【摘 要】
：
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.
【机 构】
：
The Key Laboratory of Molecular Medicine,Ministry of Education,Fudan University,Shanghai,People's R
【出 处】
：
第九届国际分子模拟与信息技术应用学术会议(ICMS&I2018)
论文部分内容阅读
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.
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