拟南芥IAA7的原核表达及稳定性分析

Prokaryotic expression and stability analysis of IAA7 protein in Arabidopsis

以p GEX–KG为基本骨架,构建了拟南芥IAA7蛋白的原核表达载体,用IPTG诱导IAA7在3种大肠杆菌表达菌株Rosetta、BL21和Tuner中表达,利用GST SefiroseTM resin亲和树脂分离纯化GST–IAA7融合蛋白,并分析重组蛋白在体外保存时的稳定性。结果表明:GST–IAA7融合蛋白在Rosetta菌株中于25℃和0.4 mmol/L IPTG诱导下表达较好;蛋白酶抑制剂苯甲基磺酰氟(PMSF)可显著延长GST–IAA7在体外保存的时间,最后利用凝血酶切除GST标签后获得了纯化的IAA7蛋白质。

Prokaryotic expression vector of IAA7 protein in Arabidopsis were constructed based on p GEX–KG in this experiment. IPTG was used to induce the expression of IAA7 protein in three Escherichia coli strains, namely, Rosetta, BL21 and Tuner. Fusion protein of GST–IAA7 was isolated and purified through GST SefiroseTM resin, and then the stability of recombination proteins was analyzed in vitro. The results showed that the expression of GST–IAA7 was higher in Rosetta with 0.4 mmol/L IPTG concentration at 25 ℃, and PMSF could significantly prolong the storage time of GST–IAA7 in vitro. Pure protein IAA7 was harvested after GST tag was cut off by thrombin.