title: Processing and assembly in vitro of engineered soybean beta-conglycinin subunits with the asparagine-glycine proteolytic cleavage site of 11S globulins.
abstract: A short interdomain sequence between the N- and C-terminal domains of beta-conglycinin, the major 7S seed storage protein of soybean, was selected as a target for insertion of amino acid residues specifically cleaved by an asparaginyl endopeptidase that processes globulins into acidic and basic chains. Modified beta-conglycinin subunits containing the proteolytic cleavage site self-assembled into trimers in vitro at an efficiency similar to that of the unmodified subunit. In contrast to the absence of cleavage of the unmodified subunits, however, the modified beta-conglycinin trimers were processed by purified soybean asparaginyl endopeptidase into two polypeptides, each the size expected for the beta-conglycinin N- and C-terminal domains, respectively. The cleavage did not alter the assembly of mutant beta-conglycinins and the cleaved mutant trimers remained stable to further proteolytic attack. To examine the possibility of coassembly between the cleaved 11S and 7S subunits, in vitro processed mutant beta-conglycinin subunits were mixed with native dissociated 11S globulin preparations. Reassembly at a high ionic condition did not induce the 7S subunits to interact with 11S subunits to form hexameric complexes. Thus, cleavage of 7S globulin subunits into acidic and basic domains may not be sufficient for hexamer assembly to occur. Biotechnological implications of the engineered proteins are discussed.
authors.affiliation: United States Department of Agriculture, Agricultural Research Service, Purdue University, West Lafayette IN 47907, USA.
authors.forename: Luiz O
authors.initials: LO
authors.lastname: Oliveira
authors.forename: Young-Woo
authors.initials: YW
authors.lastname: Nam
authors.forename: Rudolf
authors.initials: R
authors.lastname: Jung
authors.forename: Niels C
authors.initials: NC
authors.lastname: Nielsen
mesh_terms: D000595:Amino Acid Sequence; D052179:Antigens, Plant; D001483:Base Sequence; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000378:metabolism; D005916:Globulins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010940:Plant Proteins; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055314:Seed Storage Proteins; D017386:Sequence Homology, Amino Acid; D030262:Soybean Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013025:Glycine max / Q000235:genetics / Q000378:metabolism; D000099240:Asparaginyl Endopeptidase
chemical_list: D052179:Antigens, Plant; D003546:Cysteine Endopeptidases; D005916:Globulins; D010940:Plant Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D055314:Seed Storage Proteins; D030262:Soybean Proteins; D000099240:Asparaginyl Endopeptidase; C013894:beta-conglycinin protein, Glycine max