Expression of a male accessory gland peptide of Leptinotarsa decemlineata in insect cells infected with a recombinant baculovirus

• Published in: Journal of insect physiology 1998-03, Vol.44 (3), p.255-262
• Main Authors: Smid a, H.M, Schooneveld a, H, Deserno b, M.L.L.G, Put b, B, Vlak b, J.M
• Format: Article
• Language: English
• Subjects: Baculovirus; Chrysomelidae; Colorado potato beetle; insect; Laboratorium voor Entomologie; Laboratorium voor Virologie; Laboratory of Entomology; Laboratory of Virology; Leptinotarsa decemlineata; male accessory gland; PE&RC
• ISSN: 0022-1910; 1879-1611
• DOI: 10.1016/S0022-1910(97)00167-4

The male accessory glands (MAGs) of Leptinotarsa decemlineata produce an 8 kDa peptide, designated Led-MAGP, that is recognized by monoclonal antibody MAC-18. The site of synthesis, amino acid sequence and the gene encoding this peptide have been documented ( Smid and Schooneveld 1992, Smid et al. 1997). The primary structure is homologous to the N-terminal hexa-repeat section of the chicken prion protein ( Harris et al., 1991). The biological function of the Led-MAGP has yet to be determined. For further research, large amounts of Led-MAGP is required, both for the production of a more specific antiserum, as well as for application in bio-assays. This paper describes the expression of Led-MAGP in insect cells infected with recombinant baculovirus, and the production of a polyclonal antibody against this recombinant peptide. The peptide was expressed under the control of the polyhedrin promotor. The resulting product was HPLC-purified, and analysis on Western blots immuno-labelled with MAC-18 confirmed that the correct peptide was produced. Purified recombinant peptide was also analyzed by Edman degradation and mass spectrometry; this indicated that it was N-terminally blocked and that the methionine residue at position 7 was oxidized. Large scale production resulted in the formation of aggregations of Led-MAGP, nevertheless a substantial proportion remained in a soluble state and could be harvested. A polyclonal antiserum encoded #87 was produced against recombinant Led-MAGP and its specificity was tested on Western blots of authentic peptide and on LM and EM sections of MAGs. All labelling results were equal to those obtained after MAC-18 labelling. However, antiserum #87 proved to be superior compared to MAC-18, since it recognizes the MAG peptide in normally fed, sexually active males, whereas MAC-18 labelling can only be accomplished after 7 days of starvation of the males. Therefore, the new antiserum #87 enables us to study the transfer dynamics of the Led-MAGP on histological sections.