矮秆蓖麻天冬氨酸蛋白酶基因的克隆、表达及蛋白纯化

Gene cloning,expression and purification of Ricinus communis.L aspartic proteases

目的克隆表达矮秆蓖麻(Ricinus communis L)天冬氨酸蛋白酶(aspartic protease,AP)基因(RcAP)并进行蛋白纯化。方法Q-PCR法验证高秆及矮秆蓖麻六叶期叶片RcAP基因表达量;提取矮秆蓖麻六叶期叶片总RNA,PCR扩增RcAP基因,克隆至pETM30载体中,构建重组表达质粒pETM30-RcAP[含谷胱甘肽-S-转移酶(glutathione-Stransferase,GST)标签],转化至E.coli DH5α感受态细胞中,优化IPTG诱导浓度及温度,SDS-PAGE分析目的蛋白的可溶性;采用亲和层析纯化重组蛋白GST-RcAP。结果矮秆蓖麻RcAP基因表达量为高秆蓖麻RcAP基因的3.7倍;经PCR及双酶切鉴定,重组表达质粒pETM30-RcAP构建正确;最适诱导条件为0.1 mmol/L IPTG 16℃诱导;重组蛋白GST-RcAP以可溶形式表达,相对分子质量约为62000;纯化的重组蛋白GST-RcAP浓度为6 mg/mL。结论成功构建了重组表达质粒pETM30-RcAP,获得了可溶性重组蛋白GST-RcAP,本研究为解析RcAP蛋白晶体结构、探索矮化基因与表型的联系奠定了理论基础并提供了试验依据。

Objective To clone and express Ricinus communis.L aspartic protease(RcAP)gene and purify the expressed protein.Methods Q-PCR method was used to verify the expression levels of RcAP gene in the leaves of high and low stem castors at six leaf stage.The total RNA was extracted from the leaves of low stem castor at six leaf stage,with which the RcAP gene was amplified by PCR,and cloned into vector pETM30.The constructed recombinant plasmid pETM30-RcAP with glutathione-S-transferase(GST)tag was transformed to competent E.coli DH5αand induced with IPTG.The IPTG concentration and temperature for induction were optimized,and the expressed protein was analyzed for solubility by SDS-PAGE.The recombinant protein GST-RcAP was purified by affinity chromatography.Results The expression level of RcAP gene in low stem castor was 3.7 times of that in high stem castor.PCR and restriction analysis proved that recombinant plasmid pETM30-RcAP was constructed correctly.The optimal IPTG concentration and temperature for induction were 0.1 mmol/L and 16℃respectively.The expressed protein,with a relative molecular mass of about62000,existed in a soluble form,of which the purity reached 6 mg/mL after purification.Conclusion Recombinant plasmid pETM30-RcAP was successfully constructed,and soluble recombinant protein GST-RcAP was obtained.This study provided theoretical and experimental bases for analyzing the crystal structure of RcAP protein and exploring the relationship between dwarfing gene and phenotype.