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

Abstract

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. Insect Biochem. Physiol. 42:245–252, 1999. © 1999 Wiley‐Liss, Inc.

Mutational analysis of the C-terminus in ion transport peptide (ITP) expressed inDrosophila Kc1 cells

“…1B,C,E,F) and may indicate a post-translational modification of one or more amino acids common to all these expressed peptides (Tables 1,2). The amino acid sequence of the first 22 residues of both bands was identical, as reported by Pfeifer et al (1999). Figure 1B and C compare Western blots of supernants from Kc1 cells transformed with preproITP cDNAs in which successive C-terminal amino acids were removed.…”

“…Membranes were probed with antibodies made to amino acids 57-85 (underlined in Table 1) of the preproITP sequence at 1:10,000 dilution. The amount of expressed peptides was determined by densitometry as detailed in Macins et al (1999) and Pfeifer et al (1999). Band density on Western blots was first shown to be a linear function of concentration and then compared to known amounts of synITP.…”

“…SL2, Sf9, and Ld652Y lines failed to transiently express ITP in quantities that could be detected on Western blots (detection limit 3 pmol). However, supernatants from transformed Drosophila Kc1 cells were very active in the bioassay, and contained a peptide (KcITP 75 ) that was correctly cleaved at the N-terminus and co-migrated with synITP in Western blots (Pfeifer et al, 1999).…”

Mutational analysis of the n-terminus inSchistocerca gregaria ion-transport peptide expressed inDrosophila Kc1 cells

“…Drosophila Kc1 cells transfected with pre-proITP secreted a peptide (KcITP) that was very active in the locust ileal bioassay and had the same time course of stimulation as native ITP. KcITP was correctly cleaved at the N-terminus, correctly folded, and co-migrated with SchgrITP and synITP on Western blots (King et al, 1999;Pfeifer et al, 2001). Site-directed mutagenesis and expression of KcITP mutants (Wang et al, 2000) indicated that amidated leucine (residue 72) at the C-terminus is essential for receptor stimulation.…”

“…The widely used baculovirus-Sf9 cell expression system was found to process preproITP incorrectly . This led us to use the plasmid vector p2Zop2F to transfect several cultured insect cell-lines (Pfeifer et al, 2001). Drosophila Kc1 cells transfected with pre-proITP secreted a peptide (KcITP) that was very active in the locust ileal bioassay and had the same time course of stimulation as native ITP.…”