The Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University, Shanghai, People's Republic of China.
PLoS One. 2012;7(8):e42207. doi: 10.1371/journal.pone.0042207. Epub 2012 Aug 7.
A novel recombinant hirudin, RGD-hirudin, inhibits the activity of thrombin and the aggregation of platelets. Here, we successfully expressed (15)N, (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 (15)N and (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 (15)N, (13)C and (1)H resonance assignments of the r-RGD-Hirudin. The (15)N-(1)H HSQC spectrum of uniformly (15)N, (13)C-labeled RGD-hirudin allowed successful assignment of the signals. Examples of the quality of the data are provided for the (15)N-(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.
一种新型重组水蛭素 RGD-水蛭素，可抑制凝血酶的活性和血小板的聚集。在这里，我们成功地在发酵罐中用毕赤酵母表达了（15）N、（13）C 标记的 RGD-水蛭素。随后对蛋白质进行纯化，以获得足够的量用于结构和功能研究。用 HPLC 和 MALDI-TOF 质谱对纯化的蛋白质进行了表征。分析表明，该蛋白质是纯的，并且均匀地标记了（15）N 和（13）C。生物测定表明，标记蛋白的抗凝血酶活性和抗血小板聚集能力与未标记的 RGD-水蛭素相同。多维异核 NMR 光谱已用于确定 r-RGD-Hirudin 的几乎完整的骨架（15）N、（13）C 和（1）H 共振分配。均匀（15）N、（13）C 标记的 RGD-水蛭素的（15）N-（1）H HSQC 谱允许成功分配信号。（15）N-（1）H 相关谱和 CBCA（CO）NH、CBCANH 和 HNCO 实验的选定平面提供了数据质量的示例。这些结果为进一步研究 RGD-水蛭素与凝血酶和血小板的结构-功能关系提供了基础。
