LHCGR胞外结构域蛋白的生信分析、原核表达及纯化

Bioinformatics analysis,prokaryotic expression and purification of extracellular domain protein of LHCGR

目的为获得小鼠LHCGR胞外结构域蛋白(LHCGR-1),分析其结构及理化性质,为今后研究该蛋白功能及筛选与之相互作用的小分子化合物奠定基础。方法根据NCBI中小鼠LHCGR氨基酸序列合成目的基因Lhcgr-1,经生物信息学分析后,将该基因插入至表达载体pET-28a(+),并转化至BL21(DE3)感受态细胞中;经IPTG诱导,表达重组蛋白pET-28a-LHCGR-1,利用SDS-PAGE和Western Blot进行鉴定,之后对该蛋白进行纯化,并通过分子对接技术预测该蛋白与LH、CG的相互作用。结果预测LHCGR-1蛋白分子量为40749.08,由367个氨基酸组成,等电点理论值为5.47;氨基酸序列中有29个丝氨酸位点、10个苏氨酸位点和5个酪氨酸位点;成功构建了表达载体pET-28a-LHCGR-1并获得纯化的目的蛋白;分子对接结果表明目的蛋白可以与LH、CG通过多种相互作用,形成稳定的复合物,有利于其发挥原有的生物学功能。结论成功构建了小鼠胞外结构域蛋白LHCGR-1在大肠杆菌内的原核表达体系并获得了纯化蛋白,分子对接预测该蛋白具有生物学功能,提示该蛋白可以用于后续研究。

Objective In order to obtain mouse LHCGR extracellular domain protein(LHCGR-1),the structure and physicochemical properties of LHCGR-1 protein were analyzed,which laid a foundation for the study of the function of this protein and the screening of small molecule compounds interacting with it.Method The target gene Lhcgr-1 was synthesized according to the amino acid sequence of mouse LHCGR in NCBI.After bioinformatics analysis,the gene was inserted into the expression vector pET-28a(+)and transformed into BL21(DE3)receptor cells.The recombinant protein pET-28a-LHCGR-1 was induced by IPTG and identified by SDS-PAGE and Western Blot.After that,the protein was purified,and the interaction between the protein and LH and CG was predicted by molecular docking technique.Results The molecular weight of the LHCGR-1 protein was estimated to be 40749.08,composed of 367 amino acids,and the theoretical isoelectric point value was 5.47.There were 29 serine sites,10 threonine sites and 5 tyrosine sites in the amino acid sequence.The recombinant plasmid pET-28a-LHCGR-1 was constructed successfully and the purified target protein was obtained.The molecular docking results showed that the target protein could form stable complexes with LH and CG through various interactions,so as to play its original biological functions.Conclusion The prokaryotic expression system of mouse extracellular domain protein LHCGR-1 in Escherichia coli was successfully constructed and the purified protein was obtained.The biological function of the protein was predicted by molecular linkage,suggesting that the protein could be used in subsequent studies.