摘要
[目的]建立一种可工业化的3-甾酮-Δ^(1)-脱氢酶(KstD)上游纯化的新方法。[方法]实验以T4噬菌体中的T4溶菌酶为基础,对其进行改造,利用重组溶菌酶与高分子聚合物聚乙烯亚胺(PEI)偶联应用于表达KstD的大肠杆菌工程菌的细胞破碎以及初步纯化。[结果]成功构建了MBP-SN-T4L溶菌酶,其最佳表达条件为18℃、0.4 mmol/L IPTG下诱导表达4 h。表达后的纯化程序为:50 mmol/L Tris—HCl缓冲溶液中溶菌酶浓度为10 g/L,含有KstD酶的大肠杆菌菌泥浓度100 g/L,pH 7.0,反应30 min后加入终浓度为0.25%的聚乙烯亚胺,搅拌分离。[结论]成功建立了一套应用于工业快速大批量生产蛋白质的上游纯化方法,获得目的蛋白含量约为传统纯化方式所得蛋白的96.23%。
[Objective]To establish a new upstream purification method of 3-sterone-Δ^(1)-dehydrogenase(KstD)for industrial proteins.[Method]Based on the T4 lysozyme from T4 phage,the recombinant lysozyme coupled with polymer polyethyleneimine was used for the cell fragmentation and preliminary purification of E.coli engineered bacterium containing the target protein.[Result]MBP-SN-T4L lysozyme was successfully constructed,and the optimal conditions for its expression were 18 C,0.4 mmol/L IPTG for 4 h.After expression,the purification procedure was as follows:50 mmol/L Tris-HCl buffer solution with 10 g/L lysozyme,100 g/L E.coli bacterial sludge containing KstD enzyme,pH 7.0,and the reaction was carried out for 30 min with the addition of polyethyleneimine at the final concentration of 0.25%,and then stirred to separate.[Conclusion]A set of upstream purification methods for proteins applied to industrial production was successfully established,and the content of the target protein obtained was about 96.23% of that obtained with the traditional purification method.
作者
崔雨
柯志强
苏正定
CUI Yu;KE Zhiqiang;SU Zhengding(College of Life Science and Health Engineering,Hubei University of Technology,Wuhan 430068;School of Pharmacy,Xinjiang University,Urumqi 830046,China)
出处
《生物技术》
2025年第3期290-295,共6页
Biotechnology
