Expression of Schistocerca gregaria ion transport peptide (ITP) and its homologue (ITP-L) in a baculovirus/insect cell system.
We expressed an N-terminally extended Schistocerca gregaria ion transport peptide (ScgITP) and its homologue (ion transport peptide-like; ITP-L) in insect Sf9 cells using baculovirus expression vectors. Antibodies raised against peptide fragments of ITP and ITP-L were used to detect and characterize the baculovirus expressed peptides (bacITP, bacITP-L). Biological activity of the expressed peptides was assayed using the highly specific bioassay for native ITP, namely the increase in ileal short-circuit current which is a measure of active Cl- transport. BacITP and bacITP-L expression was optimal in Sf9 cells infected at a multiplicity of infection of 1, grown in Grace's medium, and harvested 2-3 days after infection. Western blots showed that bacITP was 2 kDa larger than native or synthetic ITP. This difference was not due to glycosylation and could in part be attributed to post-translational cleavage of the ITP propeptide at a site 11 amino acids upstream of the cleavage site used by S. gregaria to produce native ITP. BacITP stimulated ileal short-circuit current but is significantly less active (270-fold) than synthetic ITP (synITP) possibly as a result of the N-terminal extension. Production of bacITP-L permitted us to show that it is not stimulatory in the bioassay but reduces the synITP response in vitro and thus may have some potential for enhancing the effectiveness of biological control agents such as baculoviruses.
Ring
M
M
Department of Zoology, University of British Columbia, Vancouver, Canada.
Meredith
J
J
Wiens
C
C
Macins
A
A
Brock
H W
HW
Phillips
J E
JE
Theilmann
D A
DA
D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors; D006110:Grasshoppers / Q000235:genetics / Q000378:metabolism*; D007301:Insect Hormones / Q000235:genetics*; D019476:Insect Proteins; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000235:genetics*; D017924:Nucleopolyhedroviruses; D011499:Protein Processing, Post-Translational; D018411:Spodoptera
D002352:Carrier Proteins; D007301:Insect Hormones; D019476:Insect Proteins; D009479:Neuropeptides; C079361:ion-transport peptide, Schistocerca gregaria