title: The de novo designed nutritive protein MB-1Trp does not resist proteolytic degradation in alfalfa leaves.
abstract: We previously reported on a de novo designed protein "milk bundle-1Trp" (MB-1Trp) as a source of selected essential amino acids (EAA) for ruminant feeding. Here, we attempt to express this de novo designed protein in alfalfa. The microbial version of the gene encoding the protein was modified in order to achieve two expression strategies in transgenic alfalfa plants. Chimeric MB-1Trp genes alone or fused to a signal peptide and an endoplasmic reticulum retention sequence were introduced into alfalfa via Agrobacterium-mediated transformation. Polymerase chain reaction and reverse transcriptase polymerase chain reaction analysis performed on individual transgenic lines demonstrated that the MB-1Trp gene was correctly integrated and transcribed into mRNA. However, under our conditions, it was impossible to detect MB-1Trp protein expression in any of the transgenic plants analyzed. In order to assess MB-1Trp stability in alfalfa, Escherichia coli-derived MB-1Trp was incubated with proteins extracted from leaves of a non-transgenic plant. This study revealed a high susceptibility of mature MB-1Trp to alfalfa proteases, which may have contributed to its lack of accumulation.
authors.affiliation: Plant Biology Research Group, Département de chimie-biologie, Université du Québec à Trois-Rivières, C.P. 500, Trois-Rivières, Que., Canada G9A5H7.
authors.forename: Habib
authors.initials: H
authors.lastname: Khoudi
authors.forename: Marc
authors.initials: M
authors.lastname: Beauregard
mesh_terms: D000601:Amino Acids, Essential / Q000096:biosynthesis*; D010450:Endopeptidases / Q000378:metabolism*; D000455:Medicago sativa / Q000235:genetics* / Q000378:metabolism*; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012231:Rhizobium / Q000235:genetics
chemical_list: D000601:Amino Acids, Essential; D010940:Plant Proteins; D011994:Recombinant Proteins; D010450:Endopeptidases