FEBS 28607 FEBS Letters 571 (2004) 67–73 Functional characterization of recombinant batroxobin, a snake venom thrombin-like enzyme, expressed from Pichia pastoris Weon-Kyoo Youa,b,1, Won-Seok Choia,b,1, You-Seok Koha, Hang-Cheol Shinc, Yangsoo Jangd, Kwang-Hoe Chunga,d,* aBiobud Co., Ltd., R&D Center, Seoul 120-110, Republic of Korea bDepartment of Biochemistry, College of Science, Yonsei University, Seoul 120-749, Republic of Korea cDepartment of Bioinformatics and Life Science, Soongsil University, Seoul 156-743, Republic of Korea dCardiovascular Research Institute and BK21 Project for Medical Sciences, Yonsei University, College of Medicine, Seoul 120-752, Republic of Korea Received 18 March 2004; revised 10 June 2004; accepted 25 June 2004 Available online 2 July 2004 Edited by Judit Ovadi have been identified and cloned from various snake venoms Abstract A thrombin-like enzyme of Bothrops atrox moojeni venom, batroxobin, specifically cleaves fibrinogen a chain, [4,5]. Batroxobin (EC 3.4.21.29) was known to be a thrombin- resulting in the formation of non-crosslinked fibrin clots. The like enzyme from Bothrops atrox moojeni venom and its cDNA cDNA encoding batroxobin was cloned, expressed in Pichia was also cloned [6,7]. In contrast to thrombin, which converts pastoris and the molecular function of purified recombinant fibrinogen into fibrin by cleavage of fibrinogen A and B chains, protein was also characterized. The recombinant batroxobin had batroxobin only splits off fibrinopeptide A [6,7]. Additionally, an apparent molecular weight of 33 kDa by SDS–PAGE thrombin activates various blood coagulation proteins such as analysis and biochemical activities similar to those of native factors V, VIII, and XIII as well as platelets, endothelial cells, batroxobin. The purified recombinant protein strongly converted and smooth muscle cells which are closely related to hemostatic fibrinogen into fibrin clot in vitro, and shortened bleeding time system, whereas batroxobin has no direct effects on these fac- and whole blood coagulation time in vivo. However, it did not tors or cells [1]. The enzyme has a defibrinogenating effect and is make any considerable alterations on other blood coagulation factors. Several lines of experimental evidence in this study currently being used clinically for the treatment of various suggest that the recombinant batroxobin is a potent pro- thrombotic diseases including deep vein thrombosis, myocar- coagulant agent. dial infarction, pulmonary embolism, and acute ischemic stroke Ó 2004 Published by Elsevier B.V. on behalf of the Federation of [8–10]. European Biochemical Societies. Batroxobin contains 231 amino acids and 12 cysteine resi- dues which are involved in six disulfide bonds. There are two Keywords: Fibrin clot; Fibrinogen; Recombinant batroxobin; putative glycosylation sites in the polypeptide chain of bat- Pro-coagulant; Thrombin-like enzyme roxobin [6,7,11]. Although recombinant thrombin-like en- zymes of various snake venoms have been expressed in Escherichia coli, these recombinant proteins usually formed insoluble inclusion bodies [12–15]. It has proven difficult to 1. Introduction refold inclusion bodies of thrombin-like enzymes as function- ally active and soluble proteins [16,17]. Snake venoms contain various components which affect the In the present study, we report the cloning, expression and mammalian hemostatic system as either pro-coagulants or anti- functional characterization of recombinant batroxobin, a coagulants [1–3]. These components interact with diverse pro- snake venom thrombin-like enzyme, from Pichia pastoris. This teins of the blood coagulation cascade and the fibrinolytic is the first report demonstrating that the recombinant yeast- pathway [2,3]. Generally, they are classified into several groups produced batroxobin might be developed as a clinical pro- as follows: (i) fibrinogen clotting enzymes; (ii) fibrino(geno)lytic coagulant agent for the treatment of thrombotic diseases. enzymes; (iii) plasminogen activators; (iv) prothrombin activators; (v) factor V, X activators, (vi) hemorrhagins; and (vii) platelet aggregation inhibitors [3]. Thrombin-like enzymes, 2. Materials and methods which are serine proteases with fibrinogen clotting activity, 2.1. Materials Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– * Corresponding author. Fax: +82-2-365-1878. PAGE) gel, molecular weight marker, and Pichia Expression Kit were E-mail address: [email protected] (K.-H. Chung). obtained from Invitrogen (Carlsbad, CA). Human fibrinogen, throm- bin, biotin, H-D-Ile-Phe-Lys-p-nitroanilide, N-Benzoyl-Phe-Val-Arg-p- 1 The first two authors contributed equally to this work. nitroanilide, and N-tosyl-Gly-Pro-Arg-p-nitroanilide were from Sigma Chemical Co. (St. Louis, MO). H-D-Ile-Pro-Arg-p-nitroanilide (S- Abbreviations: APTT, activated partial thromboplastin time; PAS, 2288), H-D-Phe-Pip-Arg-p-nitroanilide (S-2238), H-D-Pro-Phe-Arg-p- periodic acid-Schiff’s reagent; PBS, phosphate-buffered saline; PCR, nitroanilide (S-2302), and H-D-Val-Leu-Arg-p-nitroanilide (S-2266) polymerase chain reaction; PT, prothrombin time; SDS–PAGE, were from Chromogenix (Milano, Italy). Heparin–Sepharose and sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TT, Phenyl–Sepharose were products of Amersham Biosciences (Uppsala, thrombin time Sweden). Yeast nitrogen base was obtained from Becton Dickinson 0014-5793/$22.00 Ó 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. doi:10.1016/j.febslet.2004.06.060 68 W.-K. You et al. / FEBS Letters 571 (2004) 67–73 (Sparks, MD). Native batroxobin was from Pentapharm (Basel, was then overlaid on a fibrinogen (0.7%, w/v)-agarose (0.5%, w/v) plate Switzerland). and incubated at 37 °C. Fibrinogen clotting assay was measured as previously described [20]. After recombinant and native batroxobins 2.2. Construction of expression plasmid for batroxobin were added to 0.5 ml of human fibrinogen (0.5 mg/ml) in 20 mM Tris– The full-length cDNA of batroxobin (GenBank Accession HCl (pH 7.5), clotting was monitored by changes in turbidity at No. J02684) was kindly donated by Dr. Yamashina [7]. The cDNA 405 nm using a DU650 spectrophotometer (Beckman Coulter, Inc. encoding batroxobin for yeast expression was cloned by polymerase Chaska, MN). chain reaction (PCR) with the N-terminal primer 50-CTCGAGA- 0 AAAGAGTCATTGGAGGTGATG-3 and the C-terminal primer 2.6. Measurement of plasma clotting time in vitro 0 5 -TTCACGGGCTCGCAGTTTTATTTCCTGCAATAATGCTC- Rat plasma was obtained from whole blood by centrifugation. Re- 0 3 . The PCR product was cloned into pGEM-T Easy vector combinant and native batroxobins were treated in the plasma, and (Promega, Madison, WI) and sequenced. The amplified cDNA of then plasma clotting time was measured by using an Automated Co- batroxobin was transferred into P. pastoris expression vector, agulation Laboratory (ACL) 100 Instrument (Instrumentation Labo- pPIC9 (Invitrogen). The pPIC9-batroxobin vector was digested ratory Company, Milano, Italy). with SalI (NEB, Beverly, MA) restriction enzyme. This linearized plasmid was transformed into electro-competent GS115 cells (Gene Pulser, Bio-Rad, Hercules, CA). Methanol utilization 2.7. Substrate specificity of batroxobin and thrombin ability of the Hisþ pPIC9-batroxobin transformants was analyzed To investigate substrate specificity of recombinant batroxobin, na- by replica selection on minimal dextrose and on minimal methanol tive batroxobin, and thrombin, various synthetic substrates (100 lM) plate. Finally, Hisþ and Mutþ colony was selected for batroxobin were incubated with thrombin (60 nM) or each batroxobin (120 nM) in expression. 0.2 ml of 20 mM Tris–HCl (pH 7.5). The hydrolytic reaction was monitored at 405 nm by the formation of p-nitroaniline and lKat 2.3. Expression of recombinant batroxobin by fermentation value was calculated as described previously [21]. The chromogenic Fermentation was performed using a BIOSTATÒ B fermenter (B. substrates examined were: H-D-Ile-Phe-Lys-p-nitroanilide, N-Benzoyl- Braun Biotech International, Melsungen, Germany) equipped with Phe-Val-Arg-p-nitroanilide, N-tosyl-Gly-Pro-Arg-p-nitroanilide, H-D- software control (MFCS/Win 2.0) of temperature (30 °C), agitation Ile-Pro-Arg-p-nitroanilide (S-2288), H-D-Phe-Pip-Arg-p-nitroanilide (1000 rpm), pH, anti-foam addition, supplemental oxygen addition, (S-2238), H-D-Pro-Phe-Arg-p-nitroanilide (S-2302), and H-D-Val-Leu- and feeding rate (glycerol and methanol). Stock cells were grown in Arg-p-nitroanilide (S-2266). 200 ml of yeast nitrogen base media for 24 h. The cultured cells were transferred to a fermenter containing 1.5 liter of basal salt-glycerol 2.8. Measurements of bleeding time and whole blood coagulation time The bleeding time was measured as previously described [22]. media (H3PO4, 27 ml/l; CaSO4 Á 2H2O, 0.9 g/l; K2SO4, 18 g/l; Sprague–Dawley rats (n ¼ 5, 250–300 g) were pretreated by the MgSO4 Á 7H2O; 15 g/l, KOH, 4.13 g/l; and glycerol, 40 g/l) supple- mented with 4.4 ml/l trace mineral solution. The pH of culture media intravenous injection of recombinant and native batroxobins (1 NIH unit/kg) in phosphate-buffered saline (PBS). After 1 h, the tail was maintained at 5.0 with 12.5% (w/v) NH4OH during fermentation. Growth in batch mode was continued until the dissolved oxygen of rats was transfected at 5 mm from the tip and 1.5 cm of distal concentration increased as a result of carbon source limitation (20 h). portion was vertically immersed in PBS. The whole blood coagula- A 50% glycerol containing 12 ml/l trace mineral solution and supple- tion time was measured by modified method in the previous report mental oxygen was automatically administered to maintain the dis- [23]. Rats (n ¼ 5) were pretreated by intravenous injection of re- solved oxygen concentration above 40% for 10 h. After carbon source combinant and native batroxobins (2 NIH unit/kg) in PBS. After 1 h, starvation (30 min) for complete glycerol consumption, methanol in- the coagulation time of 0.5 ml citrated whole blood containing 10 duction was initiated.