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Cloning and functional expression of B chains of beta-bungarotoxins from Bungarus multicinctus (Taiwan banded krait).

ABSTRACT: The cDNA species encoding the B chains (B1 and B2) of beta-bungarotoxins (beta-Bgt) were constructed from the cellular RNA isolated from the venom glands of Bungarus multicinctus (Taiwan banded krait). The deduced amino acid sequences of the B chains were different from those determined previously by a protein sequencing technique. One additional Arg residue is inserted between Val-19 and Arg-20 of the B1 chain. Similarly the insertion of one additional Val residue between Val-19 and Arg-20 of the B2 chain is noted. Thus the B chains should comprise 61 amino acid residues. Moreover, the residues at positions 44-46 are Gly-Asn-His, in contrast with a previous result showing the sequence His-Gly-Asn. Instead of Asp, the residues at positions 41 and 43 are Asn. The B chain was subcloned into the expression vector pET-32a(+) and transformed into Escherichia coli strain BL21(DE3). The recombinant B chain was expressed as a fusion protein and purified on a His-Bind resin column. The yield of affinity-purified fusion protein was increased markedly by replacing Cys-55 of the B chain with Ser. However, the isolated B(C55S) chain became insoluble in aqueous solution after removal of the fused protein from the affinity-purified product, suggesting that protein-protein interactions might be crucial for stabilizing the structure of the B chain. The B(C55S) chain fusion protein showed activity in blocking the voltage-dependent K+ channel, but did not inhibit the binding of beta-Bgt to synaptosomal membranes. These results, together with the finding that modification of His-48 of the A chain of beta-Bgt caused a marked decrease in the ability to bind toxin to its acceptor proteins, suggest that the B chain is involved in the K+ channel blocking action observed with beta-Bgt, and that the binding of beta-Bgt to neuronal receptors is not heavily dependent on the B chain.

SUBMITTER: Wu PF

PROVIDER: S-EPMC1219665 | biostudies-other | 1998 Aug

REPOSITORIES: biostudies-other

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Project description:Backgroundβ-defensins have attracted considerable research interest because of their roles in protecting hosts from various pathogens. This study was conducted to investigate the expression profiles of the porcine β-defensin 114 (PBD114) in different breeds and in response to infections. Moreover, the function of PBD114 protein was partially investigated.MethodsSix Tibetan pigs (TP) and six DLY (Duroc×Landrace×Yorkshire) pigs were slaughtered to explore the expression profiles of PBD114 in different breeds and tissues. For infection models, sixteen DLY pigs were divided into two groups and challenged either with sterile saline or E. coli K88. The recombinant protein PBD114 (rPBD114) was obtained by using a heterologous expression system in E. coli.ResultsPBD114 gene was highly expressed in tissues such as the intestine, liver, spleen, and thymus. Interestingly, the expression level of PBD114 gene was higher in the TP pigs than in the DLY pigs (P < 0.05), and was significantly elevated upon E. coli K88 challenge (P < 0.05). The nucleotide sequences of PBD114 from Tibetan and DLY pigs was identical, and both showed a 210-bp open reading frame encoding a 69-amino acid mature peptide. To explaore the function of PBD114 protein, PBD114 gene was successfully expressed in E. coli Origami B (DE3) and the molecular weight of the rPBD114 was estimated by SDS-PAGE to be 25 kDa. The rPBD114 was purified and mass spectrometry verified the protein as PBD114. Importantly, rPBD114 showed antimicrobial activities against E. coli DH5α and E. coli K88, and the minimal inhibitory concentrations (MICs) were 64 and 128 μg/mL, respectively. Hemolytic and cytotoxicity assays showed that rPBD114 did not affect cell viability under physiological concentrations.ConclusionsPBD114 is an infection response gene that is differentially-expressed between different porcine breeds and tissues. The antimicrobial activity of PBD114 protein, against pathogens such as the E. coli K88, suggested that it may serve as a candidate for the substitution of conventionally used antibiotics.

Project description:BackgroundEndo-1,4-β-mannanase is an enzyme that can catalyze the random hydrolysis of β-1, 4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans and has a number of applications in different biotechnology industries. Penicillium oxalicum is a powerful hemicellulase-producing fungus (Bioresour Technol 123:117-124, 2012); however, few previous studies have focused on the cloning and expression of the endo-1,4-β-mannanase gene from Penicillium oxalicum.ResultsA gene encoding an acidophilic thermostable endo-1,4-β-mannanase (E.C. 3.2.1.78) from Penicillium oxalicum GZ-2, which belongs to glycoside hydrolase family 5, was cloned and successfully expressed in Pichia pastoris GS115. A high enzyme activity (84.4 U mL(-1)) was detected in the culture supernatant. The recombinant endo-1,4-β-mannanase (rPoMan5A) was tagged with 6 × His at its C-terminus and purified using a Ni-NTA Sepharose column to apparent homogeneity. The purified rPoMan5A showed a single band on SDS-PAGE with a molecular mass of approximately 61.6 kDa. The specific activity of the purified rPoMan5A was 420.9 U mg(-1) using locust bean gum as substrate. The optimal catalytic temperature (10 min assay) and pH value for rPoMan5A are 80 °C and pH 4.0, respectively. The rPoMan5A is highly thermostable with a half-life of approximately 58 h at 60 °C at pH 4.0. The K m and V max values for locust bean gum, konjac mannan, and guar gum are 7.6 mg mL(-1) and 1425.5 μmol min(-1) mg(-1), 2.1 mg mL(-1) and 154.8 μmol min(-1) mg(-1), and 2.3 mg mL(-1) and 18.9 μmol min(-1) mg(-1), respectively. The enzymatic activity of rPoMan5A was not significantly affected by an array of metal ions, but was inhibited by Fe(3+) and Hg(2+). Analytical results of hydrolytic products showed that rPoMan5A could hydrolyze various types of mannan polymers and released various mannose and manno-oligosaccharides, with the main products being mannobiose, mannotriose, and mannopentaose.ConclusionOur study demonstrated that the high-efficient expression and secretion of acid stable and thermostable recombinant endo-1, 4-β-mannanase in Pichia pastoris is suitable for various biotechnology applications.

Project description:BACKGROUND: Plasmodium vivax is the most widespread human malaria parasite. However, genetic information about its pathogenesis is limited at present, due to the lack of a reproducible in vitro cultivation method. Sequencing of the Plasmodium vivax genome suggested the presence of a homolog of deoxyhypusine synthase (DHS) from P. falciparum, the key regulatory enzyme in the first committed step of hypusine biosynthesis. DHS is involved in cell proliferation, and thus a valuable drug target for the human malaria parasite P. falciparum. A comparison of the enzymatic properties of the DHS enzymes between the benign and severe Plasmodium species should contribute to our understanding of the differences in pathogenicity and phylogeny of both malaria parasites. RESULTS: We describe the cloning of a 1368 bp putative deoxyhypusine synthase gene (dhs) sequence from genomic DNA of P. vivax PEST strain Salvador I (Accession number AJ549098) after touchdown PCR. The corresponding protein was expressed and functionally characterized as deoxyhypusine synthase by determination of its specific activity and cross-reactivity to human DHS on a Western blot. The putative DHS protein from P. vivax displays a FASTA score of 75 relative to DHS from rodent malaria parasite, P. yoelii, and 74 relative to that from the human parasite, P. falciparum strain 3D7. The ORF encoding 456 amino acids was expressed under control of IPTG-inducible T7 promoter, and expressed as a protein of approximately 50 kDa (theoretically 52.7 kDa) in E. coli BL21 DE3 cells. The N-terminal histidine-tagged protein was purified by Nickel-chelate affinity chromatography under denaturing conditions. DHS with a theoretical pI of 6.0 was present in both eluate fractions. The specific enzymatic activity of DHS was determined as 1268 U/mg protein. The inhibitor, N-guanyl-1, 7-diaminoheptane (GC7), suppressed specific activity by 36-fold. Western blot analysis performed with a polyclonal anti-human DHS antibody revealed cross-reactivity to DHS from P. vivax, despite an amino acid identity of 44% between the proteins. CONCLUSION: We identify a novel DHS protein in the more benign malaria parasite,P. vivax, on the basis of specific enzymatic activity, cross-reactivity with a polyclonal antibody against human DHS, and amino acid identity with DHS homologs from the rodent malaria parasite, P. yoelii, and human P. falciparum strains.

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Cloning and functional expression of B chains of beta-bungarotoxins from Bungarus multicinctus (Taiwan banded krait).

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