Expression, refolding, and activity of a recombinant nonhemorrhagic snake venom metalloprotease

Snake venoms are rich sources of proteases that strongly affect the vascular system, by promoting blood coagulation, hemorrhage, and fibrinolysis. Hemorrhagic activity is mostly due to the enzymatic action of metalloproteases on capillary basement membrane components, such as collagen IV, laminin, and fibronectin. A few low-molecular-weight snake venom metalloproteases (svMP) have been described as being devoid of hemorrhagic activity, but they have strong direct-acting fibrinolytic activity that could be very helpful in thrombosis therapy. We have developed an expression system for production of a recombinant svMP from a cDNA (ACLPREF) coding for a small metalloprotease (ACLF) with three disulfide bonds from an Agkistrodon contortrix laticinctus (broad-banded copperhead) venom gland cDNA library. The mature protein-coding region was amplified by PCR and subcloned into the pET28a vector, and the resulting plasmid was used to transform BL21(DE3) Escherichia coli cells. Culture of the transformants at either 37 or 20 degrees C led to the overexpression of an insoluble and inactive 30-kDa protein after 1.0 mM IPTG induction. The expressed protein (rACLF) was recovered from inclusion bodies with 6 M buffered urea solution and purified on a nickel-Sepharose column followed by gel filtration chromatography, both under denaturing conditions. After treatment with dithiothreitol, protein refolding was performed by gradual removal of the denaturing agent by dialysis. The refolded recombinant protein was active in fibrin-agarose plates. The purified protein achieved a conformation similar to that of the native enzyme as judged by circular dichroism analysis.     

Expression,refolding, and in vitro activation of a recombinant snake venom pro-metalloprotease

Metalloproteases comprise a family of Zn(2+)-endopeptidases that degrade most components of the extracellular matrix. Snake venoms are rich sources of metalloproteases, which also digest fibrinogen as well as fibrin, and in some cases, induce hemorrhage. A few low-molecular weight snake venom metalloproteases (svMPs) have been described as being devoid of hemorrhagic activity, but they have strong direct-acting fibrinolytic activity. This property could be very helpful in thrombosis therapy. ACLF is a fibrinolytic, non-hemorrhagic metalloprotease from the venom of the North American snake Agkistrodon contortrix laticinctus. We have developed an expression system for production of a recombinant pro-ACLF from a clone (ACLPREF) isolated from a venom gland cDNA library. The coding region including both the pro-enzyme domain and the mature protein domain was amplified by PCR and subcloned into the pET28a vector and the new plasmid was used to transform BL21(DE3) Escherichia coli cells. Culture of the transformants at 37 degrees C led to the overexpression of an insoluble 48kDa protein after induction with 1.0mM IPTG. The expressed protein was recovered from inclusion bodies with 6M buffered urea and purified by affinity chromatography under denaturing conditions. After dithiothreitol treatment, protein refolding was performed by gradual removal of the denaturing agent by dialysis. The pro-enzyme underwent auto-activation during refolding and it was active on fibrinogen and on a synthetic substrate. To control the activation step, the denaturing agent was rapidly removed to keep the protein in an unprocessed form, followed by later addition of Ca(2+) and Zn(2+) ions. This allowed controlling the enzyme activation, when it is needed.     

Fibrinogenolytic proteases isolated from the snake venom of Taiwan habu: serine proteases with kallikrein-like and angiotensin-degrading activities

Two venom proteases with fibrinogenolytic activity were isolated from the venom of Taiwan habu (Trimeresurus mucrosquamatus), one major crotalid snake species in Taiwan. The purified enzymes showed a strong beta-fibrinogenolytic activity, cleaving the beta-chain of fibrinogen molecules specifically. They also showed strong kallikrein-like activity in vitro, releasing bradykinin from kininogen. The purified enzymes did not coagulate human plasma, yet decreasing fibrinogen levels in plasma and prolonging bleeding without formation of fibrin clots, indicating that both proteases have specificities different from thrombin and the thrombin-like proteases of snake venom reported previously. They also exhibit amidase activity against N-benzoyl-Pro-Phe-Arg-p-nitroanilide, which is a specific synthetic substrate for kallikrein-like proteases. Their stability at high temperatures was examined and found to be more stable when compared with ancrod and thrombin. Intravenous injection of either protease was shown to lower blood pressure in experimental rats. Most noteworthy is the observation that the proteases can cleave angiotensin I and release bradykinin from plasma kininogen in vitro, which is a strong vasodilator and probably responsible for the in vivo hypotensive effect of these venom proteases.     

Isolation and characterization of a protein C activator from the moccasin snake venom

The protein C activator detectable in the venom of the Southern Copperhead snake (Agkistrodon contortrix contortrix) was isolated by a combination of chromatofocusing on PBE-94 in the range pH9-7 and gel-filtration on Sephadex G-100 column. The peak protein C activator from Sephadex G-100 column appeared as double diffuse bands with apparent molecular weight of 37,700 and 31,400 after electrophoresis in the presence of sodium dodecylsulfate and 2-mercaptoethanol. The isolated enzyme does not clot human fibrinogen and when mixed with normal plasma generates activity of Protein C. It can be used for the measurement of protein C functional activity.     

Evidence for heterogeneous forms of the snake venom metalloproteinase jararhagin: a factor contributing to snake venom variability

The reprolysin subfamily of metalloproteinases includes snake venom metalloproteinases (SVMP) and mammalian disintegrin/metalloproteinase. These proteins are synthesized as zymogens and undergo proteolytic processing resulting in a variety of multifunctional proteins. Jararhagin is a P-III SVMP isolated from the venom of Bothrops jararaca. In crude venom, two forms of jararhagin are typically found, full-length jararhagin and jararhagin-C, a proteolytically processed form of jararhagin that is composed of the disintegrin-like and cysteine-rich domains of jararhagin. To better understand the structural and mechanistic bases for these forms of jararhagin in the venom of B. jararaca and the source of venom complexity in general, we have examined the jararhagin forms isolated from venom and the autolysis of isolated jararhagin under the conditions of varying pH, calcium ion concentration, and reducing agents. From our results, jararhagin isolated from venom appears as two forms: a predominant form that is stable to in vitro autolysis and a minor form that is susceptible to autolysis under a variety of conditions including alkaline pH, low calcium ion concentrations, or reducing agent. The autolysis site for production of jararhagin-C from isolated jararhagin was different from that observed for jararhagin-C as isolated from crude venom. Taken together, these data lead us to the conclusion that during the biosynthesis of jararhagin in the venom gland at least three forms are present: one form which is rapidly processed to give rise to jararhagin-C, one form which is resistant to processing in the venom and autolysis in vitro, and one minor form which is susceptible to autolysis under conditions that promote destabilization of its structure. The presence of these different forms of jararhagin contributes to greater structural and functional complexity of the venom and may be a common feature among all snake venoms. The biological and biochemical features in the venom gland responsible for these jararhagin isoforms are currently under investigation.     

Molecular characterization of BjussuSP-I, a new thrombin-like enzyme with procoagulant and kallikrein-like activity isolated from Bothrops jararacussu snake venom

A thrombin-like enzyme, named BjussuSP-I, isolated from Bothrops jararacussu snake venom, is an acidic single-chain glycoprotein with M(r)=61,000, pI approximately 3.8 and 6% sugar. BjussuSP-I shows high proteolytic activity upon synthetic substrates, such as S-2238 and S-2288. It also shows procoagulant and kallikrein-like activity, but is unable to act on platelets and plasmin. These activities are inhibited by specific inhibitors of this class of enzymes. The complete cDNA sequence of BjussuSP-I with 696bp encodes open reading frames of 232 amino acid residues, which conserve the common domains of thrombin-like serine proteases. BjussuSP-I shows a high structural homology with other thrombin-like enzymes from snake venoms where common amino acid residues are identified as those corresponding to the catalytic site and subsites S1, S2 and S3 already reported. In this study, we also demonstrated the importance of N-linked glycans to improve thrombin-like activity of BjussuSP-I toxin.     

Expression of cDNA for batroxobin, a thrombin-like snake venom enzyme

The cloned cDNA for batroxobin has been expressed in E. coli. Batroxobin could only be obtained as intracellular aggregates of fusion proteins, fused with a small peptide. To obtain the mature batroxobin, the recognition sequence for thrombin was inserted between the peptide and the mature batroxobin. This fusion protein accumulated in an insoluble form and could easily be purified. After site-specific cleavage of the fusion protein with thrombin, recombinant batroxobin was isolated by preparative electrophoresis. Batroxobin with enzymatic activity was obtained by the refolding of recombinant batroxobin.     

Preparation of Thermus thermophilus holo-chaperonin-immobilized microspheres with high ability to facilitate protein refolding

Carboxylated poly(styrene/acrylamide) (P(St/AAm)-H) microspheres with different acrylamide contents were prepared by emulsifier-free emulsion polymerization. Thermus thermophilus holo-chaperonin (cpn) was covalently immobilized onto these microspheres with high yield. The T. thermophilus holo-cpn-immobilized microspheres were used for refolding of guanidine hydrochloride (Gdn-HCl)-denatured enzymes and showed sufficiently high ability to facilitate refolding of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (G6PD) and pig heart lactate dehydrogenase (LDH) at 30 degrees C and Bacillus stearothermophilus LDH at 60 degrees C. The specific ability of T. thermophilus holo-cpn-immobilized microspheres increased with increasing immobilization amount and reached plateau at around 10-15 mg/m(2). When the immobilization amounts of T. thermophilus holo-cpn were approximately 10 mg/m(2), the microspheres retained sufficiently high ability to facilitate protein refolding during repeated use. Therefore, the P(St/AAm)-H microspheres on which approximately 10 mg/m(2) of T. thermophilus holo-cpn is immobilized are very effective for refolding of various (Gdn-HCl)-denatured enzymes over a wide temperature range.     

Identification of a kallikrein-like latent serine protease in human erythrocyte membranes

We have discovered and characterized a kallikrein-like latent serine protease in intact human erythrocytes and ghosts. The enzyme is activatable by trypsin. The solubilized enzyme has esterolytic activity with a pH optimum of 9; but the membrane-associated activity increases almost linearly up to pH 10. The activated enzyme releases kinin from bovine low molecular weight kininogen. Enzyme activity is inhibited by TosLysCH2Cl , phenylmethylsulfonyl fluoride, aprotinin and amiloride, and weakly by soybean or lima bean trypsin inhibitor. It is inhibited by Co2+, Zn2+ and Mn2+ but is stimulated by Fe2+, deoxycholate and phospholipase A2. An erythrocyte membrane protein (Mr = 88,000) with an active site serine residue was identified with [14C]-diisopropylphosphorofluoridate labeling. Consistent with the finding of tryptic activation of the latent erythrocyte serine protease, trypsin treatment reduced the density of labeling of this protein and revealed a lower molecular weight form (Mr = 64,000). Possible relationships between the activity of this newly identified serine protease and events such as erythrocyte membrane ion fluxes might be of interest.     

Expression of human insulin-like growth factor I in bacteria: use of optimized gene fusion vectors to facilitate protein purification

Several fusions between the gene for human insulin-like growth factor I (IGF-I) and the genes for different IgG-binding fragments of staphylococcal protein A were assembled and compared regarding expression, secretion, and purification of the peptide hormone. After IgG affinity purification of the fusion proteins from the growth medium of Staphylococcus aureus or Escherichia coli, native IGF-I was released by cleavage of an Asn-Gly peptide bond with hydroxylamine. An optimized expression system based on a modified synthetic IgG-binding domain (z), resistant to hydroxylamine, gave the highest yield of fusion protein. After cleavage, the hormone could be separated from the IgG-binding moiety and from noncleaved fusion protein by a second passage through the IgG affinity column. The biological activity and the purity of the IGF-I obtained were confirmed by a radioreceptor assay, N-terminal sequence analysis, polyacrylamide gel electrophoresis, isoelectric focusing, and high-performance liquid chromatography.     

Inhibitor-assisted refolding of protease: a protease inhibitor as an intramolecular chaperone

Pleurotus ostrearus proteinase A inhibitor 1 (POIA1), which was discovered as a protease inhibitor, is unique in that it shows sequence homology to the propeptide of subtilisin, which functions as an intramolecular of a cognate protease. In this study, we demonstrate that POIA1 can function as an intramolecular chaperone of subtilisin by in vitro and in vivo experiments. The specific cleavage between POIA1 and the mature region of subtilisin BPN' occurred in a refolding process of a chimera protein, and Bacillus cells transformed with a chimera gene formed a halo on a skim milk plate. The mutational analyses of POIA1 in the chimera protein suggested that the tertiary structure of POIA1 is required for such a function, and that an increase in its ability to bind to subtilisin BPN' makes POIA1 a more effective intramolecular chaperone.     

Adaptive evolution in the snake venom Kunitz/BPTI protein family

Snake venoms are rich sources of serine proteinase inhibitors that are members of the Kunitz/BPTI (bovine pancreatic trypsin inhibitor) family. However, only a few of their gene sequences have been determined from snakes. We therefore cloned the cDNAs for the trypsin and chymotrypsin inhibitors from a Vipera ammodytes venom gland cDNA library. Phylogenetic analysis of these and other snake Kunitz/BPTI homologs shows the presence of three clusters, where sequences cluster by functional role. Analysis of the nucleotide sequences from the snake Kunitz/BPTI family shows that positive Darwinian selection was operating on the highly conserved BPTI fold, indicating that this family evolved by gene duplication and rapid diversification.     

Expression of the murine leukemia virus protease in fusion with maltose-binding protein in Escherichia coli

The protease of murine leukemia virus (MLV) was cloned into pMal-c2 vector, expressed in fusion with maltose-binding protein (MBP), and purified to homogeneity after Factor Xa cleavage of the chimeric protein. Substantial degradation of the fusion protein was observed during expression, which severely diminished the yield. The degree of degradation of the fusion protein was even more pronounced when a single-chain form of the MLV protease was cloned after the gene coding for MBP. To increase the yield, a hexahistidine tag with an additional Factor Xa cleavage site was cloned after the protease and nickel chelate affinity chromatography was used as the first purification step. The modified procedure resulted in substantially higher yield as compared to the original procedure. The degradation of hexahistidine-tagged active site mutant MLV protease was very low and comparable to that obtained with hexahistidine-tagged MBP, but purified MLV protease alone was not able to degrade purified MBP, suggesting that during expression the active MLV protease may activate bacterial proteases which appear to be responsible for the degradation of the fusion proteins.     

Isolation and characterization of "Reprotoxin", a novel protein complex from Daboia russelii snake venom

In snake venoms, non-covalent protein-protein interaction leads to protein complexes with synergistic and, at times, distinct pharmacological activities. Here we describe a new protein complex containing phospholipaseA(2) (PLA(2)), protease, and a trypsin inhibitor. It is isolated from the venom of Daboia russelii by gel permeation chromatography, on a Sephadex G-75 column. This 44.6kDa complex exhibits only phospholipase A(2) activity. In the presence of 8M urea it is well resolved into protease (29.1kDa), PLA(2) (13kDa), and trypsin inhibitor (6.5kDa) peaks. The complex showed an LD(50) of 5.06mg/kg body weight in mice. It inhibited the frequency of spontaneous release of neurotransmitter in hippocampal neurons. It also caused peritoneal bleeding, and edema in the mouse foot pads. Interestingly, the complex caused degeneration of both the germ cells and the mouse Leydig cells of mouse testis. A significant reduction in both the diameter of the seminiferous tubules and height of the seminiferous epithelia were observed following intraperitoneal injection of the sub-lethal dose (3mg/kg body weight). This effect of the toxin is supported by the increase in the activities of acid and alkaline phosphatases and the nitric oxide content in the testes, and a decrease in the ATPase activity. Because of its potent organ atrophic effects on the reproductive organs, the toxin is named "Reprotoxin". This is the first report demonstrating toxicity to the reproductive system by a toxin isolated from snake venom.     

The snake venom protein botrocetin acts as a biological brace to promote dysfunctional platelet aggregation

Botrocetin is a snake venom protein that enhances the affinity of the A1 domain of plasma von Willebrand factor (vWF) for the platelet receptor glycoprotein Ibalpha (GPIbalpha), an event that contributes to bleeding and host death. Here we describe a kinetic and crystallographic analysis of this interaction that reveals a novel mechanism of affinity enhancement. Using high-temporal-resolution microscopy, we show that botrocetin decreases the GPIbalpha off-rate two-fold in both human and mouse complexes without affecting the on-rate. The key to this behavior is that, upon binding of GPIbalpha to vWF-A1, botrocetin prebound to vWF-A1 makes no contacts initially with GPIbalpha, but subsequently slides around the A1 surface to form a new interface. This two-step mechanism and flexible coupling may prevent adverse alterations in on-rate of GPIbalpha for vWF-A1, and permit adaptation to structural differences in GPIbalpha and vWF in several prey species.