Expression, Purification, and Mass Spectrometric Analysis of .sup.15N, .sup.13C-Labeled RGD-Hirudin, Expressed in Pichia pastoris, for NMR Studies

• Published in: PloS one Vol. 7; no. 8; p. e42207
• Main Authors: Huang, Yinong, Zhang, Yanling, Wu, Yi, Wang, Jue, Liu, Xingang, Dai, Linsen, Wang, Longsheng, Yu, Min, Mo, Wei
• Format: Journal Article
• Language: English
• Published: Public Library of Science 07.08.2012
• Subjects: Mass spectrometry; Nuclear magnetic resonance spectroscopy; Thrombin
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0042207

A novel recombinant hirudin, RGD-hirudin, inhibits the activity of thrombin and the aggregation of platelets. Here, we successfully expressed .sup.15 N, .sup.13 C-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 .sup.15 N and .sup.13 C. 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 .sup.15 N, .sup.13 C and .sup.1 H resonance assignments of the r-RGD-Hirudin. The .sup.15 N-.sup.1 H HSQC spectrum of uniformly .sup.15 N, .sup.13 C-labeled RGD-hirudin allowed successful assignment of the signals. Examples of the quality of the data are provided for the .sup.15 N-.sup.l H 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.