Expression, purification, and mass spectrometric analysis of 15N, 13C-labeled RGD-hirudin, expressed in Pichia pastoris, for NMR studies.

Yinong Huang,Min Yu,Jue Wang,Yi Wu,Xingang Liu,Linsen Dai,Wei Mo,Longsheng Wang,Yanling Zhang

doi:10.1371/journal.pone.0042207

Yinong Huang, Min Yu + Show 7 more

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https://doi.org/10.1371/journal.pone.0042207

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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 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.

Highlights

Hirudin, an antithrombotic substance produced by the salivary glands of the medicinal leech (Hirudo medicinalis) [1,2], is the most potent and specific thrombin inhibitor currently known
It blocks the thrombin-mediated conversion of fibrinogen to fibrin in clot formation, and, unlike heparin, it is a direct thrombin inhibitor (DTI) [3], which is not inactivated by platelet factor 4 (PF4) [4,5]
NH4OH was used as the nitrogen source and for controlling the pH; unlabeled glycerol and methanol were used as the carbon source

Summary

Introduction

An antithrombotic substance produced by the salivary glands of the medicinal leech (Hirudo medicinalis) [1,2], is the most potent and specific thrombin inhibitor currently known. It blocks the thrombin-mediated conversion of fibrinogen to fibrin in clot formation, and, unlike heparin, it is a direct thrombin inhibitor (DTI) [3], which is not inactivated by platelet factor 4 (PF4) [4,5]. RGD-hirudin is a novel bi-functional molecule that contains the Arg-Gly-Asp (RGD) adhesion site recognition sequence The abilities of this protein to inhibit thrombin and the aggregation of platelets were confirmed in our previous study [8]. Purified 15N-labeled RGD-hirudin was prepared in our lab [10]

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