title: 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 (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.
authors.affiliation: The Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University, Shanghai, People's Republic of China.
authors.forename: Yinong
authors.initials: Y
authors.lastname: Huang
authors.forename: Yanling
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yi
authors.initials: Y
authors.lastname: Wu
authors.forename: Jue
authors.initials: J
authors.lastname: Wang
authors.forename: Xingang
authors.initials: X
authors.lastname: Liu
authors.forename: Linsen
authors.initials: L
authors.lastname: Dai
authors.forename: Longsheng
authors.initials: L
authors.lastname: Wang
authors.forename: Min
authors.initials: M
authors.lastname: Yu
authors.forename: Wei
authors.initials: W
authors.lastname: Mo
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000991:Antithrombins / Q000494:pharmacology; D002247:Carbon Isotopes; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006629:Hirudins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007553:Isotope Labeling / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D008970:Molecular Weight; D009587:Nitrogen Isotopes; D010843:Pichia / Q000378:metabolism*; D010974:Platelet Aggregation / Q000187:drug effects; D013696:Temperature
chemical_list: D000991:Antithrombins; D002247:Carbon Isotopes; D006629:Hirudins; D009587:Nitrogen Isotopes; C544959:RGD-hirudin; D000244:Adenosine Diphosphate