猪环鸟苷酸-腺苷酸合成酶cGAS基因的原核表达及其表达产物的生物活性分析

Prokaryotic expression and activity analysis of porcine cyclic GMP-AMP synthase

为获得具有生物学活性的环鸟苷酸-腺苷酸合成酶(cyclic GMP-AMP synthase,cGAS)蛋白与第二信使分子2′3′-cGAMP,构建了猪源cGAMP合成酶pcGAS基因的原核表达载体pET-28a-SUMO-pcGAS,将其转化至大肠杆菌Rosetta(DE3),并通过优化诱导条件实现了可溶性表达;利用镍柱亲和层析对表达的重组蛋白产物进行初步纯化,随后切除SUMO标签蛋白,再用亲和层析进一步纯化,获得了纯度较高的重组蛋白pcGAS;将获得的重组蛋白pcGAS通过体外酶促反应生物合成了2′3′-cGAMP,且合成产物能够诱导启动细胞Ⅰ型IFNs的产生。结果表明,pcGAS重组蛋白和2′3′-cGAMP均具有生物活性,这为后续抗病毒、抗肿瘤药物以及免疫增强剂的开发奠定了基础。

Cyclic GMP-AMP synthase(c GAS)has been recently discovered as a novel cytosolic DNA sensor.Upon activation by cytosolic DNA,cGAS catalyzes the formation of the endogenous second messenger 2′3′-cGAMP to activate STING-TBK1-IRF3 axis,leading to type Ⅰ interferons(IFNs)production and an innate immune response.Here we reported that the prokaryotic expression vector p ET-28 a-SUMO-pcGAS was constructed and transformed into E.coli Rosetta(DE3)and the soluble recombinant protein pcGAS was expressed in medium by optimizing the conditions. Then, the recombinant protein pcGAS was purified via nickel column affinity chromatography, and the SUMO tag protein was removed using ULP protease by affinity chromatography to further obtain higher purity pcGAS protein. The recombinant pcGAS protein was used to synthesize 2′3′-cGAMP in vitro, which was confirmed to induce the typeⅠIFNs production at the cellular level. The results indicated that pcGAS recombinant protein and 2′3′-cGAMP have biological activity, which lay the foundation for the further development of anti-viral and anti-tumor drugs and immunopotentiators.