Isolation of Cloned Antidiuretic Factor Ion Transport Peptide in the Tobacco Hookworm, Manduca sexta

Abstract

Manduca sexta, the tobacco hornworm, is a garden pest that consumes leaves of the Solanaceae family. Water absorption in the colon of Manduca sexta is regulated by a hormone called ion transport peptide (ITP), which is vital to their survival. Producing ITP in vitro may assist in discovering the ITP receptor, a suspected key regulator in Manduca sexta metabolism, which may also lead to the development of an insect pesticide. The ITP gene was expressed in S. cerevisiae and in E. coli. A C4 reversed-phase column and a chitin resin column were used to purify the protein, followed by SDS-PAGE and mass spectrophotometric analysis to detect the presence of the protein. Preliminary mass spec. results lead us to believe that ITP was possibly produced in both S. cerevisiae and E. coli cells. Analysis will be repeated in order to confirm by mass spectrometric analysis, providing evidence that ITP hormone was produced by the S. cerevisiae and E. coli cells and that ITP was successfully transformed. The production of ITP may lead to the discovery of the ITP receptor which will allow for the development of a more specific Manduca pesticide, as well as elucidate the mechanism of insect ITP-antidiuretic signaling.