大肠杆菌T蛋白PDH结构域的高效表达纯化与活性测定

High expression, purification and bioactivity of prephenate dehydrogenase domain in E. coli T-protein

目的构建高效表达菌株,以大量表达大肠杆菌T蛋白的预苯酸脱氢酶(PDH),为研究T蛋白双功能结构域CM和PDH的相互协同作用以及T蛋白抑制剂对其影响提供活性蛋白材料。方法利用pGEX高效融合表达系统将PDH与谷光甘肽疏基转移酶(GST)编码序列在表达宿主BL21实现高效表达,并利用对GST具有亲和作用的GlutathioneSepharose4B琼脂糖珠进行亲和层析以对其分离纯化。结果表达产物达菌体总蛋白的40%,可溶性目的产物得量为240mg·L-1,利用亲和层析法纯化后纯度达92.5%,GSTPDH融合蛋白的PDH的比活为38U·mg-1。结论本法成功构建高效表达PDH菌株,表达量高,纯化度高,GSTPDH酶活性正常,为进一步研究奠定基础。

OBJECTIVE: To clone and highly express prephenate dehydrogenase(PDH) domain in E. coli T-protein for the further study of synrgistic action with chorismate mutase domain. METHODS: The pGEX-4T-1 vector was employed for the high expression of prephenate dehydrogenase domain. The N-terminal GST tagged fusion protein was purified by Glutathione Sepharose 4B. RESULTS: High expression of soluble target protein was obtained by IPTG(isoprogyl-β-D-thiogalactoside), which accounted for approximately 40% of germ proteins. The fusion protein was obtained with the purity up to 92.5% after being purified by affinity chromatography. Bioactivity assay showed that the specific activity of cloned prephenate dehydrogenase domains was 38 U&middotmg-1. CONCLUSION: The prephenate dehydrogenase domain expressed was high, easy to be purified and displayed good catalytic activity. All these made a good ground for the further investigation of molecular synergistic action between prephenate dehydrogenase and chorismate mutase domain in T-protein in the process of new drug development.