Expression of functional pentameric heat-labile enterotoxin B subunit of Escherichia coli in Saccharomyces cerevisiae.
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.
Lim
Jung-Gu
JG
Institute for Molecular Biology and Genetics, Research Center of Bioactive Materials, Chonbuk National University, Jeonju 561-756, Korea.
Kim
Jung-Ae
JA
Chung
Hea-Jong
HJ
Kim
Tae-Geum
TG
Kim
Jung-Mi
JM
Lee
Kyung-Ryul
KR
Park
Seung-Moon
SM
Yang
Moon-Sik
MS
Kim
Dae-Hyuk
DH
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*
D004768:Enterotoxins; D021382:Protein Sorting Signals; D021122:Protein Subunits; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C034993:ganglioside receptor; D000681:Amylases