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

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. 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.