Poster: Metabolic Engineering

Abs # 997: Engineering the 2S methionine-rich protein of Brazil nut (Bertholletia excelsa H. B. K.) for reduced allergenic activity

Presenter:	Chen, Da-Ming , dmchen@zju.edu.cn
Authors	Chen, Da-Ming  (A) (C)  Wong, Ho-Wan  (A)   Lam, Christopher W.K. (B)   Sun, Samuel S.M. (A)
Affiliations:	(A): Department of Biology, The Chinese University of Hong Kong,Hong Kong (B): Department of Chemical Pathology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong (C): Department of Horticulture, College of Agriculture and Biotechnology, Zhejiang University, China

We had demonstrated earlier that the content of essential amino acid methionine in transgenic seed protein could be significantly increased through expressing a Brazil nut gene encoding the 2S albumin protein exceptionally rich in methionine (BNMRP). Unfortunately, the BNMRP was subsequently identified as a major allergen in Brazil nut, hampering the potential use of this protein in nutrition enhancement. As an initiator of this crop improvement strategy, we are interested in pursuing the allergenic issues related to genetically modified food crops. In this study, we identified at least 7 linear epitopes in the BNMRP by immunodetection and generation of serial deletions and overlapping recombinant peptides covering the entire length of this protein. The epitope with the strongest IgE reactivity was mapped to the C-terminal region of the large subunit. The methionine-rich regions in the large subunit were also found to harbor IgE-binding ability. Amino acid substitution analysis of the epitopes showed that in each epitope, mutation of key amino acids to alanine could significantly reduce or eliminate IgE binding. We also demonstrated that arginine residues in BNMRP play an important role in defining the IgE reactivity of BNMRP. A modified BNMRP with dramatically reduced IgE-binding activity and increased methionine content was produced successfully by mutating the identified epitopes. Transformation of this and further engineered BNMRP variants into plants such as Arabidopsis, tobacco, and soybean has been carried out. This study renews our effort to enhance the nutritional quality of plants, especially legumes, through transgenic approach. (Supported by a RGC grant CUHK4351/98M).