Expression Of Recombinant Reversible Hirudin(rRH) In Pichia Pastoris And Study On Its Biological Properties

Hirudin is known as the strongest anticoagulant as far. The affinity of hirudin withthrombin is so high to form irreversible complexes in the molar ratio of1:1. Thedeficiencies of hirudin in clinical trial, such as: bleeding, relying on renal clearance andlacking of antagonist limit its wide usage as heparin except HIT and vein thrombosisprevention in hip or knee replacement operation. However, Bivalirudin derived fromhirudin, could be degraded by thrombin gradually after combining with it. Thecarboxyl-terminal of arginine of Bivalirudin is digested so that the combined thrombinre-exposes and fibrin regenerates. The metabolism of Bivalirudin in vivo relys mainly onenzymatic degradation, which could remarkably reduce the patient’s kidney burden. Thisstudy designed a new variant of hirudin variant1by relpaced45-52amino acid sequencesand computed theoretically with Bivalirudin specific sequences which could be recognisedand cleaved by bound thrombin, to reconstruct an reversible hirudin.Objective:To obtain a candidate, recombinant reversible hirudin (rRH), expressed by Pichia Pastoris,which would be predicted to be cleaved by bound thrombin.Method:⑴Calculating the HV1structure by computer modelling, and obtaining the best onetheoretically, named rRH.⑵Expressing rRH in Pichia pastoris. The cDNA of rRH was synthesized by Takara.Then, the fragment of rRH, digested by Xho I and Not I, was cloned into plasmidpPIC9which was digested by the same enzyme. The constructed plasmid pPIC9-rRH,digested by Sal I and Sac I, was cloned into plasmid pPIC9K. Linearized the plasmidpPIC9K-rRH was transformed into Pichia pastoris GS115through electroporation.⑶Using Geneticin to Screen high copies and using MD and MM plates to indentify thephenotype.⑷Obtaining a high-level expression strain and expressing target protein in30L fermentor by methanol induction.⑸rRH was purified by Phenyl F.F. and reverse phase C18. And using LC-MS andpeptide mass to analyze the amio acid sequences of target protein.⑹Using the methods of fibrinogen clot and chromozyme to determine the antithrombinactivity of rRH.⑺Using the methods of RP-HPLC and chromozyme to determine the binding dynamic ofrRH.Result:⑴The cDNA of rRH was cloned into pPIC9K and the plasmid pPIC9K-rRH wastransformed into Pichia Pastoris GS115successfully.⑵The t ransformants were identified as Mut+by MD and MM plates.⑶The purify of rRH was above98%, which molecular weight of LC-MS and peptidemapping are identical with theoretical value.⑷The antithrombin of rRH is approximately14000ATU, which is determined by themethod of fibrinogen clot and chromozyme.⑸The binding dynamic of rRH with thrombin showed that the reversible ability of rRH isabout20%-40%.