No abstract

Although the Escherichia coli heat-labile enterotoxin B subunit (LTB) has already been expressed in several different systems, including prokaryotic and eukaryotic organisms, studies regarding the synthesis of LTB into oligomeric structures of pentameric size in the budding yeast Saccharomyces cerevisiae have been limited. Therefore, this study used a functional signal peptide of the amylase 1A protein from rice to direct the yeast-expressed LTB towards the endoplasmic reticulum to oligomerize with the expected pentameric size. The expression and assembly of the recombinant LTB were confirmed in both the cellfree extract and culture media of the recombinant strain using a Western blot analysis. The binding of the LTB pentamers to intestinal epithelial cell membrane glycolipid receptors was further verified using a GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). On the basis of the GM1-ELISA results, pentameric LTB proteins comprised approximately 0.5-2.0% of the total soluble proteins, and the maximum quantity of secreted LTB was estimated to be 3 mg/l after a 3-day cultivation period. Consequently, the synthesis of LTB monomers and their assembly into biologically active oligomers in a recombinant S. cerevisiae strain demonstrated the feasibility of using a GRAS microorganism-based adjuvant, as well as the development of carriers against mucosal disease.

^Äëíê~Åí= The neutralizing epitope (K-COE) of the spike protein from a Korean strain of porcine epidemic diarrhea virus (PEDV) has been shown to prevent and foster an immune response to PED, when orally adjusted. The cell surface of the budding yeast, p~ÅÅÜ~êçãóÅÉë=ÅÉêÉîáëá~É, was engineered to anchor the K-COE on the outer layer of the cell, and consequently, the altered yeast was applied as a dietary complement for animal feed, with immunogenic functions. In this study, the K-COE gene (hJ`lb) of the Korean strain of PEDV with the signal peptide of rice amylase 1A (o~ãóN^), was fused with the gene encoding the carboxyterminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin, a mating associated protein that is anchored covalently to the cell wall. The glyceraldehyde-3-phosphate dehydrogenase (dma) promoter was selected in order to direct the expression of the fusion construct, and the resulting recombinant plasmid was then introduced into pK=ÅÉêÉîáëá~É. The surface display of K-COE was visualized via confocal microscopy using a polyclonal antibody against K-COE as the primary antibody, and FITC (fluorescein isothiocyanate)-conjugated goat anti-mouse IgG as the secondary antibody. The display of the K-COE on the cell surface was further verified via Western blot analysis using the cell wall fraction after the administration of α-1,3-glucanase/PNGase F/β-mannosidase treatment. © KSBB

A DnaJ-like gene, Cpdj1, a molecular chaperone and regulator of Hsp70 in Cryphonectria parasitica, was characterized. The protein product of Cpdj1 gene consists of 379 amino acids with a predicted molecular mass of 40.6 kDa and a pI of 7.79. The deduced protein sequence revealed preservation of the conserved hall-mark J-region and exhibited high homolo y to all known DnaJ-like proteins. Disruption of the Cpdj1 gene resulted in slow growth and produced colonies characterized by retarded growth and deep orange color. Accordingly, reduced virulence of the Cpdj1-null mutant was observed. This reduced growth rate was magnified when the Cpdj1-null mutant was cultured under heat-stress conditions. Reduced conidiation was also observed in the Cpdj1-null mutant, indicating that Cpdj1 gene, although not essential for cell viability, is required for appropriate cellular processes including growth and sporulation. Northern analysis showed that Cpdj1 was constitutively expressed, and when the culture was subject to high temperature, a strong induction of the transcript was observed. No significant difference in the expression and induction pattern of Cpdj1 was observed between virus-free EP155/2 and virus-infected hypovirulent UEP1 strains. However, further severe defects in mycelia growth and conidiation were observed in the hypovirus-infected Cpdj1-null mutant suggesting that the presence of Cpdj1 is required for mycelia growth and sporulation of the hypovirus-infected strain.

Trichoderma species are among the primary producers of β-mannanase, an enzyme that catalyses the hydrolysis of β-1, 4-glycosidic linkages in mannans and heteromannans. In this study, a Trichoderma species producing high mannanase activity was identified as Trichoderma longibrachiatum based on sequence analysis of its rDNA internal transcribed spacer region. The open reading frame of the gene encoding for β-mannanase of T. longibrachiatum, man1 is 1,441 bp and is separated by two introns. The MAN1 amino acid sequence showed 95% identity to Trichoderma reesei β-mannanase. Domain analysis classified MAN1 as a member of glycosyl hydrolase family 5 and detected the presence of a carbohydrate-binding domain family 1 at its C-terminus. The recombinant mannanase, rMAN1, was successfully expressed as a ~60 kDa extracellular recombinant protein in Pichia pastoris and was verified via western blotting analyses. The specific activity of the purified rMAN1 was 1416.18 U/mg. The optimal rMAN1 activity was recorded at 55°C and pH 5. The enzyme was stable with 30 min preincubation at temperatures ranging from 4 to 50°C. The enzyme was stable at pH 4 to 7 but became progressively unstable at pH values below 3 and above 8. rMAN1 had a high affinity towards locust bean gum as a substrate, with a K m value of 0.95 mg/ml.