新霉素磷酸转移酶基因npt Ⅱ的原核表达、蛋白纯化及其活性鉴定

Prokaryotic Expression,Purification and Activity Assay of Neomycin Phosphotransferase Ⅱ(nptⅡ) Gene

通过PCR方法从pCAMBIA1305载体上克隆了新霉素磷酸转移酶基因nptⅡ的全编码序列,并插入到原核表达载体pET30a(+)中,构建了重组质粒pET30a-nptⅡ。将重组质粒转化大肠杆菌BL21(DE3)宿主菌,经IPTG诱导,获得了相对分子量为35kD的表达蛋白,约占全菌总蛋白的45%。表达蛋白以可溶性和包涵体两种形式存在。用5mmol/L二硫苏糖醇和1%十二烷基肌氨酸钠变性溶解包涵体,经透析后复性获得了可溶性重组蛋白。将可溶的表达蛋白用Ni 2+-NTA亲和层析纯化,获得纯化的NPTⅡ蛋白,电泳谱带扫描分析表明蛋白纯度达95%以上。体外活性检测显示,NPTⅡ蛋白具有良好的生物活性,在浓度30μg/mL以上时可使卡拉霉素失去抑菌活性。

Neomycin phosphotransferaseⅡ（nptⅡ） gene,cloned from a pCAMBIA1305 vector by PCR method,was inserted into a pET30a（＋） vector to construct the recombinant vector pET30a-nptⅡ.Then the recombinant vector was transformed into an Escherichia coli strain BL21（DE3） and the expression of the NPTⅡ protein was successfully induced by adding isopropyl-β-D-thiogalactopyranoside（IPTG）.The NPTⅡ fusion protein was produced in the form of the solubility or inclusion body with molecular weight around 35 kD,and a yield of approximately 45%.The inclusion bodies were solubilized and denatured by addition of 5 mmol/L dithiothreitol（DDT） and 1% sodium lauroyl sarcosine（SKL）,then renatured by dialysis.The solubilized proteins were purified by Ni2＋-NTA affinity chromatography at approximately 95% purity.An in vitro assay revealed that,the NPTⅡ protein can inactivate the kanamycin at concentrations up to 30 μg/mL.