|Chronis, Demosthenis - UNIV OF MISSOURI|
Submitted to: University of Missouri Molecular Biology Week
Publication Type: Abstract Only
Publication Acceptance Date: March 4, 2002
Publication Date: N/A
Technical Abstract: Soybeans contain significant amounts of protein and oil. Because of the high content of seed proteins, they have been promoted as a good protein source for both humans and livestock. However, soybean seed proteins are deficient in the sulfur-containing amino acids, cysteine and methionine. Attempts are currently being made to increase the overall content of these two essential amino acids in soybean seeds by expressing heterologous seed proteins that are rich in sulfur amino acids. We are pursuing an additional approach to improve the nutritional quality of soybean seed proteins by manipulating the enzymes that are involved in sulfur metabolism. Cysteine synthase [EC 220.127.116.11] catalyzes the synthesis of L-cysteine from O-acetyl-L-serine and hydrogen sulfide. We have isolated a full-length cDNA (1281 bp) encoding the cytosolic isoform of cysteine synthase from a developing soybean seeds. Nucleotide sequence analysis of the cDNA revealed a single open-reading frame of 978 bp encoding a 34 kD protein. The amino acid sequence of the 34 kD protein shows significant homology with other plant and bacterial cysteine synthase. The authenticity of the isolated cDNA was also confirmed by the functional complementation of an E. coli auxotrophic mutant. Analysis by RT-PCR revealed that cysteine synthase mRNA was abundant at early stages of seed development. The coding region of soybean cysteine synthase was cloned into pET28a vector for overexpression in E. coli. Induction of E.coli with isopropyl-beta-thiogalactopyranoside (IPTG) resulted in overexpression of a recombinant cysteine synthase with N-terminal histidine tag. Antibodies are currently being raised against the purified recombinant cysteine synthase for further biochemical and immunological studies.