CAT）,the PTD is a short peptide with a sequence of RKKRRQRRR.The fusion gene CAT-PTD-SOD was firstly constructed by overlap PCR,and then put the gene transformation into E.coli express strains Rosetta 2（DE3）.This strain was confirmed to express CAT-PTD-SOD by SDS-PAGE,the analysis of the enzymatical activity of CAT and SOD as well as the separated fractions of the cell lysate.Its expression level could amount to 8.9% of the total protein,and majority of the fusion protein was found soluble.The stability of CAT-PTD-SOD did not decline within 20 min in 0.033 mol·L-1 or even 0.067 mol·L-1 H2O2 solution.