EXPRESSION OF A MALE ACCESSORY-GLAND PEPTIDE OF LEPTINOTARSA-DECEMLINEATA IN INSECT CELLS INFECTED WITH A RECOMBINANT BACULOVIRUS

The male accessory glands (MAGs) of Leptinotarsa decemlineata produce an 8 kDa peptide, designated Led-MAGP, that is recognized by monoclona l antibody MAC-is. 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 hexarepeat section of the chicken prion protein (Harris et al., 1991). The biological function of the Led-MAGP has yet to be dete rmined. For further research, large amounts of Led-MAGP is required, b oth for the production of a more specific antiserum, as well as for ap plication in bio-assays. This paper describes the expression of Led-MA GP in insect cells infected with recombinant baculovirus, and the prod uction of a polyclonal antibody against this recombinant peptide. The peptide was expressed under the control of the polyhedrin promotor. Th e resulting product was HPLC-purified, and analysis on Western blots i mmune-labelled with MAC-18 confirmed that the correct peptide was prod uced. Purified recombinant peptide was also analyzed by Edman degradat ion and mass spectrometry; this indicated that it was N-terminally blo cked and that the methionine residue at position 7 was oxidized. Large scale production resulted in the formation of aggregations of Led-MAG P, nevertheless a substantial proportion remained in a soluble state a nd could be harvested. A polyclonal antiserum encoded #87 was produced against recombinant Led-MAGP and its specificity was tested on Wester n blots of authentic peptide and on LM and EM sections of MAGs. All la belling results were equal to those obtained after MAC-18 labelling. H owever, antiserum #87 proved to be superior compared to MAC-is, since it recognizes the MAG peptide in normally fed, sexually active males, whereas MAC-18 labelling can only be accomplished after 7 days of star vation of the males. Therefore, the new antiserum #87 enables us to st udy the transfer dynamics of the Led-MAGP on histological sections. (C ) 1998 Elsevier Science Ltd. All rights reserved.