水稻OsAAA1蛋白的原核表达载体构建及其可溶性表达研究

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.

Research on the Construction and Soluble Expression of Prokaryotic Expression Vector of OsAAA1 Protein from Rice

To express and purify the activated enzyme protein OsAAA1 from rice by exogenous expression vector, the prokaryotic expression vector of pET-32a-OsAAA1 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 SDS-PAGE and Western Blot analysis. The results indicated that the recombinant vector pET-32a-OsAAA1 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 pET-32a-OsAAA1 was successfully constructed, and the soluble protein of OsAAA1 was obtained, which would lay the foundation for its further research.