Expression of Schistocerca gregaria ion transport peptide (ITP) and its homologue (ITP-l) in a baculovirus/insect cell system
| Autor: | J. Meredith, D. Theilmann, H. W. Brock, John E. Phillips, Mark Ring, C. Wiens, A. Macins |
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| Rok vydání: | 1998 |
| Předmět: |
Glycosylation
Blotting Western Genetic Vectors Molecular Sequence Data Gene Expression Sf9 Peptide Grasshoppers Spodoptera Biology Biochemistry Cell Line chemistry.chemical_compound Multiplicity of infection immune system diseases hemic and lymphatic diseases Animals Bioassay Amino Acid Sequence Cloning Molecular Molecular Biology chemistry.chemical_classification Neuropeptides Biological activity Molecular biology Nucleopolyhedroviruses In vitro Amino acid chemistry Insect Hormones Insect Science Insect Proteins Biological Assay Carrier Proteins Protein Processing Post-Translational |
| Zdroj: | Insect Biochemistry and Molecular Biology. 28:51-58 |
| ISSN: | 0965-1748 |
| DOI: | 10.1016/s0965-1748(97)00096-9 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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