title: Analysis of an insect neuropeptide, Schistocerca gregaria ion transport peptide (ITP), expressed in insect cell systems.
abstract: We have produced an active form of Schistocerca gregaria ion transport peptide (ITP) in an insect cell expression system. Transformed Drosophila Kc1 cells secreted a form of ITP into the cell culture medium that was proteolytically cleaved correctly at the amino (N)-terminus. Concentrated culture supernatant from transformed Kc1 and Hi5 cells had high biological activity when tested on isolated locust ilea. Conversely, ITP expressed by baculovirus-infected Sf9 cells was larger in size and had decreased specific activity compared to ITP produced by Kc1 cells due to incorrect cleavage of the peptide at the N-terminus in the baculovirus system. This demonstrates how processing of the secreted foreign protein (ITP) expressed under the late polyhedrin promoter is compromised in a baculovirus-infected cell. Transient transformation of Kc1 cells results in supernatants containing two forms of ITP; one form (A) co-elutes with synthetic ITP and the other form (B) has reduced electrophoretic mobility. In contrast, in stably transformed Kc1 cell supernatant, ITP is expressed in a single form, which has the same electrophoretic mobility and specific biological activity as form A produced by transiently transformed Kc1 cells. Arch.
authors.affiliation: Department of Zoology, University of British Columbia, Vancouver, BC V6T1Z4 Canada.
authors.forename: T A
authors.initials: TA
authors.lastname: Pfeifer
authors.forename: D
authors.initials: D
authors.lastname: Hegedus
authors.forename: Y J
authors.initials: YJ
authors.lastname: Wang
authors.forename: Y
authors.initials: Y
authors.lastname: Zhao
authors.forename: J
authors.initials: J
authors.lastname: Meredith
authors.forename: H W
authors.initials: HW
authors.lastname: Brock
authors.forename: J E
authors.initials: JE
authors.lastname: Phillips
authors.forename: T A
authors.initials: TA
authors.lastname: Grigliatti
authors.forename: D A
authors.initials: DA
authors.lastname: Theilmann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006110:Grasshoppers / Q000235:genetics / Q000502:physiology*; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000737:chemistry / Q000235:genetics*; D020539:Sequence Analysis, Protein
chemical_list: D002352:Carrier Proteins; D019476:Insect Proteins; D009479:Neuropeptides; C079361:ion-transport peptide, Schistocerca gregaria