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目的:构建微丝相关蛋白hHBRK1截短体和突变体原核表达载体,并表达及纯化融合蛋白。方法:采用常规PCR并结合定点突变技术,对hHB rk1基因进行缺失和点突变;利用限制性内切酶将PCR产物克隆至原核表达载体pGEX-4T;IPTG诱导融合蛋白表达,通过谷胱甘肽-琼脂糖纯化技术分离纯化GST融合蛋白。结果与结论:构建了包括氨基端缺失截短体hHBRK1-ΔN、羧基端缺失截短体hHBRK1-ΔC和点突变蛋白hHBRK1-S56G57在内的原核表达质粒,分离获得较高纯度的重组hHBRK1突变体融合蛋白,W estern杂交证实纯化蛋白为GST融合蛋白。本实验为进一步研究hHBRK1的相互作用蛋白及其可能的结合位点提供了基础。
OBJECTIVE: To construct the prokaryotic expression vector of hHBRK1 truncate and mutant of microfilament-related protein, and to express and purify the fusion protein. Methods: The hHB rk1 gene was deleted and mutated by conventional PCR combined with site-directed mutagenesis. The PCR product was cloned into the prokaryotic expression vector pGEX-4T by restriction endonuclease. The fusion protein was induced by IPTG, Separation and purification of GST fusion protein by agarose purification. RESULTS AND CONCLUSION: Prokaryotic expression plasmids including hHBRK1-ΔN, hHBRK1-ΔC, hHBRK1-ΔC and hHBRK1-S56G57 were constructed, and the recombinant hHBRK1 mutant with higher purity was isolated The fusion protein, W estern hybridization, confirmed that the purified protein was a GST fusion protein. This experiment provides the basis for further study of the interacting proteins of hHBRK1 and its possible binding sites.