Preparation And Characterization Of A Recombinant Thrombin-like Enzyme From Gloydius Shedaoensis

Snake venom thrombin-like enzymes belong to the fibrinogenolytic enzyme family and are serine proteases. They do not activate factor VIII and possess the fibrinogenolytic ability to hydrolyze fibrinogen into non-crossing linked fibrin, which are then eliminated by succeeding blood fibrinolytic system. They are applied as anti-coagulates due to their "defibrinogenation" in the blood. Snake venom thrombin-like enzyme preparations, gloshedobin, from the venom of the rare arboreal pit-viper, Gloydius shedaoensis, has been applied in clinic therapy for many years. However, upon the recent domestic decree of forbidding snake capture, production of these enzymes from snake venoms faces big challenge. Therefore, genetic engineering is one of the best alternatives to solve this problem. In this work, the preparation of recombinant gloshedobin in soluble and active form, and its biochemical properties in particular fibrinogenolytic activity was investigated.Firstly, cloning and expression of soluble and active gloshedobin in E. coli was investigated. The solubility tendency of recombinant gloshedobin with various commercial fusion proteins was predicted using Wilkinson-Harrison protein solubility predication model, which showed that recombinant gloshedobin fused with NusA, TrxA or GST tags at its N terminus had higher solubility. NusA, TrxA and GST were thus chosen for facilitating the expression of soluble gloshedobin in Escherichia coli. Accordingly, three expression vectors, pQYNusA-Glo-a, pQYGST-Glo-a and pQYTrxA-Glo-a were constructed and then transformed into Escherichia coli BL21-Gold (DE3). pQYNusA-glo-a was determined to be the best by both SDS-PAGE analysis and Western blotting assay.Secondly, separation and purification of the recombinant gloshedobin was performed. (1) Removal of nucleic acids contaminants from crude recombinant gloshedobin samples by artificial nuclease R5 was investigated: Under 400nm UV-light, 1 mM of R5 could efficiently eliminate nucleic acids contaminants without impairing the activity of recombinant gloshedobin. Since the cost of synthesis is low, artificial nuclease R5 shows application potentials in the production of protein/enzymes; (2) Four-step column chromatographic purification of the recombinant gloshedobin with high purity: The column chromatographies included hydrophobic interaction chromatography on Phenyl Sepharose FF, ion exchange chromatography on Q Sepharose HP and Mono Q, and size exclusion chromatography on Superdex 200 were used. And the purified recombinant gloshedobin with the activity yield of 0.1mg/L were obtained, which appeared as one single band on SDS-PAGE. Its apparent molecular weight was determined to be 90 kDa and its specific amidolytic activity was 506 U/mg.Thirdly, biochemical characterization of recombinant gloshedobin was investigated: (1) Enzymatic properties: The recombinant gloshedobin exhibited fibrinogen clotting, fibrinogenolytic and amidolytic activities. By using N-a-benzoyl-DL-arginine p-nitroanilide (BApNA) as model substrate, its optimum temperature and pH for amidolytic activity were 40℃and pH 8.0, respectively. The recombinant gloshedobin preferentially cleaved bovine fibrinogen's Aα-chain, then Bβ-chain, but did not cleaveγ-chain at all, which was dose-dependent and time-dependent. (2) Effects of serine proteases inhibitors, reducing agents, chelator and thrombin inhibitors, on the amidolytic activity of recombinant gloshedobin were determined. Serine protease inhibitors PMSF (1 mM) and TPCK (1 mM) inhibited the amidolytic activity of recombinant gloshedobin by 100 % and 77 %, respectively, indicating recombinant gloshedobin belongs to serine proteases family. Inhibitors including benzamidine, 3-aminobenzamidine, 4-aminobenzamidine and imidazole inhibited <15 % amidolytic activity of recombinant gloshedobin. EDTA did not affect recombinant gloshedobin, suggesting gloshedobin is not metalloproteinase. Reducing agents dithiothreitol andβ-mercaptoethanol did not affect recombinant gloshedobin, suggesting recombinant gloshedobin is highly stable. Thrombin natural inhibitors such as heparin also did not affect this enzyme's amidolytic activity, suggesting recombinant gloshedobin differs from thrombin in structure; (3) Effects of serine proteases inhibitors, reducing agents, chelator and thrombin inhibitors on the fibrinogenolytic activity of recombinant gloshedobin were determined by using SDS-PAGE analysis: none of the serine protease inhibitors except PMSF had any effect on the fibrinogenolytic activity of recombinant gloshedobin, suggesting Ser182 is a key amino acid catalytic residue for both amidolytic and fibrinogenolytic activities. The convalent bond formed between PMSF and Ser182 was predicted to hamper the approach of substrates according to homology modeling. However, TPCK did not affect the fibrinogenolytic activity but affect amidolytic activity. EDTA hampered the activity, suggesting that metal ion(s), in particular transition metal ions, played a role in the fibrinogenolytic process. The inhibitory effect of EDTA might relate to the removal of divalent metal ions by EDTA causing the configuration change of substrate fibrinogen. Reducing agents dithiothreitol andβ-mercaptoethanol did not affect recombinant gloshedobin, suggesting recombinant gloshedobin is highly stable. The fact that heparin as well as hirudin did not show any effect on the fibrinogenolytic activity of recombinant gloshedobin allowed the combined application of recombinant gloshedobin with heparin or hirudin in the clinical treatment of heparin-associated thrombocytopenia and cardiopulmonary diseases; (4) Effects of metal ions on recombinant gloshedobin were determined by amidolytic activity and SDS-PAGE analysis: Gloshedobin's amidolytic activity toward BApNA was greatly inhibited by 1 mM of transition metal ions in the order: Fe²⁺>Cu²⁺≈Zn²⁺>Hg²⁺>>Ni²⁺. The addition of EDTA can reverse this inhibitory effect to >80 % of the original amidolytic activity which could be inhibited by transition metal ions. Other ions such as Co²⁺ and Mn²⁺ inhibited the amidolytic activity of recombinant gloshedobin by 38 % and 25 %, respectively. Besides, its fibrinogenolytic activity was inhibited by divalent transition metal ions in the order: Cu²⁺≈Ni²⁺>Zn²⁺>Co²⁺. Other ions such as Ca²⁺ and Mg²⁺ have almost no effect on either activity. According to the Irving-Williams crystal field theory and gloshedobin 3-D structure model generated by homology modeling, the inhibitory effects by metal ions like Zn²⁺ might be due to four intermolecular coordination bonds formed between Zn²⁺ and oxygen atom in the side chain of Ser182, Zn²⁺ and nitrogen atom in the imidazole ring of His43, Zn²⁺ and oxygen atoms in two water molecules.In conclusion, the production of recombinant gloshedobin in a soluble and active form was achieved by the strategy of fusing NusA tag at its N-terminal. Artificial nuclease R5 proved to be highly efficient in the removal of nucleic acids contaminants from crude recombinant gloshedobin samples and non-impairment on the recombinant gloshedobin's activity, showing its application potentials in the field of biochemical engineering. The recombinant gloshedobin with high purity was obtained by four-step column chromatographic purification, which exhibited high amidolytic and fibrinogenolytic activities. The effects of serine protease inhibitors, reducing agents, chelator, thrombin inhibitors and divalent metal ions on recombinant gloshedobin were systematically investigated and the inhibitory mechanisms were also proposed using homology modeling techniques. The data obtained in this study might provide essential assistances in the preparation and clinic application of recombinant thrombin-like enzyme.