Analysis of an insect neuropeptide, Schistocerca gregaria ion transport peptide (ITP), expressed in insect cell systems

✍ Scribed by T.A. Pfeifer; D. Hegedus; Y.-J. Wang; Y. Zhao; J. Meredith; H. W. Brock; J. E. Phillips; T.A. Grigliatti; D.A. Theilmann

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 166 KB
• Volume: 42
• Category: Article
• ISSN: 0739-4462
• DOI: 10.1002/(sici)1520-6327(199912)42:4<245::aid-arch3>3.0.co;2-w

✦ Synopsis

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.