Expression of a functional recombinant C-type lectin-like protein lebecetin in the human embryonic kidney cells.

Lebecetin is an anticoagulant C-type lectin-like protein that was previously isolated from Macrovipera lebetina venom and described to consist of two subunits (alpha and beta). It was reported to potently prevent platelet aggregation by binding to glycoprotein Ib and to exhibit a broad spectrum of inhibitory activities on various integrin-mediated functions of tumor cells, including adhesion, proliferation, and cell migration. This study aimed to investigate the structure-function of lebecetin. Accordingly, the cDNA of each subunit was cloned and separately or jointly expressed in the human embryonic kidney cells using two vectors with different selectable tags. The immunofluorescence analysis of transfected cells revealed significant expression levels and co-localization of the two lebecetin subunits. The recombinant proteins were efficiently secreted and purified using metal-chelating affinity chromatography. We found that the Lebecetin alpha and beta subunits were produced as a mixture of homodimers and heterodimers and that the heterodimerization represents a prerequisite for functioning.

High-level expression, purification, characterization and structural prediction of a snake venom metalloproteinase inhibitor in Pichia pastoris.

Snake venom metalloproteinase inhibitor BJ46a is from the serum of the venomous snake Bothrops jararaca. It has been proven to possess the capacity to inhibit matrix metalloproteinases (MMPs), likely based on its structural similarity to MMPs. This report describes the successful expression, purification, and characterization of the recombinant protein BJ46a in Pichia pastoris. Purified recombinant protein BJ46a was found to inhibit MMPs. Structural modeling was completed and should provide the foundation for further functional research. To our knowledge, this is the first report on the large scale expression of BJ46a, and it provides promise as a method for generation of BJ46a and investigation of its potential use as a new drug for treatment of antitumor invasion and metastasis.

Engineered mammalian vector to express EGFP-tagged proteins as biomarkers.

Due to its specialized post-translational machinery, mammalian cells represent an interesting and not fully explored system to express snake toxins. Therefore, in this work, we built up a new mammalian expression vector that enhances the feasibility to use mammalian cells to express proteins as biomarkers. Among the modifications, an Igκ signal peptide and a 6xHis tag were inserted into this vector in order to drive the protein to the supernatant and simplify its purification, respectively. In addition, to facilitate selection of high producing clones and also tag proteins which may function as a biomarker, the sequence of enhanced green fluorescent protein (EGFP) was added. The efficiency of the resulting vector (pToxEGFP) was tested by cloning and expressing the viper venom disintegrin echistatin (Ech) that due to its affinity to integrin αvß3 was tested as a molecular marker. Expression of EGFP-Ech was achieved in CHO-DXB11 cells resulting in a yield of 22 mg/L. The binding activity of this chimera protein was successfully achieved on human umbilical vein endothelial cells which highly express αvß3. The results indicate that pToxEGFP may constitute an efficient and versatile expression vector to express tagged proteins with potential biomarker activity.

Inhibition of proliferation of ECV304 cells by a disintegrin from Chinese green tree viper.

A novel disintegrin, stejnin, was purified from the Trimeresurus stejnegeri venom by gel filtration and reverse phase high performance liquid chromatography. The molecular weight of stejnin was determined to be 7428 Da by MALD-TOF MS analysis. The cDNA encoding the precursor of stejnin was cloned from the venom gland. From the deduced amino acid sequence, stejnin is composed of 71 amino acid residues contains the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Stejnin strongly inhibited ADP- and ristomycin-induced human platelet aggregation with IC50 of 45 and 50 nM, respectively. Stejnin also possessed potent inhibited cell proliferation of ECV304 cells.

Microvesicles in the venom of Crotalus durissus terrificus (Serpentes, Viperidae).

Microvesicles with electron-dense content are consistently observed by transmission electron microscopy on the luminal face of secretory cells of venom glands of viperid snakes. In this work, we evaluated their presence in Crotalus durissus terrificus venom glands and also in freshly collected venom. Microvesicles were found in the venom glands mainly in regions of exocytosis. They ranged from 40 to 80 nm in diameter. Freeze-fracture replicas of the glands revealed particles on the cytoplasmic leaflet (P-face) of these vesicles, suggesting that they carry transmembrane proteins. Vesicles separated by ultracentrifugation from cell-free venom were similar in size and structure to the microvesicles observed in the glands. A fine fuzzy coat surrounded each microvesicle. The function of these venom vesicles is still unknown, but they may contribute to inactivation of stored venom components, or their activation after the venom is released.

[Fermentation and purification of Echistatin fusion protein expressed in Escherichia coli].

Lots of studies of Echistatin (Ecs) have proved its wide use in many aspects. However, the low yield of Ecs has impeded the relative researches of the protein. To establish the high-level expression system of Ecs, the fermentation and purification process of Ecs fusion protein expressed in E. coli were optimized. The Ecs gene was introduced into vector pTXB1 and placed under the control of highly efficient T7 promoter system. The cloned Ecs gene was expressed in E. coli BL21 (DE3) as soluble form. The Ecs production and biomass accumulation were optimized by examining medium composition, point of induction and induction time in fed-batch fermentation. Biomass accumulation was greatly affected by medium gradient, reaching 50.3g/L in 2 x YT medium. Ecs production was found to increase to 35% of total protein with 75g/L biomass accumulation after induced for 4h. Purification of Ecs from supernatant of sonication was done using one-step chromatographic procedure with chitin affinity chromatography and DTF cleavage, resulting in yields of 28mg/L and > 90% purity. The bioactivity of purified Ecs was determined and the result showed that purified Ecs could inhibit the aggregation of platelet in vitro with similar bioactivity to wild Ecs. This optimized method is readily scaled up for the expression and purification of Ecs in sufficient quantities for further structural and biological studies and applications.

Factor X activator from Vipera lebetina venom is synthesized from different genes.

Vipera lebetina venom contains specific coagulant Factor X activator (VLFXA) that cleaves the Arg52-Ile53 bond in the heavy chain of human factor X. VLFXA is a glycoprotein that is composed of a heavy chain (HC) and two light chains (LC) linked by disulfide bonds. The complete amino acid sequences of the three chains of the factor X activator from V. lebetina snake venom are deduced from the nucleotide sequences of cDNAs encoding these chains. The full-length cDNA (2347 bp) sequence of the HC encodes an open reading frame (ORF) of 612 amino acids that includes signal peptide, propeptide and mature metalloproteinase with disintegrin-like and cysteine-rich domains. The light chain LC1 contains 123 and LC2 135 amino acid residues. Both light chains belong to the class of C-type lectin-like proteins. The N-termini of VLFXA chains and inner sequences of peptide fragments detected by liquid chromatography-electrospray ionization tandem mass spectrometry (LC MS/MS) from protein sequence are 100% identical to the sequences deduced from the cDNA. The molecular masses of tryptic fragments of VLFXA chains analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) also confirm the protein sequences deduced from the cDNAs. These are the first cloned factor X activator heavy and light chains. We demonstrate that the heavy and light chains are synthesized from different genes.

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.

Expression of gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis, in Escherichia coli.

Gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis, was expressed in Escherichia coli using expression vector pET-32a(+). The gene was expressed under T7 promotor with a fusion partner of Thx.Tag and a 6xHis.Tag at its 5' terminal. After induction by IPTG for 6 h, the recombinant enzyme was expressed in the cytoplasm. Expression at 25 degrees C gave twice the amount of recombinant gloshedobin in cytoplasm than at 37 degrees C.

Expression and processing of recombinant sarafotoxins precursor in Pichia pastoris.

Sarafotoxins are peptides isolated from the Atractaspis snake venom, with strong constrictor effect on cardiac and smooth muscle. They are structurally and functionally related to endothelins. The sarafotoxins precursor cDNA predicts an unusual structure 'rosary-type', with 12 successive similar stretches of sarafotoxin (SRTX) and spacer. In the present work, the recombinant precursor of SRTXs was sub-cloned and expressed in the yeast Pichia pastoris, and secreted to the culture medium. Characterization by SDS-PAGE, immunoblot, mass spectrometry and biological activity, suggests that intact precursor was expressed but processing into mature toxins also occurred. Furthermore, our results indicate that the correct proportion of sarafotoxin types as contained in the precursor, is obtained in the yeast culture medium. Contractile effects of the expressed toxins, on rat and Bothrops jararaca isolated aorta, were equivalent to 5x10(-10)M and 5x10(-11)M of sarafotoxin b, respectively. The enzymes responsible for the complete maturation of sarafotoxins precursor are still unknown. Our results strongly suggest that the yeast Pichia pastoris is able to perform such a maturation process. Thus, the yeast Pichia pastoris may offer an alternative to snake venom gland to tentatively identify the molecular process responsible for SRTXs release.

Ammodytoxin C gene helps to elucidate the irregular structure of Crotalinae group II phospholipase A2 genes.

Ammodytoxin C is a presynaptically neurotoxic phospholipase A2 (PLA2) expressed in the venom glands of Vipera ammodytes (subfamily Viperinae). The gene spans more than 4 kb and consists of five exons and four introns characteristic of group II phospholipase A2 genes. The first exon encodes the 5' untranslated region, the second exon encodes most of the signal peptide, while exons 3-5 encode three parts of the mature protein. Comparison of the Crotalinae and Viperinae PLA2 genes has shown that Crotalinae PLA2 retain the first intron in their mRNAs. The apparent cause of this retention is a deletion of 40 bp in the first exon of PLA2 genes of the subfamily Crotalinae, which prevents splicing of the first intron. Analysis of the secondary structure of the pre-mRNA of the ammodytoxin C gene has shown that the first exon is able to form an intra-exon hairpin which is absent in Crotalinae PLA2 pre-mRNAs. Our results indicate that this intra-exon hairpin structure is essential for the splicing of the retained first intron. Contrary to the predictions of the neutral theory of molecular evolution, the introns of all known snake venom PLA2 genes are conserved up to 90%, that is considerably more than the exons. Consequently it is proposed that highly conserved introns, in multigene families, which evolve under positive Darwinian selection, may have an important role in enabling homologous recombination.

Expression of fully active ammodytoxin A, a potent presynaptically neurotoxic phospholipase A2, in Escherichia coli.

A cDNA encoding the most presynaptically neurotoxic phospholipase A2, ammodytoxin A, from the venom of the long-nosed viper (Vipera ammodytes ammodytes) has been expressed in Escherichia coli. Ammodytoxin A was produced as a fusion protein with the 81 N-terminal residues of adenylate kinase followed by the tetrapeptide recognition site for factor Xa (IEGR) just preceding the first amino acid residue of the toxin. The fusion protein was expressed under the control of tac promoter without IPTG induction in the form of insoluble inclusion bodies. It was dissolved in guanidine hydrochloride, S-sulfonated and refolded in a reoxidation mixture including a reduced/oxidized glutathione redox couple. Ammodytoxin A was fully activated by limited hydrolysis with trypsin that preferentially cleaves the fusion protein at the factor Xa recognition site and purified by cation-exchange chromatography. The correct N-terminus was confirmed by protein sequencing. Recombinant ammodytoxin A has been proved to be indistinguishable from the native toxin in its enzymatic activity and toxicity.

Sarafotoxin expression in the bronchopulmonary tract: immunohistochemical occurrence and colocalization with endothelins.

The immunohistochemical occurrence of sarafotoxin (SRTX), a snake venom peptide under strong evolutionary control, was investigated in the pulmonary diffuse neuroendocrine system (PDNES) of newborn cats and rats. By applying the avidin-biotin-peroxidase complex method on serial lung sections, we have demonstrated its distribution and colocalization with different endothelin (ET) isoforms. A light microscopic study revealed apparent immunostaining for SRTX in neuronal components and smooth muscle tissue and in neuroepithelial bodies (NEB), while isolated neuroendocrine cells (NEC) remain unlabelled. Comparison of the SRTX reactivity pattern with that of different ET peptides on adjacent lung sections showed colocalization of SRTX-b with ET-3 in NEB, intrapulmonary ganglion cells and nerve fibres, on the one hand, and with ET-1 in airway and vascular smooth muscle cells, on the other. These findings, in addition to the remarkable functional and structural similarities between SRTX and ET peptides, suggest a common evolutionary origin and biological significance of sarafotoxin and endothelins. Moreover, this is the first time that a toxic peptide has been demonstrated in the PDNES.

Mutations of the alpha-galactosidase signal peptide which greatly enhance secretion of heterologous proteins by yeast.

The Saccharomyces carlsbergensis MEL1 gene encodes alpha-galactosidase (melibiase; MEL1) which is readily secreted by yeast cells into the culture medium. To evaluate the utility of the MEL1 signal peptide (sp) for the secretion of heterologous proteins by Saccharomyces cerevisiae, an expression vector was constructed which contains the MEL1 promoter and MEL1 sp coding sequence (MEL1sp). The coding sequences for echistatin (Echis) and human plasminogen activator inhibitor type 1 (PAI-1) were inserted in-frame with the MEL1sp. S. cerevisiae transformants containing the resulting expression vectors secreted negligible amounts of either Echis or PAI-1. Using site-directed mutagenesis, several mutations were introduced into the MEL1sp. Two mutations were identified which dramatically increased the secretion of both Echis and PAI-1 to levels similar to those achieved when using the yeast MF alpha 1 pre-pro secretory leader. In particular, increasing the hydrophobicity of the core region plus the addition of a positive charge to the N-terminal domain of the MEL1 sp resulted in the greatest increase in the secretion levels of those two proteins.

Expression and secretion of biologically active echistatin in Saccharomyces cerevisiae.

A synthetic gene coding for a platelet aggregation inhibitor, echistatin (ECS), was inserted into a Saccharomyces cerevisiae expression vector utilizing the alpha-mating factor pre-pro leader sequence and galactose-inducible promoter, GAL10. Cleavage of the pre-pro leader sequence in vivo results in the secretion of a properly processed recombinant ECS with the native N-terminal glutamic acid residue. Recombinant ECS was recovered from yeast supernatants and purified by reverse phase high performance liquid chromatography. Recombinant ECS expressed and purified from yeast was identical to native ECS in its ability to inhibit platelet aggregation.

High-level expression in Escherichia coli of a chemically synthesized gene for [Leu-28]echistatin.

A gene (Ecs) encoding a platelet aggregation inhibitor, echistatin (Ecs), has been chemically synthesized. Met at position 28 of the native protein was replaced by Leu in the recombinant Ecs. To express this synthetic gene in Escherichia coli, an expression vector, pJC264, was constructed by inserting portions of the E. coli cheB and cheY gene complex into the plasmid pUC13. High-level expression of the synthetic [Leu-28]Ecs was achieved by its fusion with the E. coli cheY gene in the expression vector. Recombinant [Leu-28]Ecs was liberated from the fusion protein by CNBr cleavage at the Met inserted between the CheY protein and [Leu-28]Ecs. The recombinant [Leu-28]Ecs was purified to homogeneity by reverse-phase high-performance liquid chromatography. The refolded [Leu-28]Ecs was identical to native Ecs in inhibiting platelet aggregation, suggesting that Met at position 28 is not essential for the biological activity of this platelet aggregation inhibitor.

Characterization of the messenger RNA population coding for components of viperid snake venom.

Total polyadenylated messenger RNA was prepared from the milked venom glands of the South African puff adder (Bitis arietans) and translated in an in vitro translation system. The products of cell free synthesis were immunoprecipitable with puff adder venom antiserum. Treatment of these cell free products with a dog pancreas microsomal membrane preparation demonstrated the presence of signal peptides. Northern blot hybridization of total puff adder venom gland mRNA to its complementary single stranded copy DNA revealed two discrete mRNA populations coding for the major components of puff adder venom. The relative amounts of these polyadenylated mRNA sequences changed during the onset of venom synthesis, suggesting mRNA deadenylation, general endonucleolytic RNA degradation and selective degradation of high molecular weight message components.

Asynchrony in the synthesis of secretory proteins in the venom gland of the snake Vipera palaestinae.

1. Venom of Vipera palastinae was subjected to isoelectrofocusing on polyacrylamide gel. The protein separation profiles were similar for different venom samples; more than 25 protein bands with a wide range of pI values could be demonstrated by this technique. 2. Labelled venom was obtained 8h after an intracardial injection of [3H]leucine. The relative radioactivities of four out of 12 main protein bands were significantly different in the venom synthesized during the 2nd day of the venom regeneration cycle as compared with the venom of the 4th day. The comparison was made in venom samples obtained from the two glands of the same snake at two different secretory stages. 3. It is concluded that the asynchronous synthesis of exportable proteins after the initiation of a new venom regeneration cycle is responsible for the non-parallel secretion of some venom proteins by the venom gland of Vipera palaestinae during the first few days after milking.