Snake Venom Thrombin-like Enzymes Research Articles

Molecular mechanism analysis of Gloydius shedaoensis venom gloshedobin interaction with inhibitors by homology modeling

Snake venom thrombin-like enzymes (SVTLEs) are widely applied in the treatment of thrombotic diseases, however, the molecular mechanism of its inhibition by synthetic and natural proteinaceous inhibitors is not yet understood. Here we investigated effects of protease inhibitors including phenylmethylsulfonil fluoride (PMSF), benzamidine (BMD) and its derivates on the activity of recombinant gloshedobin, a SVTLE from the snake Gloydius shedaoensis. The molecular inhibition mechanism was postulated by separately docking inhibitors into three-dimensional model of gloshedobin using protein C activator from Agkistrodon contortrix contortrix venom (ACC-C, which bear 78% identity with gloshedobin) as template. The analysis indicated that the strongest inhibitor, PMSF, was via a covalent bond with the catalytic Ser195, while other inhibitors showing weaker inhibitory activity were via hydrogen bond with Ser195 or non-catalytic residues.

Isolation and characterization of the thrombin-like enzyme from Cryptelytrops purpureomaculatus venom

A thrombin-like enzyme, purpurase, was purified from the Cryptelytrops purpureomaculatus (mangrove pit viper) venom using high performance ion-exchange and gel filtration chromatography. The purified sample (termed purpurase) yielded a homogeneous band in SDS-polyacrylamide gel electrophoresis with a molecular weight of 35,000. The N-terminal sequence of purpurase was determined to be VVGGDECNINDHRSLVRIF and is homologous to many other venom thrombin-like enzymes. Purpurase exhibits both arginine ester hydrolase and amidase activities. Kinetic studies using tripeptide chromogenic anilide substrates showed that purpurase is not fastidious towards its substrate. The clotting times of fibrinogen by purpurase were concentration dependent, with optimum clotting activity at 3 mg fibronogen/mL. The clotting activity by purpurase was in the following decreasing order: cat fibrinogen > human fibrinogen > dog fibrinogen > goat fibrinogen >> rabbit fibrinogen. Reversed-phase HPLC analysis of the products of action of purpurase on bovine fibrinogen showed that only fibrinopeptide A was released. Indirect ELISA studies showed that anti-purpurase cross-reacted strongly with venoms of most crotalid venoms, indicating the snake venom thrombin-like enzymes generally possess similar epitopes. In the more specific double-sandwich ELISA, however, anti-purpurase cross-reacted only with venoms of certain species of the Trimeresurus complex, and the results support the recent proposed taxonomy changes concerning the Trimeresurus complex.

Biochemical and Hemostatic Mechanism of A Novel Thrombin-Like Enzyme

Thrombin-like enzyme (TLE) plays a significant role in vessel injury hemostasis. A novel snake venom TLE (Agacutin) was purified from Agkistrodon Acutus snake venom. Structural analysis indicated that Agacutin is a heterodimer that has a MW of 29,402 Da, a pI value of 5.39, and optimum activity at 35 °C and pH 7.5. The N-terminal 15 amino acid sequences of Agacutin are DSSGWSSYEGHEYYV (small subunit) and DCSSGWSSYEEHQYY (large subunit). In vitro studies indicated that the coagulation activity of Agacutin was activated by Ca +2 or inhibited by phenylmethanesulfonyl fluoride, but not influenced by heparin or hirudin. The arginine esterase activity and fibrinogen hydrolysis result showed that Agacutin only cleaves α-subunit and releases fibrinopeptide A. In vivo studies indicated that Agacutin iv (0.01-0.05 U/kg) shortened 30.2-49% of the rabbit blood clotting time, or ip (0.5-2.0 U/kg) shortened 29.7-73.1% of the mouse tail bleeding time. Agacutin does not influence APTT, platelet or euglobulin clotting time, and activate Factor II or XIII. It converts fibrinogen into the soluble fibrin that accelerates hemostasis at wound.

Fibrin adhesive derived from snake venom in periodontal surgery: histological analysis.

Background:A new fibrin adhesive made of buffalo plasma-derived fibrinogen and a thrombin-like snake venom enzyme, has been successfully used to immobilize free gingival grafts. This case series histologically compared sutured grafts (control group) with others immobilized by using the fibrin adhesive (experimental group).Case Description:The grafts were placed in the contralateral mandibular bicuspids of 15 patients, so that each subject received one treatment of each type. Five biopsies of each group were collected at 7, 14 and 45 days of healing, which were histologically and morphometrically analyzed as regards the relative volume density of the different connective tissue components.Results:The sites in the control group presented a higher inflammatory cell density at 7 days and a tendency towards a lower collagen density. In the experimental group, the grafts had an appearance of more advanced healing. Tissue maturity characteristics progressed until 14 and 45 days, but no difference between groups could be noted at these times.Conclusions:Within the limits of the present study, it may be suggested that the alternative fibrin adhesive tested could represent an alternative to sutures in gingival grafts procedures.

A novel serine protease from the snake venom of Agkistrodon blomhoffii ussurensis

A novel serine protease, ABUSV-SPase, was isolated to homogeneity for the first time from Chinese Agkistrodon blomhoffii ussurensis snake venom, and its enzymatic and structural properties were characterized by multiple techniques. ABUSV-SPase is a stable monomeric protein with a molecular mass of 26,752.6 a.m.u. It reacts optimally with its substrate N α-tosyl- l-arginine methyl ester (TAME) at pH 7.0 and 41 °C. ESI-MS/MS analysis indicates that ABUSV-SPase is a new serine protease, sharing peptide homologies with various snake venom serine proteases, especially the snake venom thrombin-like enzymes of this group, and serine protease precursors. It is a zinc-containing protein, and although zinc is not essential for activity, its replacement by various divalent metal ions, including Mg 2+, Mn 2+, and Ca 2+, increases the TAME hydrolysis activity of the enzyme. The intrinsic fluorescences of Tyr and Trp residues of ABUSV-SPase have emission wavelengths red-shifted by 12.8 nm and 3.6 nm from those of free Tyr and Trp, respectively. The zinc ion increases the hydrophobicity of the environment of the Trp residues, increases the thermostability of the protein, and affects the protein secondary structure to stabilize the enzyme, but appears to have no direct role in its esterase hydrolysis activity.

Effective reduction of truncated expression of gloshedobin in Escherichia coli using molecular chaperone ClpB

Snake venom thrombin-like enzymes (TLEs) have been widely studied for potential therapeutic applications as anti-coagulants in the treatment of blood clotting disorders. However, due to the cysteine-rich nature of these proteins, their expressions in Escherichia coli were often impeded by inclusion body (IB) formation. The formation of truncated expression products significantly complicated the production of gloshedobin, a recently isolated TLE from the snake venom of Gloydius shedaoensis [Yang, Q., Li, M., Xu, J.Q., Bao, Y.M., Lei, X.Y., An, L.J., 2003a. Expression of gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis, in Escherichia coli. Biotechnology Letters 25, 101–104; Yang, Q., Xu, J.Q., Li, M., Lei, X.Y., An, L.J., 2003b. High-level expression of a soluble snake venom enzyme, gloshedobin, in E. coli in the presence of metal ions. Biotechnology Letters 25, 607–610]. In this work, it was found that the expression of gloshedobin was strongly dependent on the expression host. The truncated expression was reduced by 25% when the protein was expressed in E. coli BL21(DE3)pLysS instead of BL21(DE3). It was also demonstrated that co-expression of ClpB (a molecular chaperone) in BL21(DE3) enabled the expression of gloshedobin mostly in intact form without compromising expression level, while almost completely eliminating its truncation products. This suggests a new simple strategy to significantly improve the quality of protein expression in TLE production and may find its useful application for many other recombinant proteins whose expressions and/or purifications are hindered by the formation of truncation products.

The hidden sepsis marker: aPTT waveform analysis

Thromb Haemost 2008; 100: 9–10 Downey et al. published a paper in 1997 entitled ‘Novel and diagnostically applicable information from optical waveform analysis of blood coagulation in disseminated intravascular coagulation’ (1). Using a new automated coagulation analyzer, the MDA-180, the authors observed that during measurement of the aPTT, some samples became turbid immediately after addition of the calcium chloride reagent, well before clot formation. These plasma samples were mainly from intensive care unit (ICU) patients, which also fulfilled the diagnostic criteria for disseminated intravascular coagulation (DIC). The ‘biphasic waveform’ (BPW) recorded during optical measurement of the aPTT was thus introduced as a diagnostic marker for DIC (2). The authors found BPW also in some patients without DIC, and speculated that the phenomenon was related to coagulation activation or presence of pre-formed fibrin complexes. At that time, our laboratory was involved in an analysis of the effects of ancrod, a thrombin-like snake venom enzyme used for treatment of ischemic stroke (3). Ancrod induces massive intravascular fibrin formation, but none of our probands treated with ancrod developed BPW, indicating that BPW was not related to intravascular fibrin formation. Finally, Toh et al. were able to show that BPW was due to calcium-dependent complex formation between C-reactive protein (CRP) and very low density lipoprotein (VLDL) (4). Actually, this phenomenon had been described 20 years earlier. Cabana et al. showed that VLDL formed precipitating complexes with CRP, and these complexes were not observed in the presence of EDTA (5). In the same year, Hulman et al. reported that the serum of acutely ill patients agglutinated fat emulsions prepared from soybean oil designated for intravenous infusions, resulting in ‘creaming’ of the sera (6). Free calcium ions were necessary for ‘creaming’, and no ‘creaming’ was observed if citrate was added to the sera. The authors concluded that ‘creaming’ was a property of acute phase sera, and that this phenomenon is the explanation for adverse reactions to intravenous lipid emulsions, related to lipid microembolism, such as fever, shivering, precordial pains, nausea and vomiting. A ‘fat emulsion agglutination test’ was developed for measurement of CRP (7). It was later shown that the ‘creaming’ phenomenon in response to intravenous lipids was only observed in patients with highVLDL levels (8). Rowe et al. further characterized the interaction between CRP and VLDL and concluded that CRP may be related in some way to lipoprotein metabolism (9). It was also shown in experiments on rabbits that the CRP-VLDL complexes formed in acute phase serum contained predominantly β-VLDL, an abnormal apo-B containing lipoprotein low in apo-E content (10, 11). Inflammatory stimuli such as endotoxin induce the upregulation of VLDL secretion in the liver (12). Bennett et al. reported that endotoxin has a distinct effect on structural properties of VLDL, resulting in improved utilization by the heart as energy substrate (13). The authors did not consider the possibility that complex formation of CRP and VLDL might be involved. BPW is an indicator of a systemic inflammatory response, as observed in sepsis. Chopin et al. studied 187 patients with 217 episodes of systemic inflammatory response syndrome, of which 34 were classified as sepsis, 26 as severe sepsis, and 50 as septic shock. The diagnostic sensitivity and specificity of BPW for the combined group of severe sepsis and septic shock was 92% and 67%, respectively. Both CRP and procalcitonin displayed a lower diagnostic sensitivity (14). Another recent study showed a sensitivity of BPW for sepsis of 81%, with a specificity of 76% in ICU patients (15). Combination of aPTT waveform analysis with procalcitonin measurement did not increase sensitivity but raised specificity to 94%. In our own study on ICU patients, the diagnostic sensitivity of BPW for sepsis was 74%, with a specificity of 81% (16). Within the group of ICU patients with systemic inflammatory response syndrome, BPW identifies a group with increased mortality rate. In the study of Toh et al., the mortality rate of patients with BPW was 44%, compared to 26% in the patients without BPW (17). In the study of Bakhtiari et al., mortality of patients with and without BPW was 43% and 32%, respectively (18). In our study, the mortality rate of ICU patients with and without BPW was 36.8% and 13.1%, respectively (16). Use of the aPTT waveform analysis is not limited to ICU patients. Smith et al. have shown that also non-ICU patients with BPW are more likely to have positive blood cultures (19). Similarly, the study now published by Hussain et al. (20) shows that BPW can identify patients with sepsis within a specific high-risk patient group. Myelosuppressive therapy results in neutropenia

Purification, crystallization and preliminary X-ray diffraction analysis of saxthrombin, a thrombin-like enzyme fromGloydius saxatilisvenom

The snake-venom thrombin-like enzymes (SVTLEs) are a class of serine proteinases that show fibrinogen-clotting and esterolytic activities. Most TLEs convert fibrinogen to fibrin by releasing either fibrinopeptide A or fibrinopeptide B and cannot activate factor XIII. The enzymes hydrolyze fibrinogen to produce non-cross-linked fibrins, which are susceptible to the lytic action of plasmin. Because of these physiological properties, TLEs have important medical applications in myocardial infarction, ischaemic stroke and thrombotic diseases. Here, a three-step chromatography procedure was used to purify saxthrombin (AAP20638) from Gloydius saxatilis venom to homogeneity. Its molecular weight is about 30 kDa as estimated by SDS-PAGE. A saxthrombin crystal was obtained using the hanging-drop vapour-diffusion method and diffracted to a resolution limit of 1.43 A. The crystal belongs to space group C2, with unit-cell parameters a = 97.23, b = 52.21, c = 50.10 A, beta = 96.72 degrees , and the Matthews coefficient (V(M)) was calculated to be 2.13 A(3) Da(-1) with one molecule in the asymmetric unit.

Recombinant Batroxobin Expressed Highly in Pichia pastoris

Methylotrophic yeast, Pichia pastoris, was used to express recombinant batroxobin, and a technology for producing a recombinant protein was finally established. We synthesized batroxobin gene artificially by means of recursive PCR. pPIC9-batroxobin was constructed and transformed into P. pastoris GS115 (his4). Recombinant batroxobin was expressed in yeast engineering strain and purified from the culture supernatant. 10 mg of recombinant batroxobin was purified from 1 L fermentation media, exhibiting specific activity of 238 NIH units/mg, and had molecular weight of 30.55 kD. The purified recombinant protein converted fibrinogen into fibrin clot in vitro and shortened bleeding time in vivo. The study laid a foundation for the development of hemostatic of recombinant snake venom thrombin-like enzyme (SVTLE).

Insights into the substrate specificity of a novel snake venom serine peptidase by molecular modeling

The cDNA encoding BthaTL, a serine peptidase from the venom of the snake Bothrops alternatus, was cloned and sequenced. The deduced primary structure shows over 62% of identity with snake venom thrombin-like enzymes (SVTLEs), molecules with high substrate specificity toward different natural substrates. Indeed, a phylogenetic reconstruction by two different methods clustered this enzyme close to other SVTLEs. These enzymes generally affect the hemostatic system in several ways, and therefore are used as tools in pharmacology and clinical diagnosis. A three-dimensional model of BthaTL was built by homology modeling using TSV-PA ( Trimeresurus stejnegeri venom plasminogen activator) crystal structure as template. BthaTL model showed that the typical catalytic triad conformation of serine peptidases was preserved. The calcium coordination ligands were absent or adopt an unfavorable conformation, preventing interactions with metals. On the other hand, the Asp97–Arg174 saline bridge of TSV-PA was not found and its specificity determinant Phe193 is replaced by a Gly in BthaTL. The substitution of essential residues in the neighborhoods of the catalytic site cleft of BthaTL indicates that these two proteins do not share the same enzymatic specificity, what means that BthaTL will probably not activate plasminogen. Such observations may be helpful in the understanding of the molecular mechanism for substrate specificity of these enzymes.

CURRENT STATUS OF SNAKE VENOM THROMBIN-LIKE ENZYMES

Thrombin-like enzymes are one of the most important groups of enzymes present in snake venom with potential for clinical therapeutic use. The enzymes catalyze the conversion of fibrinogen into fibrin, usually similar, but not identical, to thrombin. The knowledge of their substrate specificity and catalytic mechanism has guided the development of reagents to perform coagulation tests in specimens of blood that contain heparin, and studies about their application as defibrinogenating agents in chronic and acute diseases, such as stroke, deep-vein thrombosis, myocardial infarction, and peripheral arterial thrombosis. This update will focus on the current status of snake venom thrombin-like enzymes using a perspective gained from those enzymes with structures that have been studied in detail.

A BRIEF HISTORY OF SNAKE VENOMS AFFECTING HEMOSTASIS

The earliest reports of snake venoms affecting hemostasis occur in the 17th century and first half of the 18th century with the observations of Nieremberius (1635), van Helmont (1648), Richard Mead (1702), and Geoffrey and Hunauld (1737). However, our detailed knowledge of this field of toxinology stems largely from the work in the second half of the 18th century of Felice Fontana, who is credited with being the first investigator to inject viper venom into dogs. Fontana's work foreshadowed many seminal discoveries that were to be made in the next 200 years, but a fallow period of research followed Fontana until three Britons took up investigations into snake venoms beginning in the late 19th century: Charles Martin, who worked on Australian snake venoms; Gwyn Macfarlane, who studied Russell's viper venom, initially in relation to the treatment of hemophilia; and Alistair Reid, who worked on the Malayan pit viper and was the first to suggest the use of snake venom thrombin-like enzymes in “therapeutic defibrination” as an alternative anticoagulant strategy. From the 1960s onwards, the explosion of research activity generated in part by molecular biology, has provided a vast amount of data in this field. Notably, studies on venom factors affecting platelet function are part of this more recent history, and in the last 20 years, a wide range of C-type lectins and disintegrins have been discovered. Molecular techniques have now enabled the identification of gene structure, the cloning of genes, the production of recombinant protein, and the elucidation of protein function at the molecular level. Overall, a significant number of snake venom toxins affecting hemostasis have been put to use in both the research and routine coagulation laboratory settings and show much promise in a variety of clinical situations.

Practical applications of snake venom toxins in haemostasis

Snake venom toxins affecting haemostasis have facilitated extensively the routine assays of haemostatic parameters in the coagulation laboratory. Snake venom thrombin-like enzymes (SVTLE) are used for fibrinogen/fibrinogen breakdown product assay and for the detection of fibrinogen dysfunction. SVTLE are not inhibited by heparin and can thus can be used for assaying antithrombin III and other haemostatic variables in heparin-containing samples. Snake venoms are a rich source of prothrombin activators and these are utilised in prothrombin assays, for studying dysprothrombinaemias and for preparing meizothrombin and non-enzymic forms of prothrombin. Russell's viper ( Daboia russelli) venom (RVV) contains toxins which have been used to assay blood clotting factors V, VII, X, platelet factor 3 and, importantly, lupus anticoagulants (LA). Other prothrombin activators (from the taipan, Australian brown snake and saw-scaled viper) have now been used to assay LA. Protein C and activated protein C resistance can be measured by means of RVV and Protac ®, a fast acting inhibitor from Southern copperhead snake venom and von Willebrand factor can be studied with botrocetin ® from Bothrops jararaca venom. The disintegrins, a large family of Arg-Gly-Asp (RGD)-containing snake venom proteins, show potential for studying platelet glycoprotein receptors, notably, GPIIb/IIIa and Ib. Snake venom toxins affecting haemostasis are also used in the therapeutic setting: Ancrod (from the Malayan pit viper, Calloselasma rhodostoma), in particular, has been used as an anticoagulant to achieve ‘therapeutic defibrination’. Other snake venom proteins show promise in the treatment of a range of haemostatic disorders.

Purification of A Recombinant Thrombin-like Enzyme, Gloshedobin by Egg Yolk Antibody-Coupled Adsorbents

The gloshedobin, a snake venom thrombin-like enzyme was biosynthesized in the soluble form and purified by egg yolk antibody-coupled adsorbents from E. coli. As His-tag is not favored from the point of view of high-level and soluble expression, we herein constructed a recombinant gloshedobin without His-tag and developed a novel egg yolk antibody (IgY)-immunoaffinity chromatography for its purification in a higher activity yield. The purification process involving Octyl Sepharose FF, IgY-immunoaffinity chromatography and Source Q, yielded 454.7U mg1 protein of interest and 34.8% activity recovery. The anti-gloshedobin IgY was obtained from eggs by injecting diluted gloshedobin into the breast muscle of laying hens and then purified by several steps including 3.5%, 8.5% and 12% polyethylene glycol-6000 precipitation and affinity chromatography using gloshedobin-coupled agarose gel. The obtained IgY was covalently linked to CNBr pre-activated agarose gel, Sepharose 4B FF, to yield immunoaffinity adsorbents. Both immunological and enzymatic activities of the purified enzyme were determined by western-blotting analysis and fibrinogen clotting assay, respectively.

The role of thioredoxin and disulfide isomerase in the expression of the snake venom thrombin-like enzyme calobin in Escherichia coli BL21 (DE3)

Thrombin-like enzymes (TLEs) are studied widely because of their therapeutic potential in myocardial infarction and thrombotic diseases. But they are always produced in Escherichia coli BL21 (DE3) as inclusion bodies because they are single chain Cys-rich proteins. In this work, coexpressing of thioredoxin (TrxA) largely increased the solubility of calobin, one of the TLEs from korea viper Agkistrodon caliginosus, but soluble calobin-T had poor enzyme activity. Disulfide isomerase (DsbC) was introduced into the host cell and coexpressed with calobin in the presence of TrxA, the result was that both the solubility and enzyme activity of calobin-TD were higher than those of calobin-T. Although recombinant calobins exhibited the enzyme of hydrolyzing the fibrinogen, they lost clotting activity to the substrate. Recombinant calobin-TD remained poor amidolytic activity, the effects of divalent metal cations and various inhibitors on this activity were similar to those of native calobin nevertheless, suggesting that calobin-TD exhibited the characteristics of serine protease especially of trypsin-like serine protease.

Functional characterization of recombinant batroxobin, a snake venom thrombin-like enzyme, expressed from Pichia pastoris

A thrombin-like enzyme of Bothrops atrox moojeni venom, batroxobin, specifically cleaves fibrinogen α chain, resulting in the formation of non-crosslinked fibrin clots. The cDNA encoding batroxobin was cloned, expressed in Pichia pastoris and the molecular function of purified recombinant protein was also characterized. The recombinant batroxobin had an apparent molecular weight of 33 kDa by SDS–PAGE analysis and biochemical activities similar to those of native batroxobin. The purified recombinant protein strongly converted fibrinogen into fibrin clot in vitro, and shortened bleeding time and whole blood coagulation time in vivo. However, it did not make any considerable alterations on other blood coagulation factors. Several lines of experimental evidence in this study suggest that the recombinant batroxobin is a potent pro-coagulant agent.

Corrigendum to ?S1 subsite in snake venom thrombin-like enzymes: can S1 subsite lipophilicity be used to sort binding affinities of trypsin-like enzymes to small-molecule inhibitors?? 6Bioorg. Med. Chem. 12 (2004) 25719

Corrigendum to ?S1 subsite in snake venom thrombin-like enzymes: can S1 subsite lipophilicity be used to sort binding affinities of trypsin-like enzymes to small-molecule inhibitors?? 6Bioorg. Med. Chem. 12 (2004) 25719

S1 subsite in snake venom thrombin-like enzymes: can S1 subsite lipophilicity be used to sort binding affinities of trypsin-like enzymes to small-molecule inhibitors?

Thrombin-like enzymes isolated from snake venoms comprise a group of serine proteinases responsible for many important coagulation disorders in the envenomed victims. Besides, these proteinases have great biotechnological interest as antithrombotic agents and as diagnostic tools. However, in spite of the recent overflow of snake venom thrombin-like enzymes (SVTLEs) on protein sequence databases, there is a lack of three-dimensional (3D) structural information on this family. Without such 3D structures available many aspects of the biological function and biochemical properties of these enzymes still remain obscure. Therefore, we have gone through a series of computational techniques, which enabled us to identify the set of residues involved in molecular recognition of inhibitors bound to the S1 subsite of snake venom thrombin-like enzymes (SVTLEs) and ultimately conclude that nonpolar (van der Waals) intermolecular interactions and ligand's hydrophobicity are the most important factors affecting binding affinities to the S1 subsite of a SVTLE isolated from the venom of Lachesis muta muta (Lmm-TLE). Consequently, we have proposed that S1 subsite lipophilicity may be used to sort binding affinities of trypsin-like enzymes to small molecules by showing that the inhibitory potency of several S1-directed compounds follows subsite lipophilicity among Lmm-TLE and other three homologous proteases. Noteworthy, in the course of our analyses we determined that thrombin's S1 subsite should, in fact, be considered less lipophilic than that of trypsin if we account for the presence of the sodium-controlled water channel communicating with the S1 subsite in the coagulant enzyme.

Snake venom thrombin-like enzymes: from reptilase to now.

The snake venom thrombin-like enzymes (SVTLEs) comprise a number of serine proteases functionally and structurally related to thrombin. Until recently, only nine complete sequences of this subgroup of the serine protease family were known. Over the past 5 years, the primary structure of several SVTLEs has been characterized, and now this family includes several members. Of particular interest is their possible use in pathologies such as thrombosis. The aim of the present review is to summarize the state of the art concerning the evolutionary, structural and biological features of the SVTLEs.

High-level expression of a soluble snake venom enzyme, gloshedobin, in E. coli in the presence of metal ions.

The mature gene of gloshedobin, a snake venom thrombin-like enzyme from the snake, Gloydius shedaoensis, was cloned and expressed in strain E. coli BL21 (DE3). Having been induced by IPTG, the recombinant gloshedobin was in both soluble and insoluble forms. To avoid inclusion body formation, expression was optimized at 25 degrees C. Furthermore, a 50% increase in solubilization of the target protein was obtained by adding 0.1 mM Mg2+ to the medium. The purified recombinant gloshedobin gave a 44 kDa band on SDS-PAGE gel.