湖北医药学院学报2017，Vol.36Issue(3)：214-220,7.DOI:10.13819/j.issn.1006-9674.2017.03.005
重组人白血病抑制因子在大肠杆菌中的表达及纯化
Expression and Purification of Recombinant Human Leukemia Inhibitory Factor from E.coli
王晓雯 1郗雪艳 1李小璐 2关欣 2郭阳 1宋伟 2杜伯雨 1卫荣华1
作者信息
- 1. 湖北医药学院基础医学院,湖北十堰442000
- 2. 北京协和医学院基础学院中国医学科学院基础医学研究所生化学系,北京100005
- 折叠
摘要
Abstract
Objective To prepare recombinant human leukemia inhibitory factor (rhLIF) from E.coli BL21.Methods The expression vector pET-30a-rhLIF was constructed and transformed into E.coli BL21 (DE3).After the soluble expression of untagged rhLIF was induced after IPTG addition,the three-step chromatography was employed for the purification of rhLIF protein.The activity of purified LIF was determined and compared with that of the commercial product.The integrity of N terminal and C terminal amino acid sequences of the purified rhLIF protein was verified by Edman degradation assay and Mass spectrometry analysis.Results The untagged rhLIF protein could be purified through three-step chromatography and an intermediate endotoxin removal step.The endotoxin level of purified rhLIF protein was below 1 EU/μg,which was comparable to that of the commercial product.The N-terminal integrity of purified rhLIF protein was confirmed by Edman degradation assay,and methionine modification at N-terminal amino acid was identified in a small number of target proteins.The integrity of C-terminus of rhLIF was also confirmed with mass spectrometry.Cornclusion A novel method for expression and purification of recombinant human leukemia inhibitory factor had been established.In this method,no affinity tags were involved thereby facilitating the purification process.Meanwhile,the endotoxin level could be effectively reduced to an acceptable level.It had been proved that the purified rhLIF by this method could be used for stem cell culture and other related biological studies.关键词
人白血病抑制因子/表达/纯化/大肠杆菌Key words
Leukemia inhibitory factor/Expression/Purification/E.coli引用本文复制引用
王晓雯,郗雪艳,李小璐,关欣,郭阳,宋伟,杜伯雨,卫荣华..重组人白血病抑制因子在大肠杆菌中的表达及纯化[J].湖北医药学院学报,2017,36(3):214-220,7.
