Analysis of an insect neuropeptide, Schistocerca gregaria ion transport peptide (ITP), expressed in insect cell systems.
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.
Pfeifer
T A
TA
Department of Zoology, University of British Columbia, Vancouver, BC V6T1Z4 Canada.
Hegedus
D
D
Wang
Y J
YJ
Zhao
Y
Y
Meredith
J
J
Brock
H W
HW
Phillips
J E
JE
Grigliatti
T A
TA
Theilmann
D A
DA
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
D002352:Carrier Proteins; D019476:Insect Proteins; D009479:Neuropeptides; C079361:ion-transport peptide, Schistocerca gregaria