微丝相关蛋白hHBRK1突变体的构建、表达及纯化

Construction,expression and purification of F-actin related protein hHBRK1 mutants

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, express and purify the mutant and the truncated hHBRK1 proteins. Methods： Polymerase chain reaction （PCR） and the site-directed mutagenesis technique were used for deletion and the site mutation of hHBrk1 gene. PCR products were cloned into pGEX-4T vector respectively with restrictive endonuclease. Recombination GST fusion proteins were induced with IPTG and these mutant and truncated hHBRK1 proteins were purified by glutathion Sepharose 4B affinity chromatography. Western blot was used to identify the recombination GST fusion proteins. Results and conclusion： Recombinant plasmids including hHBRK1-AN, hHBRK1-AC and hHBRK1-S56 Gs7 were constructed. These mutants and truncated hHBRK1 proteins were induced by 0.8 mmol/L IPTG, purified from supernatant of bacterial lysis and further identified with Western blot. These mutants and truncated hHBRK1 proteins would be useful in studying the interaction proteins and the function domains of hHBRK1.