牛环鸟苷酸-腺苷酸合成酶的可溶性原核表达与生物酶活性分析

Soluble prokaryotic expression and enzymatic activity analysis of bovine cyclic GMP-AMP synthase

为了体外获得具有生物学活性的牛环鸟苷酸-腺苷酸合成酶(cyclic GMP-AMP synthase,cGAS)及其催化合成产物2’3’-cGAMP,首先克隆了牛cGAS基因并分析其氨基酸结构,随后构建了其可溶性原核表达载体p ET-28a-SUMO-bcGAS,并将其转化至大肠杆菌Rosetta(DE3)感受态细胞中,经IPTG诱导表达,利用镍柱亲和层析对表达的重组蛋白进行初步纯化后切除SUMO标签蛋白,再用亲和层析进一步纯化,将获得的bcGAS蛋白用于体外酶促反应合成第二信使分子2’3’-cGAMP,并利用ELISA方法和反向高效液相色谱-质谱分析2’3’-cGAMP的产量、纯度和分子结构。结果显示,成功构建了pET-28a-SUMO-bcGAS载体,通过优化诱导条件实现了目的蛋白的可溶性表达,同时,酶促合成反应证实bcGAS具有生物活性,最后,通过ELISA和反向高效液相色谱-质谱分析2’3’-cGAMP结构正确、产量纯度较高,这为后续2’3’-cGAMP的抗病毒、抗肿瘤药物和免疫调节剂的开发奠定了基础。

To acquire high biological activity of cyclic GMP-AMP synthase(cGAS)and its catalytic products 2′3′-cGAMP in vitro,bovine cGAS gene was cloned and its amino acid composition was examined.After that,the soluble prokaryotic expression vector pET-28a-SUMO-bcGAS was constructed and transformed into Escherichia coli Rosetta(DE3),and induced by IPTG.The recombination protein bcGAS was purified by nickel column affinity chromatography.Following the removal of the SUMO lable protein using ULP protease,the recombination protein bcGAS was further purified using nickle column affinity chromatography.Additionally,the purified bcGAS protein was used to enzymatically produce the second messenger molecule 2′3′-cGAMP in vitro.This molecule was then examined using ELISA and reverse high-performance liquid chromatography-mass spectrometry.These findings demonstrated that pET-28a-SUMO-bcGAS vector was successfully constructed,and the soluble protein bcGAS was produced under optimal induction conditions.Simultaneously,the biological activity of the bcGAS was confirmed in enzymatic synthesis.Finally,the structure of 2′3′-cGAMP was correct and the yield and purity were high via ELISA and reverse high-performance liquid chromatography-mass spectrometry.These results laid the groundwork for the development of antiviral medicines,antitumor medicines,and immunological boosters.