Chinese Journal of Rice Science

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

WANG Ling; LIU Lian-meng; FU Qiang; HUANG Shi-wen*   

  1. China National Rice Research Institute, Hangzhou 310006, China; *Corresponding author, E-mail: swhuang666@sohu.com
  • Received:1900-01-01 Revised:1900-01-01 Online:2011-05-10 Published:2011-05-10

新霉素磷酸转移酶基因nptⅡ的原核表达、蛋白纯化及其活性鉴定

王 玲;刘连盟;傅 强;黄世文*   

  1. 中国水稻研究所, 浙江 杭州 310006; *通讯联系人, E-mail: swhuang666@sohu.com

Abstract: 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.

Key words: neomycin phosphotransferase gene, prokaryotic expression, inclusion body, soluble protein, purification

摘要: 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35 kD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5 mmol/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Ni2+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30 μg/mL以上时可使卡拉霉素失去抑菌活性。

关键词: 新霉素磷酸转移酶基因, 原核表达, 包涵体, 可溶性蛋白, 纯化