INTRODUCTION At the beginning of the 21 st century, cholera still remains an epidemic or endemic disease in much of the world (Sack et al., 2004). It is a severe diarrhoeal disease caused by the bacterium Vibrio cholerae. The bacterium secretes cholera toxin that is responsible for the profuse watery diarrhoea (Holmgren and Svnnerholm, 1994). The toxin, an 85 kDa protein, comprises of one cholera toxin A (CTA) and five B subunits (CTB) combined in an AB5 holotoxin. CTA is com- posed of two polypeptides (22 and 5 kDa) responsible for the toxic activity of the toxin (Lonnroth and Holmgren, 1973). The B subunit pentamers (55.6 kDa) are non-toxic and responsible for the binding of the toxin to ganglioside M1 (GM1) receptors present on the surface of intestinal epithe- lial cells (Spangler, 1992). CTB has many important scientific applications. There is a great deal of interest in the use of CTB as an adjuvant for vaccines targeted for delivery to the mucosa-associated lym- phatic tissues. On the other hand, CTB has been reported to elicit serum and secretory antibodies to antigens that are usually poor immunogens especially when presented togeth- er in chemically conjugated or genetically fused forms (Tochikubo and Yasuda, 2000). Other investigations have also suggested that CTB is an efficient mucosal carrier for the induction of peripheral immunologic tolerance (Sun et al., 1996). It is a candidate oral subunit vaccine for cholera. Oral administration of CTB effectively stimulates local and sys- temic immunologic responses, especially in humans (Jertborn et al., 1994, 2001). These responses are associated with high-level although relatively short-term (6 months), pro- tection against challenge or natural infection with Vibrio cholerae (Jertborn et al., 1988; Lebens et al., 1996). As most infections are acquired through the mucosa, it is logical to hypothesize that stimulation of mucosal immunity by vacci- nation is the most effective approach in preventing the ini- tial infection. The development of plant biotechnology has promoted the scientists to express foreign antigens in plant tissue as edible vaccine vehicles (Haq et al., 1995). CTB is among the first bacterial antigens expressed in edible plants such as potato and tomato for vaccine purposes (Jani et al., 2002). Transgenic plants are subjected to several disadvantages and limitations. Some concerns are: low-level expression of for- eign antigens, crop loss and spreading of pollen (Yu and lan- gridge, 2000). Another concern may be that the plant-pro- duced vaccines must be purified free of alkaloids and other toxic materials (Anderson, 1997). Whole recombinant yeast may offer a good alternative for this new generation of vaccines, as the whole recombi- nant yeast based vaccine against HIV has been examined recently (Stubbs et al., 2001). The yeast has remarkable potentials as vaccine vector. It is well established that whole recombinant yeast confers a potent adjuvant property against recombinant antigens (Rodríguez et al., 2002; Stubbs and Wilson, 2002). It enhances both cell mediated and Annals of Microbiology, 55 (2) 145-150 (2005) Expression of cholera toxin B subunit in Saccharomyces cerevisiae Mohsen ARZANLOU 1 , Abbas REZAEE 1 *, Nader SHAHROKHI 2 , Ahmad Zavaran HOSSINI 1 , Yoko YASUDA 3 , Kunio TOCHIKUBO 3 , Mohammad AHANGARZADEH REZAEE 1 1 Faculty of Medical Sciences, Tarbiat Modarres University, Tehran; 2 Department of Molecular Biology, Institute Pasteur of Iran, Tehran, Iran; 3 Department of Microbiology, Nagoya City University, Medical School, Mizuho-ku, Nagoya 467-8601, Japan Abstract - Cholera toxin, secreted by Vibrio cholerae, consists of A and B subunits. Cholera toxin B subunit (CTB) is used in many sci- entific researches. It has already been expressed in several bacterial and plant systems. In order to express CTB protein in Saccha- romyces cerevisiae, the expression plasmid pCTB83 was constructed by inserting ctxB gene in pYES2 shuttle vector. The new construct was transferred into S. cerevisiae cells and the ctxB gene was induced with 2% galactose. SDS-PAGE analysis showed the presence of CTB in yeast lysate and immunoblotting analysis of yeast total soluble protein indicated that the yeast-derived CTB protein was anti- genically indistinguishable from bacterial CTB protein. Quantitative ELISA showed that the maximum amount of CTB protein expressed in yeast was approximately 1.8% of total soluble protein. CTB is a subunit vaccine candidate against cholera. Since the whole recom- binant yeast has been introduced as a new vaccine formulation, expression of ctxB in S. cerevisiae may offer an effective and inex- pensive strategy to protect people against cholera in high-risk areas. Key words: Saccharomyces cerevisiae, CTB, gene expression, Vibrio cholera. * Corresponding Author: Postal Address: No. 106, Immam Zaman Alley, 95 square, Narmak, 16456, Tehran, Iran. Fax: +98 21 8013030; E-mail: abbasrezaee@yahoo.com, rezaee@modares.ac.ir