Catalase-TAT的融合表达与表征

Fused-expression and characterization of catalase-TAT

利用RT-PCR技术从人的肝细胞中克隆出成熟蛋白过氧化氢酶(catalase)的基因,通过PCR将其与TAT基因融合形成融合基因(catalase-TAT);将catalase-TAT基因构建到表达质粒pET21a(+)中,转入大肠杆菌Rosetta2(DE3);通过IPTG诱导,转化子成功表达出catalase-TAT融合蛋白.重组菌发酵菌体破碎液经过硫酸铵沉降和Sp-5pw阳离子交换色谱,分离得到电泳纯的目的蛋白,其纯化倍数为18.51倍;经理化性质分析确定了此融合蛋白的最适pH和温度稳定性;细胞实验表明融合蛋白catalase-TAT能够明显提高胞内的过氧化氢酶活性.

Human catalase gene was amplified from liver cells via RT PCR and fused to TAT. The catalase TAT was cloned into the expression vector pET21a （ ＋ ） and transformed into Rosetta2 （DE3）. After induction with IPTG, the recombinant catalase TAT fusion protein was successfully expressed. The recombinant bacteria were sonicated and the targeted protein was purified to a single band on SDS PAGE after ammonium sulfate precipitation and Sp 5pw cation exchange chromatogra phy, with the purification factor of 18.51. With physical and chemical analyses, the optimum pH and thermal stability of the fusion protein was determined.