大肠杆菌DPS表达、体外自组装及抗氧化作用研究

Expression,Self-assembly and Antioxidation of Escherichia coli DPS

DNA结合蛋白(DNA-binding proteins from starved cells,DPS)是细菌一类重要的抗逆境保护蛋白。对大肠杆菌DPS蛋白进行原核表达和纯化旨为研究其体外自组装和抗氧化活性。构建表达载体p BVDPSHis,诱导表达并纯化DPS单体蛋白,通过非变性PAGE检测DPS单体自组装情况,通过质粒在Fenton反应时的降解情况检测DPS对DNA的抗氧化保护作用。结果表明,PCR扩增得到522 bp的特异性基因片段,成功构建表达载体p BVDPSHis,诱导表达了分子量为19.5 k D的DPS单体蛋白,亲和层析法纯化的单体DPS经非变性PAGE电泳证实以多聚体蛋白形式存在,Fenton反应说明DPS具有保护质粒DNA抗氧化损伤作用。原核表达的DPS在体外可以自组装成多聚体结构具有抗氧化保护DNA作用。

DNA-binding protein from starved cells（DPS）is a key protective proteins in bacterial growth under stress environment. DPS was expressed in prokaryotic cells and purified so as to study its self-assembly activity in vitro. The dps gene was amplified by polymerase chain reaction with specific primers that was inserted by his tag at 3＇ end and the template was genome from Escherichia coli strain 0111. After digested together with Eco R I and Bam H I restrict enzymes, the dps and p BV220 were linked in order to construct p BVDPSHis expression vector. DPS expressed in E. coli by temperature induction was purified with affinity chromatography column and its self-assembly in vitro was tested with natural PAGE on the basis of protein molecular weight. DPS protection for DNA from oxidation was detected with 1.2% agarose electrophoresis. The results showed that a specific fragment with 522 bp length was acquired and p BVDPSHis vector was constructed correctly using enzyme digestion and sequencing tests. The DPS with 19.5 k D relative molecular weight on SDS-PAGE was identified with Western blotting, the purified DPS could self-assembly in vitro into polymers with much larger molecular weight than 19.5 k D in p H7.5 PBS solution and DPS protection DNA resistance to hydroxyl radicals oxidation could be demonstrated in Fenton reaction system. It was concluded that E.coli DPS expressed genetically in prokaryotic cells could correctly self-assembly to functional multi-polymers and had a ability of protection DNA from ROS oxidant damage in vitro.