文献类型：期刊文献

英文题名：Improving the bioactivity of rHirudin with boronophenylalanine site-specific modification

作者：Xin, Xiujuan[1];Liu, Wenshe[2];Sun, Liyun[1]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Texas A&M Univ, Dept Chem, College Stn, TX 77840 USA

年份：2015

卷号：11

期号：5

起止页码：3774

外文期刊名：MOLECULAR MEDICINE REPORTS

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000353849500081)】；

基金：The authors are grateful to Dr K.Odoi of A&M University for reviewing the manuscript. This study was supported by the Fundamental Research Funds for the Central Universities of China (nos. WF1114045 and 222201314027) and the Open Project for the State Key Laboratory of Bioreactor Engineering (no. 2060204).

语种：英文

外文关键词：antithrombin activity; boronophenylalanine; proliferation inhibition; recombinant Hirudin; site-specific modification

摘要：To improve the bioactivity of recombinant (r) Hirudin, the orthogonal pair MjB(Tyr)RS/tRNA(Tyr) CUA (made up of the boronophenylalanine, tRNA and tRNA synthetase), was selected to incorporate boronophenylalanine site-specifically into rHirudin at the 63 sites in an Escherichia coli system in response to the TAG codon. Following fusion with the gIII signal peptide and a hexahistidine tag, the modified protein was secreted into Luria-Bertani culture medium and purified by nickel-nitrilotriacetic acid affinity chromatography following a gel filtration column. In a 200 ml flask, the yield of boronophenylalanine-modified hirudin was 10 mg l(-1) and that of rHirudin was 19 mg l(-1). The authenticity of the purified proteins was verified using matrix-assisted laser desorption ionization time of flight mass spectroscopy and antithrombin activity assays. The results revealed that the antithrombin activity of the boronophenylalanine-modified hirudin to human thrombin was more enhanced than that of rHirudin. The modified hirudin demonstrated stronger proliferation inhibiting ability on fibroblast L929 cells compared with that of rHirudin.