目的在大肠埃希菌中表达重组鲎血G因子,并检测其酶活性。方法根据大肠埃希菌偏好性优化G因子成熟肽编码序列,将G因子α亚基及β亚基克隆至原核表达载体pET32a-sumo上,分别构建pET32a-sumo/factorG-α及pET32a-sumo/factorG-β原核表达质粒,转化E. coli BL21(DE3),IPTG诱导表达;产物经Ni-NTA亲和层析,SUMO蛋白酶切割融合伴侣,获得重组目的蛋白factorG-α及factorG-β;将二者等摩尔质量重组获得复合物G,荧光底物Boc-Glu-(OBzl)-Gly-Arg-MCA检测其酶活性。结果经PCR及测序鉴定,重组质粒pET32a-sumo/factorG-α及p ET32a-sumo/factorG-β构建正确;表达蛋白均以包涵体的形式存在沉淀中,表达量占菌体总蛋白的30%及45%;纯化的factorG-α及factorG-β蛋白相对分子质量约为74 000和31 000,每1 L LB培养基酶切回收蛋白分别为0. 7和1. 7 mg。factorG-α及factorG-β蛋白与荧光底物几乎不反应,而复合物G可与荧光底物反应产生较高的荧光强度。结论成功表达了具有酶活性的鲎血G因子,为进一步探讨鲎血G因子的生物学功能及新型真菌感染检测试剂盒开发奠定了基础。
Objective To express recombinant Tachypleus tridentatus factor G in E.coli and determine its enzyme activity.Methods According to codon preference of E.coli,the alpha and beta subunit genes of factor G were optimized and cloned into the prokaryotic expression vector p ET32 a-sumo.The constructed recombinant plasmids pET32 a-sumo/factorG-αand p ET32 a-sumo/factorG-βwere transformed to E.coli BL21(DE3)and induced with IPTG.The expressed protein was purified by Ni-NTA affinity chromatography,while the tag protein was digested with SUMO protein to obtain recombinant target proteins factorG-αand factorG-β.Recombinant factor G was obtained by isomolar mass recombination,and determined for enzyme activity by using a fluorogenic peptide substrate Boc-Glu-(OBzl)-Gly-Arg-MCA.Results PCR and sequencing showed that recombinant plasmids pET32 a-sumo/factorG-αand pET32 a-sumo/factorG-βwere constructed correctly.The expressed protein was in a form of inclusion body,which contained 30%and 45%of total somatic protein.The relative molecular masses of purified factorG-αand factorG-βwere about 74 000 and 31 000,of which the recoveries in 1 L of LB medium were 0.7 and 1.7 mg,respectively.FactorG-αand factorG-βshowed little reaction with fluorescent substrate.However,recombinant factor G showed strong reaction with the substrate.Conclusion Recombinant factor G with enzyme activity was expressed successfully,which laid a foundation of further study on the biological function of Tachypleus tridentatus factor G and the development of a new detection kit for fungal infection.
Chinese Journal of Biologicals
Tachypleus tridentatus factor G