Expression of a synthetic E. coli heat-labile enterotoxin B sub-unit (LT-B) in maize Rachel Chikwamba 1,2 , Jennifer McMurray 1 , Huixia Shou 1 , Bronwyn Frame 1 , Sue Ellen Pegg 1,5 , Paul Scott 4 , Hugh Mason 3 and Kan Wang 1, * 1 Plant Transformation Facility, Department of Agronomy, Iowa State University, Ames, IA 50011–1010, USA; 2 Interdisciplinary Genetics Program, Iowa State University, Ames, IA 50011, USA; 3 Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, NY 14853–1801, USA; 4 USDA-ARS, Department of Agronomy, Iowa State University, Ames, IA 50011–1010, USA; 5 Current address: Western Illinois University, Macomb, IL 61455, USA; *Author for correspondence (e-mail: kanwang@iastate.edu; fax: 515–294-2299) Received 7 March 2002; accepted in revised form 28 June 2002 Key words: Functional antigen, Gene expression, LT-B, Zea mays Abstract We have produced the B subunit of the enterotoxigenic Escherichia coli (ETEC) heat-labile enterotoxin (LT-B) in transgenic maize seed. LT-B is a model antigen that induces a strong immune response upon oral administra- tion and enhances immune responses to conjugated and co-administered antigens. Using a synthetic LT-B gene with optimized codon sequence, we examined the role of promoters and the SEKDEL endoplasmic reticulum retention motif in LT-B accumulation in callus and in kernels. Two promoters, the constitutive CaMV 35S pro- moter and the maize 27 kDa gamma zein promoter, which directs endosperm-specific gene expression in maize kernels, regulated LT-B expression. Ganglioside-dependent ELISA analysis showed that using the constitutive promoter, maximum LT-B level detected in callus was 0.04% LT-B in total aqueous-extractable protein (TAEP) and 0.01% in R 1 kernels of transgenic plants. Using the gamma zein promoter, LT-B accumulation reached 0.07% in R 1 kernels. The SEKDEL resulted in increased LT-B levels when combined with the gamma zein promoter. We monitored LT-B levels under greenhouse and field conditions over three generations. Significant variability in gene expression was observed between transgenic events, and between plants within the same event. A maxi- mum of 0.3% LT-B in TAEPwas measured in R 3 seed of a transgenic line carrying CaMV 35S promoter/LT-B construct. In R 3 seed of a transgenic line carrying the gamma zein promoter/LT-B construct, up to 3.7% LT-B in TAEP could be detected. We concluded that maize seed can be used as a production system for functional anti- gens. Abbreviations: BSA – bovine serum albumin, CaMV 35S – Cauliflower Mosaic Virus 35S RNA Promoter, CTAB – cetyltrimethylammonium bromide, EDTA – ethylene-diamine tetraacetic acid, ELISA – enzyme-linked immu- nosorbent assay, G M1 – galactosyl-N-acetylgalactosamyl-sialyl-galactosylglucosyl ceramide, LT-B – heat-labile toxin from E. coli, PBS – phosphate-buffered saline, PMSF – phenylmethanesulfonyl fluoride, SDS-PAGE – so- dium dodecyl sulfate polyacrylamide gel electrophoresis, Tris – Tris hydroxymethyl aminomethane Introduction Recombinant DNA technology and advances in plant transformation have expanded the utility of plants be- yond the traditional uses of food, feed and fiber. Plants are now used as a source of proteins for indus- trial, pharmaceutical, and other purposes (Krebbers et al. 1993; Pen et al. 1993; Austin et al. 1994). Produc- tion of recombinant proteins in transgenic plants is emerging as a competitive and safe alternative to the traditional protein expression systems. This is partic- ularly relevant for the production of monoclonal anti- 253 Molecular Breeding 10: 253–265, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.