SnapTag-AmDH融合蛋白的表达纯化及其活性检测
首发时间:2020-04-08
摘要:目的:构建SnapTag与胺脱氢酶(amine dehydrogenase, AmDH) 的融合蛋白表达载体,并进行重组蛋白的原核表达、纯化、表征及活性检测。方法:针对目的基因SnapTag片段利用PCR 技术进行扩增,并用限制性内切酶将载体pET28-AmDH进行线性化,将目的基因SnapTag片段与线性化pET28-AmDH载体进行同源重组。将测序验证正确的质粒转化至大肠杆菌BL21 (DE3)中进行表达,用考马斯亮蓝染色方法对诱导表达的目的蛋白进行检测,对纯化的融合蛋白进行活性检测。结果:PCR扩增产物及消化的载体具有正确的分子量,重组蛋白质粒测序结果与目标质粒相符,在SDS-PAGE中的过表达条带分子量约在52 KDa处,与融合蛋白分子量相符。活性测试显示具有AmDH的催化活性。结论:成功构建了重组蛋白pET28a-SnapTag-AmDH的表达载体,并将重组蛋白在大肠杆菌BL21(DE3)中进行原核表达,进一步纯化和表征了重组蛋白,并检测到蛋白活性。
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Expression and purification of SnapTag-AmDH fusion protein and activity detection
Abstract:Objective: To obtain an expression vector of SnapTag-amine dehydrogenase (AmDH) fusion protein, and express, purify, characterize the recombinant protein. Methods: The SnapTag fragment of the target gene was amplified by PCR technology, and the vector pET28-AmDH was linearized with restriction enzymes. The target gene SnapTag fragment was homologously recombined with the linearized pET28-AmDH vector. The plasmid verified by sequencing was transformed into E. coli BL21 (DE3) for expression. Coomassie brilliant blue staining was used to detect the expression of the target protein, and the purified fusion protein was tested for activity. Results: The PCR amplification product and digested vector have correct molecular weights. The sequencing results of the recombinant protein plasmid were consistent with the target sequence. The molecular weight of the overexpressed band in SDS-PAGE was about 52 KDa, which was consistent with the molecular weight of the fusion protein. The activity test showed the catalytic activity of AmDH. Conclusion: The recombinant protein pET28a-SnapTag-AmDH expression vector was successfully constructed, and the recombinant protein was expressed in E. coli BL21 (DE3). The recombinant protein was further purified and characterized to be the target product with expected activity.
Keywords: SnapTag amine dehydrogenase AmDH fusion protein expression
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SnapTag-AmDH融合蛋白的表达纯化及其活性检测
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