Methionine and crysteine-rich proteins in soybean (Glycine max): identification, purification and gene cloning

This study aimed to enhance the protein quality of soybean by increasing the sulfur amino acids methionine and cystine of soybean and the possible applications in other legumes by recombinant DNA techniques. The strategy used was to increase the biosynthesis of endogenous sulfur-rich proteins which should have at least deleterious effect on plant biology. This had become very important with the recent evidence that the transgenic soybean containing Brazil nut MRP is allergenic. The methionine-rich proteins (MRP) were localized in the 8 kD band of the albumin fraction of soybean seed. Sufficient amounts of three methionine-rich proteins (2D-1, 2D-2, 2D-3) were purified by sequential affinity chromatography and 2D-SDS-PAGE. On the other hand, a cystine-rich protein (CRP-1) was purified through size exclusion chromatography followed by anion exchange through FPLC and later SDS-PAGE. This facilitated the analysis of the protein profile and the N-terminus sequence of each protein, from which degenerate primers were designed for use in the Polymerase Chain Reaction (PCR). Complimentary DNA (cDNA) fragments specific to 2D-1 and CRP-1 were amplified and cloned. DNA sequence analysis of the PCR fragment showed that 2D-1 contains 8.6 percent methionine and 8.6 percent cysteine, for a total of 17.2 percent sulfur amino acids. It was found to be present as 2 copies in the soybean genome. A search in the Genebank database showed that it shares homology with DNA binding proteins (53 percent) in Arabidopsis thaliana and a sulfur-rich protein in lupine seeds (60 percent). CRP-1 was analyzed to contain 13.7 percent cysteine and 2.8 percent methionine (total of 16.5 percent), was found to be a single copy gene and homologous to maturation-associated and dehydrin-like proteins in other plants. A mid-maturation soybean seed cDNA library was screened using the 2D-1 and CRP-1 PCR fragments as probes to isolate corresponding cDNA clones. Ten positive clones hybridizing to 2D-1 and one for CRP-1 were identified Antibody to 2D-1 was also raised in mice, as confirmed by a Western analysis on a 2D-SDS-PAGE blot of purified and crude protein samples