title: Design of genetically modified soybean proglycinin A1aB1b with multiple copies of bioactive peptide sequences.
abstract: The peptide IIAEK derived from beta-lactoglobulin has a hypocholesterolemic activity greater than that of beta-sitosterol. To create food proteins with multiple copies of this valuable peptide sequence, we introduced tandem multimers of the nucleotide sequence encoding the peptide into DNA regions corresponding to the five variable regions of soybean glycinin A1aB1b subunit, and expressed the mutants in Escherichia coli. The expression level and solubility of the five mutants, each containing four IIAEK sequences in each of the variable regions, were compared. Overall, the expression level and solubility of the mutants with four IIAEK sequences in the variable regions IV and V were the best followed by II > III > I. Further, introduction of the fifth IIAEK sequence to the variable region IV did not decrease expression level and solubility. Increasing the number of IIAEK to 7 and 10 slightly decreased expression level, while their solubility decreased to as low as 40 and 1%, respectively. Various mutations were combined to get a mutant containing as many IIAEK sequences as possible. Some of the resulting mutants were expressed in the soluble form. The mutant containing eight IIAEK from the combination of variable regions IV and V (IV-4 + V-4) showed the best balance of the expression level and solubility, followed by the combination of variable regions II and III (II-4 + III-4). The soluble fractions of these mutants were purified by hydrophobic, gel filtration and ion-exchange column chromatography. Yields of IIAEK peptide released by in vitro digestion with trypsin from both mutants were around 80%. This is the first report that a large amount of a physiologically active peptide could be introduced into food protein.
authors.affiliation: Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Krisna
authors.initials: K
authors.lastname: Prak
authors.forename: Yukie
authors.initials: Y
authors.lastname: Maruyama
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Maruyama
authors.forename: Shigeru
authors.initials: S
authors.lastname: Utsumi
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D030262:Soybean Proteins / Q000235:genetics*; D013025:Glycine max / Q000235:genetics*; D014357:Trypsin / Q000737:chemistry
chemical_list: D010455:Peptides; D030262:Soybean Proteins; C405345:proglycinin A(1a)B(1b), soybean; D004247:DNA; D014357:Trypsin