CAJ | 학술논문

为使用外源表达载体表达并纯化有活性的水稻ATP酶蛋白OsAAA1，构建了pET-32a-OsAAA1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pET-32a-OsAAA1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pET-32a-OsAAA1原核表达载体并获得了可溶性OsAAA1目的蛋白，为其后续研究奠定了基础。
위사용외원표체재체표체병순화유활성적수도ATP매단백OsAAA1，구건료pET-32a-OsAAA1원핵표체재체병진행체외IPTG유도표체화Ni＋주친화층석순화。이용SDS-PAGE화Western Blot검측료목적단백。결과표명：장성공구건적pET-32a-OsAAA1원핵표체재체，전화도대장간균Origami균주후，재70～100 KD지간검측도가용성목적단백대，병우화료유도표체적교괄온도、IPTG유도농도화유도표체시간。고성공구건료pET-32a-OsAAA1원핵표체재체병획득료가용성OsAAA1목적단백，위기후속연구전정료기출。
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.