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

Abstract 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