Synthesis of Soybean Agglutinin in Bacterial and Mammalian Cells

摘要: The cDNA of soybean agglutinin (SEA), a glycoprotein lectin, obtained from the mRNA seeds at mid-maturation, was cloned in lambda gt 10 phage and subcloned pUC-8 plasmid. Probing with fragment lectin gene [Vodkin, L. O., Rhodes, P. R. & Goldberg, B. (1983) Cell 34, 1023-1031] afforded clone 1012 nucleotides containing complete coding region 858 for precursor to agglutinin. deduced amino acid sequence contains 253 residues mature an hydrophobic N-terminal signal peptide 32 acids. Expression Escherichia coli or led accumulation large quantities inclusion bodies, which SEA isolated small yield (up 1 mg/l). It identical native hemagglutinating activity carbohydrate specificity, oligomeric structure, but, because it not glycosylated, its subunit mass lower by 2 kDa. Our findings show that pre-SEA is processed into form bacteria, that, contrary what has been suggested [Nagai, K. Yamaguchi, H. (1993) J. Biochem. (Tokyo) II3, 123-125], glycosylation essential folding nor assembly biologically active tetramer. To obtain recombinant secreted form, pTM1 vector construct inserted vaccinia virus. When monkey BS-C-1 cells were infected virus, using double expression protocol, growth medium, immunoaffinity chromatography similar bacteria. Except activity, product indistinguishable all properties tested. also susceptible digestion endo-beta-N-acetylglucosaminidase ii N-glycanase caused decrease kDa gave same results on blot analysis, indicating too single oligomannose unit.