水稻OsAAA1蛋白的原核表达载体构建及其可溶性表达研究

Research on the Construction and Soluble Expression of Prokaryotic Expression Vector of Os AAA1 Protein from Rice

为使用外源表达载体表达并纯化有活性的水稻ATP酶蛋白Os AAA1,构建了p ET-32a-Os AAA1原核表达载体并进行体外IPTG诱导表达和Ni＋柱亲和层析纯化.利用SDS-PAGE和Western Blot检测了目的蛋白.结果表明：将成功构建的p ET-32a-Os AAA1原核表达载体,转化到大肠杆菌Origami菌株后,在70~100 KD之间检测到可溶性目的蛋白带,并优化了诱导表达的较适温度、IPTG诱导浓度和诱导表达时间.故成功构建了p ET-32a-Os AAA1原核表达载体并获得了可溶性Os AAA1目的蛋白,为其后续研究奠定了基础.

To express and purify the activated enzyme protein Os AAA1 from rice by exogenous expression vector,the prokaryotic expression vector of p ET-32a-Os AAA1 was constructed and the expression of fusion protein was induced by IPTG and purified by affinity chromatography on a Ni2 ＋column. The expression of the fusion protein was detected by SDSPAGE and Western Blot analysis. The results indicated that the recombinant vector p ET-32a-Os AAA1 was constructed and transformed into E. coli Origami successfully and a soluble fusion protein was expressed at 70 ~ 100 KD. The temperature,concentration of IPTG induction and induction time were also optimized. So the prokaryotic expression vector of p ET-32 aOs AAA1 was successfully constructed,and the soluble protein of Os AAA1 was obtained,which would lay the foundation for its further research.