Abstract
The main toxin of pathogenic Escherichia coli responsible for infant diarrhea and Traveler’s diarrhea is E. coli heat-labile enterotoxin (LT), and it is the cause of death for several hundred thousand children under 5 yr old every year, particularly in underdeveloped areas (1,2). LT is a bacterial adenosine phosphate (ADP)-ribosylating exotoxin composed of a single A subunit (27 kDa) and five monomeric B subunits (11.6 kDa). Subunit A is the toxin portion, causing diarrhea with ADP-ribosyl-transferase activity. Subunit B (LTB) is the receptor-binding site, which can bind to the GM1 ganglioside present on the eukaryotic cell membrane and has no pathogenic activity (3–5). LT holotoxin and its B subunit are highly immunogenic, invoking neutralizing antibody and blocking the receptor-binding activity and diarrhea-causing activity in immunized individuals (6,7). Furthermore, LTB has been shown to be the most powerful mucosal immunogen and mucosal adjuvant, markedly strengthening the immunoresponse to coadministered antigens (8,9). In addition, LTB has an important role in the activity and control of immunomodulation, whereby it will result in a Th1 reaction switching to a Th2 reaction (10,11). Consequently, much attention has been focused on LTB preparation using genetic engineering techniques. However, recombinant LTB (rLTB) has been expressed mainly in E. coli, in which the recombinant protein is secreted into the periplasm (see Note 1). To obtain the purified target protein, it is necessary to destruct the cell wall, which makes purification more difficult. Cholerae vibrio can express and efficiently secrete rLTB into the culture medium, and a low-pathogenic mutant strain has been used to prepare rLTB (12). Marine vibrio, an organism closely related to C. vibrio, is very weakly pathogenic to humans and is similar to C. vibrio in terms of its expression and secretion of rLTB, with the ability to express this foreign protein very efficiently and secrete the recombinant protein into the media in its natural conformation (13–15). Therefore, the use of M. vibrio for LTB expression is much more efficient. The methods for LTB expression in M. vibrio and purification of the secreted protein using Sephacryl S-100 chromatography are described.
Keywords
- Genetic Engineering Technique
- Marine Vibrio
- Eukaryotic Cell Membrane
- Purify Target Protein
- Infant Diarrhea
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Wang, Y., Si, L. (2005). Expression of Recombinant LTB Protein in Marine vibrio VSP60. In: Smales, C.M., James, D.C. (eds) Therapeutic Proteins. Methods in Molecular Biology™, vol 308. Humana Press. https://doi.org/10.1385/1-59259-922-2:043
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DOI: https://doi.org/10.1385/1-59259-922-2:043
Publisher Name: Humana Press
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