title: Expression and biological activity of the cystine knot bioinsecticide PA1b (Pea Albumin 1 Subunit b).
abstract: The PA1b (Pea Albumin 1, subunit b) peptide is an entomotoxin extract from Legume seeds with lethal activity on several insect pests, such as mosquitoes, some aphids and cereal weevils. This 37 amino-acid cysteine-rich peptide has been, until now, obtained by biochemical purification or chemical synthesis. In this paper, we present our results for the transient production of the peptide in Nicotiana benthamiana by agro-infiltration, with a yield of about 35 µg/g of fresh leaves and maximum production 8 days after infiltration. PA1b is part of the PA1 gene which, after post-translational modifications, encodes two peptides (PA1b and PA1a). We show that transforming tobacco with the PA1b cDNA alone does not result in production of the toxin and, in fact, the entire cDNA is necessary, raising the question of the role of PA1a. We constructed a PA1-cassette, allowing for the quick "cut/paste" of different PA1b mutants within a conserved PA1 cDNA. This cassette enabled us to produce the six isoforms of PA1b which exist in pea seeds. Biological tests revealed that all the isoforms display similar activity, with the exception of one which is inactive. The lack of activity in this isoform led us to conclude that the amphiphilic nature of the peptide is necessary for activity. The possible applications of this expression system for other cysteine-rich biomolecules are discussed.
authors.affiliation: BF2I (Biologie Fonctionnelle, Insectes et Interactions), INRA - INSA-Lyon, Villeurbanne, France.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Eyraud
authors.affiliation: BF2I (Biologie Fonctionnelle, Insectes et Interactions), INRA - INSA-Lyon, Villeurbanne, France.
authors.forename: Lamis
authors.initials: L
authors.lastname: Karaki
authors.affiliation: BF2I (Biologie Fonctionnelle, Insectes et Interactions), INRA - INSA-Lyon, Villeurbanne, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Rahioui
authors.affiliation: BF2I (Biologie Fonctionnelle, Insectes et Interactions), INRA - INSA-Lyon, Villeurbanne, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Sivignon
authors.affiliation: BF2I (Biologie Fonctionnelle, Insectes et Interactions), INRA - INSA-Lyon, Villeurbanne, France.
authors.forename: Pedro
authors.initials: P
authors.lastname: Da Silva
authors.affiliation: BF2I (Biologie Fonctionnelle, Insectes et Interactions), INRA - INSA-Lyon, Villeurbanne, France.
authors.forename: Yvan
authors.initials: Y
authors.lastname: Rahbé
authors.affiliation: BF2I (Biologie Fonctionnelle, Insectes et Interactions), INRA - INSA-Lyon, Villeurbanne, France.
authors.forename: Corinne
authors.initials: C
authors.lastname: Royer
authors.affiliation: BF2I (Biologie Fonctionnelle, Insectes et Interactions), INRA - INSA-Lyon, Villeurbanne, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Gressent
mesh_terms: D000595:Amino Acid Sequence; D061046:Biological Control Agents; D018076:DNA, Complementary; D015870:Gene Expression; D057927:Hydrophobic and Hydrophilic Interactions; D007306:Insecticides / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000737:chemistry* / Q000378:metabolism; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D020033:Protein Isoforms / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism; D014118:Toxins, Biological / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
chemical_list: D061046:Biological Control Agents; D018076:DNA, Complementary; D007306:Insecticides; D010940:Plant Proteins; D020033:Protein Isoforms; D021122:Protein Subunits; D011994:Recombinant Proteins; D014118:Toxins, Biological