title: Expression of de novo high-lysine alpha-helical coiled-coil proteins may significantly increase the accumulated levels of lysine in mature seeds of transgenic tobacco plants.
abstract: We have designed protein molecules based on an alpha-helical coiled-coil structure. These proteins can be tailored to complement nutritionally unbalanced seed meals. In particular, these proteins may contain up to 43% mol/mol of the essential amino acid lysine. Genes encoding such proteins were constructed using synthetic oligonucleotides and the protein stability was tested for in vivo by expression in an Escherichia coli model system. A protein containing 31% lysine and 20% methionine (CP 3-5) was expressed in transgenic tobacco seeds utilizing the seed specific bean phaseolin and soybean beta-conglycinin promoters. Both promoters provided a level of expression in the mature transgenic tobacco seeds which resulted in a significant increase in the total lysine content of the seeds. Several of these transgenic lines were analyzed for three generations to determine the stability of gene expression. Plants transformed with the soybean beta-conglycinin promoter/CP 3-5 gene consistently expressed the high-lysine phenotype through three generations. However, expression of the high-lysine phenotype in plants transformed with the bean phaseolin/CP 3-5 was variable. This is the first report of a significant increase in seed lysine content due to the seed-specific expression of a de novo protein sequence.
authors.affiliation: E.I. Dupont de Nemours, Experimental Station, Wilmington, DE 19880-0402, USA.
authors.forename: S J
authors.initials: SJ
authors.lastname: Keeler
authors.forename: C L
authors.initials: CL
authors.lastname: Maloney
authors.forename: P Y
authors.initials: PY
authors.lastname: Webber
authors.forename: C
authors.initials: C
authors.lastname: Patterson
authors.forename: L T
authors.initials: LT
authors.lastname: Hirata
authors.forename: S C
authors.initials: SC
authors.lastname: Falco
authors.forename: J A
authors.initials: JA
authors.lastname: Rice
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D007887:Fabaceae / Q000235:genetics; D005822:Genetic Vectors; D008239:Lysine / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D030821:Plants, Genetically Modified; D010946:Plants, Medicinal; D010947:Plants, Toxic; D011401:Promoter Regions, Genetic; D017433:Protein Structure, Secondary; D012639:Seeds / Q000254:growth & development / Q000378:metabolism; D014026:Nicotiana / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
chemical_list: D010940:Plant Proteins; C048826:phaseolin protein, Phaseolus vulgaris; D008239:Lysine