Expression, Purification, and Mass Spectrometric Analysis of 15N, 13C-Labeled RGD-Hirudin, Expressed in Pichia pastoris, for NMR Studies
A novel recombinant hirudin,RGD-hirudin,inhibits the activity of thrombin and the aggregation of platelets.Here,we successfully expressed 15N,13C-labeled RGD-hirudin in Pichia pastoris in a fermenter.The protein was subsequently purified to yield sufficient quantities for structural and functional studies.The purified protein was characterized by HPLC and MALDI-TOF mass spectroscopy.Analysis revealed that the protein was pure and uniformly labeled with 15N and 13C.A bioassay showed that the anti-thrombin activity and the anti-platelet aggregation ability of the labeled protein were the same as those of unlabeled RGD-hirudin.Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone 15N,13C and 1H resonance assignments of the r-RGD-Hirudin.The 15N-1H HSQC spectrum of uniformly 15N,13C-labeled RGD-hirudin allowed successful assignment of the signals.Examples of the quality of the data are provided for the 15N-lH correlation spectrum,and by selected planes of the CBCA(CO)NH,CBCANH,and HNCO experiments.These results provide a basis for further studies on the structure-function relationship of RGD-hirudin with thrombin and platelets.
Yinong Huang Yanling Zhang Yi Wu Jue Wang Xingang Liu Linsen Dai Longsheng Wang Min Yu Wei Mo
The Key Laboratory of Molecular Medicine,Ministry of Education,Fudan University,Shanghai,People”s Re Center of Analysis and Measurement,Fudan University,Shanghai,People”s Republic of China
国内会议
第九届国际分子模拟与信息技术应用学术会议(ICMS&I2018)
太原
英文
142-149
2018-05-01（万方平台首次上网日期，不代表论文的发表时间）