Rhodococcus rhodochrous J1腈水合酶在大肠杆菌中的表达策略  

Strategy for over-expression of nitrile hydratase from Rhodococcus rhodochrous J1 in Escherichia coli

作　　者：余越春 崔文璟[1] 刘义[1] 周哲敏[1] 

机构地区：[1]江南大学生物工程学院,江南大学工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《工业微生物》2014年第2期14-19,共6页Industrial Microbiology

基　　金：国家自然科学基金(No.31070711)

摘　　要：针对红球茵低分子量腈水合酶(L-NHase)在重组茵中难以表达这一问题,通过对其α亚基及调控蛋白NhlE基因的核糖体结合位点和α,β亚基间隔序列的长度进行改造,构建了重组表达栽体,实现了L-NHase及其调控蛋白NhlE在E.COli BL21(DE3)中过量表达。通过培养条件优化,得到最佳表达条件为:37℃培养茵体浓度(DD_600)到1.0时,加入终浓度为0.1 g/L的COCl_2·6H_2O,0.6 mmol/,L的IPTG,然后在24℃下诱导表达24 h。最终得到的重组蛋白粗酶液的活性为(109.9±5.5)U/mg。采用Strep-tag/Strep-Tactin亲和层析简化了L-NHase的纯化方法,本研究结果为一些难于异源重组表达的多亚基蛋白质的表达具有一定的借鉴意义。In order to obtain the active and soluble L-NHase of Rhodococcus rhodochrous J1 by E. coli expression system, the recombinant expression vector was constructed by substituting a stronger ribosome binding site sequence ahead of its c~ subunit and its activator genes, and by optimizing the length of interval sequence between ot,fl-subunit genes. The optimi- zation of fermentation conditions for L-NHase expression was investigated. The optimal induction conditions were deter- mined as follows： 24＂12, 24 h, 0.6 mmol/L IPTG, and 0.1 g/L CoCI2 ~ 6H20 （final concentration）. The highest level of L-NHase activity in cell-free extracts was up to （109.9 ＋ 5.5 ）U/rag. Based on the over-expression of L-NHase, the purification strategy by fusion of a strep-tag to the C-terminus of/3 subunit was simplified. These results were helpful for the heterologous expression of muhi-subunit proteins.

关 键 词：腈水合酶 核糖体结合位点 调控蛋白 重组表达 蛋白质纯化 

分 类 号：Q78[生物学—分子生物学]