title: Expression of functional pentameric heat-labile enterotoxin B subunit of Escherichia coli in Saccharomyces cerevisiae.
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 cell-free 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.
authors.affiliation: Institute for Molecular Biology and Genetics, Research Center of Bioactive Materials, Chonbuk National University, Jeonju 561-756, Korea.
authors.forename: Jung-Gu
authors.initials: JG
authors.lastname: Lim
authors.forename: Jung-Ae
authors.initials: JA
authors.lastname: Kim
authors.forename: Hea-Jong
authors.initials: HJ
authors.lastname: Chung
authors.forename: Tae-Geum
authors.initials: TG
authors.lastname: Kim
authors.forename: Jung-Mi
authors.initials: JM
authors.lastname: Kim
authors.forename: Kyung-Ryul
authors.initials: KR
authors.lastname: Lee
authors.forename: Seung-Moon
authors.initials: SM
authors.lastname: Park
authors.forename: Moon-Sik
authors.initials: MS
authors.lastname: Yang
authors.forename: Dae-Hyuk
authors.initials: DH
authors.lastname: Kim
mesh_terms: D000681:Amylases / Q000235:genetics; D015152:Blotting, Northern; D015153:Blotting, Western; D054307:Enterotoxigenic Escherichia coli / Q000235:genetics*; D004768:Enterotoxins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D012275:Oryza / Q000235:genetics; D011485:Protein Binding; D021382:Protein Sorting Signals; D021122:Protein Subunits / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011956:Receptors, Cell Surface / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
chemical_list: D004768:Enterotoxins; D021382:Protein Sorting Signals; D021122:Protein Subunits; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C034993:ganglioside receptor; D000681:Amylases