Expression Of Recombinant Antithrombotic Fusion Protein In Pichia Pastoris

Nemtode anticoagulant peptides(NAPs),which are isolated from adult Ancylostoma caninum,are a family of antithrombotic non-enzymatic peptides of low molecular weights.The important members of NAPs identified by now include NAP1,NAPc2,NAPc3,NAPc4,NAP5,NAP6 and NAP7,etc.Among these,NAPc2 and NAP5 shows the highest anticoagulant acivity.The NAP5,a peptide consisting of 77 amino acids with a molecular weight of 8.7kD,inhibits the coagulantion Factor X/Xa to prevent the generation of prothrombinase through its direct bingding to targeted activity site.Previous studies have revealed the high efficacy of NAP5 to the treatment and prevention of acute arterial of venous thromboembolism,including Sepsis,diffuse interior-vessel coagulation(DIC),and postoperative venous thromoembolism in patients undergoing total knee replacement,indicating its encouraging potential in the development of novel anticoagulant.However,NAP5 could only inhibit the generation of prothrombinase.It can't inhibit the activity of thrombinase. Hirudin(HV) , an acidic peptide secteted from salivary glands of Hirudo medicinalis is the most effective inhibitor of thrombin.The 12 peptide comes from C'of Hirudin ,named Hirudin 12 peptide,is considered to be the smallest peptide which has the activity of inhibiting thrombin. Hirudin 12 peptide can also bind directly to thrombin, inhibit fibrin-bound, especially fluid-phase thrombin.Though the peptide is very short,it shows a high activity of inhibiting thrombin. Even worse, there are rare antagonists of bleeding induced by Hirudin 12 peptide.But its half life time is very short and will be easily degradated in vivo.In order to eke out the two anticoagulant peptides and use their advantage,we fused a FXa recognition sequence between NAP5 and Hirudin 12 peptide to maintain the activity of the two peptides and increase the anticoagulant activity, while increasing the side effect of fusion protein in blood also. In addition, we fused Arg-Gly-Asp (RGD) sequence into C'of Hirudin 12 peptide to elevate its activity of inhibiting platelet aggregation to increase its activity of preventing arteriothromobosis. Furthermore, we added a 6×His-Tag to make its separation and purification conveniently, which provided an efficient methed to produce a great deal of Antithrombotic Fusion Protein.In this study, the recombination antithrombotic fusion protein gene was successfully cloned by PCR with 8 primers designed according to the amino acid sequences of our fusion protein and the codon preference of Pichia pastoris, and an expression vector was constructed by ligating the recombination antithrombotic fusion protein gene into the Pichia pastoris vector pPIC9K, which was transfered into Pichia pastoris GS115. Then a positive clone, which was identified as Mut+, was obtained with G418 selection, and expressed efficiently, stably and secretely in suitable conditions.The conditioned medium was concentrated with Millipore ultrafiltration and the interest protein was purified by affinity chromatography. The fusion protein was identified by tryptic digestion, western blotting. At last,we get 1.35mg of fusion protein.When the concentration of fusion protein was 0.10μg/ml, the activity of the platelet aggregation was saturated basically.The value of PT,APPT showed increase as the increasing concentration of fusion protein.While, the value were decreased after dealed with the protein by FXa.That shows that the NAP5 has activity.Compared with hirudin,the protein degrested by FXa has a good activity of inhibiting thrombin. Finally ,by testing the platelet aggregation, prothrombintime(PT), activated partial thromboplastintime(APTT) and thrombin titration, we found that the fusion protein has a high activity of NAP5,inhibiting thrombin of Hirudin 12 peptide and the platelet aggregation.