动物学研究2012，Vol.33Issue(2)：144-150,7.DOI:10.3724/SP.J.1141.2012.02144
人TFF2基因的原核表达与纯化
Bacterial expression and purification of biologically active human TFF2
摘要
Abstract
Human trefoil factor 2 (hTFF2) is considered as one of the most important initiators of mucosal healing in the gastrointestinal tract by promoting cell migration and suppressing apoptosis.However,it is hard to obtain hTFF2 from human tissue and many recombinant hTFF2 produced in vitro exist as fusion proteins.The purpose of the present study was to produce native hTFF2 while maintaining its biological activities.The open reading frame of hTFF2 was inserted into a pET-32a(+) expression vector,and hTFF2-TRX fusion protein was successfully expressed in Escherichia coli and purified by Nickel-nitrilotriacetic acid affinity chromatography and reverse-phase HPLC steps.The recombinant fusion protein (purity＞95％) was cleaved by Factor Xa at 23 C to release hTFF2.After removal of Factor Xa and undigested fusion proteins,hTFF2 was purified and identified by SDS-PAGE and Western blotting.The yield of recombinant hTFF2 was about 5 mg/L.The recombinant hTFF2 could promote IEC-6 cells migration and in vitro wound healing via the activation of ERK 1/2.Recombinant hTFF2 could also inhibit apoptosis of HCT-116 cells induced by 50 μmol/L ceramide.In summary,our results showed that the recombinant hTFF2 was expressed in E.coli and successfully purified after cleavage with the fusion partner with high yield while maintaining its biological activities.Recombinant hTFF2 might be useful for investigating the molecular mechanism of hTFF2 and development of hTFF2-related drugs.关键词
TFF2/表达/细胞迁移/抗凋亡/伤口修复Key words
TFF2/Expression/Cell migration/Anti-apoptosis/Wound healing分类
生物科学引用本文复制引用
庄永辉,李思熳,余果宇,张勇,向阳,邹浩,李文辉..人TFF2基因的原核表达与纯化[J].动物学研究,2012,33(2):144-150,7.基金项目
This work was supported by grants from the National Basic Research Program of China (973 Program,2010CB529800),the Chinese National Natural Science Foundation (81160302,30870304),the "Western Light Project" from the Chinese Academy of Sciences (Y102291081),and the Science and Technology Department of Yunnan Province (2011C1139)“973”项目(2010CB529800) （973 Program,2010CB529800）
国家基金委面上项目(81160302,30870304) （81160302,30870304）
中国科学院“西部之光”(Y102291081) （Y102291081）
