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棉花GhCOMT3基因的原核表达、纯化及鉴定

DOI: 10.7668/hbnxb.20102.S1.003, PP. 12-16

Keywords: GhCOMT3,原核表达,蛋白纯化,Westernblotting

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Abstract:

为了深入研究GhCOMT3基因的功能,将GhCOMT3基因构建到原核表达载体pET-28a中,并转化到大肠杆菌BL21(DE3)中。用0.2mmol/LIPTG诱导融合蛋白表达,结果发现,16℃诱导12h、30℃诱导3h和37℃诱导3h后融合蛋白均以包涵体的形式表达;30℃诱导的蛋白表达量最大,之后对包涵体进行变性溶解,进行SDS-PAGE检测,再经过蛋白标记亲和层析柱(HisTrapTMHP)纯化得到变性的pET-28a-GhCOMT3融合蛋白,用SDS-PAGE和Westernblotting检测纯化效果,鉴定表达产物,目的蛋白相对分子量约为39.119kDa,检测结果与预期一致。结果表明,pET-28a-GhCOMT3蛋白在大肠杆菌中获得了高效表达产物,为在蛋白水平上研究GhCOMT3基因功能奠定了基础。

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