疏棉状嗜热丝孢菌脂肪酶在大肠杆菌中的表达与纯化  

Expression and Purification of Thermomyces Lanuginosus Lipase in Escherichia coli

作　　者：王烃 郭国光 时健[1] 郝兵[1] 王慧[1] 张守涛[1] 

机构地区：[1]郑州大学生命科学学院,河南郑州450001

出　　处：《生物技术》2014年第4期31-36,共6页Biotechnology

摘　　要：目的:实现疏棉状嗜热丝孢菌脂肪酶(Thermomyces lanuginosus Lipase,TLL)在大肠杆菌中的可溶性表达,并建立有效的纯化方法。方法:根据疏棉状嗜热丝孢菌脂肪酶序列和大肠杆菌密码子的偏爱性,合成了疏棉状嗜热丝孢菌脂肪酶DNA序列,克隆至大肠杆菌表达载体PET-32a(+)中,通过筛选得到阳性重组载体,并转化入大肠杆菌Rosetta(DE3),诱导表达并利用SDS-PAGE电泳检测重组脂肪酶表达情况,并通过Ni柱亲和层析对目的蛋白TLL进行纯化,透析脱盐后,TEV酶切重组融合蛋白,再利用亲和层析纯化得到酶切后的脂肪酶,检测其酶活。结果:得到了可溶性表达的TLL,检测酶切前后脂肪酶的比活性:酶切前达5.7×106U/mg,酶切后达8.9×105U/mg。结论:实现了疏棉状嗜热丝孢菌脂肪酶的可溶性表达,并得到有效纯化,同时证明TEV酶切目的蛋白对其活性影响微小,为之后对其进行环化打下基础。Objective： Achieved Thermomyces Lanuginosus Lipase effectively expression in E. coli, and established effective purification method. Method：According to the DNA sequence of Thermomyces lanuginosus Lipase and preference of E. coli codon sequences and re- quirements of sorting enzyme cyelization, designed a optimized Thermomyces lanuginosus Lipase DNA sequence for synthesis, then cloned the sequence into the E. coli expression vector PET - 32a （ ＋ ）. After that, screening positive vector and then transformed into E. coli Rosetta,induced expression and SDS - PAGE electrophoresis to detect recombinant lipase expression situation and purified target protein TLL by affinity chromatography, after dialysis desalination, TEV enzyme cleaved the recombinant lipase and purified the recombinant after cleavage, detected the activity. Result： Got the soluble expression of TLL, Detected the activity of lipase both cleavage and not： the activity of the lipase without cleavage was up to 5.7 x 106U/rag,the activity of the lipase after cleavage was up to 8.9 × 105U/mg. Conclusion： The TLL was expressed in soluble situation, and got effective purification,it also proved the activity of the TLL after cleaved and not had little difference,which made the foundation of its cyclization for other days.

关 键 词：大肠杆菌 疏棉状嗜热丝孢菌 脂肪酶 表达 纯化 环化 

分 类 号：Q786[生物学—分子生物学]