钙激活酶激活蛋白基因的克隆及其在原核生物中表达及条件的优化

Cloning of Calpain Activator Gene and the Optimized Expression Condition in Prokaryotes

构建钙激活酶激活蛋白基因(UK114)的原核表达载体并优化其表达条件,为其高效表达提供试验依据。以UK114 cDNA为模板,通过PCR方法扩增钙激活酶激活蛋白基因,将其克隆到原核表达载体pGEX-4T-3中,酶切及测序鉴定重组体。将构建好的重组质粒转化大肠埃希菌BL21(DE3),用IPTG进行诱导表达,在保持菌种不改变的前提下,分别改变IPTG的浓度、培养时间、菌体浓度、培养温度等来优化表达条件。结果显示,原核表达载体pGEX-4T-3-UK114成功构建,可在大肠埃希菌BL21(DE3)中诱导表达,得到相对分子质量约40 kD的GST-UK114融合蛋白。在IPTG浓度为0.3 mmol/L,诱导温度为32℃,诱导时间为4 h,菌体密度OD600为0.6的条件下,目的蛋白表达量最高。试验成功构建原核表达载体pGEX-4T-3-UK114且获高效表达,为研究UK114生物学活性及产品开发提供了试验基础。

：The aim of the study was designed to optimize Calpain activator（UK114） expression in prokaryotes for the high effective expression. The UKll4 cDNA was obtained by RT-PCR, and cloned into prokaryotic expression vector pGEX-4T-3-UK114, then recombinant vector pGEX 4T-3-UKl14 was constructed, and transformed into E. coli BL21 （ DE3）. The expression of fusion protein GST-UK114 was induced with IPTG, then purified and characterized. The recombinant prokaryotic expression vector was successfully constructed by the restriction enzymes digestion and gene sequencing. Fusion protein GST-UK114 was expressed in solubility in E. coli BL21 （DE3）in SDS-PAGE gel. The content of fusion protein was 31% and the molecular weight was 40 kD. Through optimization, the satisfactory expression condi- tion was obtained, as follows, including IPTG concentration 0.3 mmol/L; induced time of 4 hours ; OD600 0.6 ; cultured temperature 32 ℃ The plasmid pGEX-4T-3-UK114 was successfully constructed and expressed in prokaryotes with high efficiency. The results provide a foundation for studing the biological activity of UK114 protein.