Insulin-like peptide 3 expressed in the silkworm possesses intrinsic disulfide bonds and full biological activity

Abstract

Insulin-like peptide 3 (INSL3) is a member of the relaxin/insulin superfamily and is expressed in testicular Leydig cells. Essential for fetal testis descent, INSL3 has been implicated in testicular and sperm function in adult males via interaction with relaxin/insulin-like family peptide receptor 2 (RXFP2). The INSL3 is typically prepared using chemical synthesis or overexpression in Escherichia coli followed by oxidative refolding and proteolysis. Here, we expressed and purified full-length porcine INSL3 (pINSL3) using a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system. Biophysical measurements and proteomic analysis revealed that this recombinant pINSL3 exhibited the correct conformation, with the three critical disulfide bonds observed in native pINSL3, although partial cleavage occurred. In cAMP stimulation assays using RXFP2-expressing HEK293 cells, the recombinant pINSL3 possessed full biological activity. This is the first report concerning the production of fully active pINSL3 without post-expression treatments and provides an efficient production platform for expressing relaxin/insulin superfamily peptides.

Development of SpyTag/SpyCatcher-Bacmid Expression Vector System (SpyBEVS) for Protein Bioconjugations Inside of Silkworms

“…One key merit of the bacmid expression vector system (BEVS) is its capability of expressing multiple proteins of interest (POIs) simultaneously via co-infection/co-transfection of several recombinant bacmids together [4]. Recently, BEVS using lepidopteran insects, e.g., silkworm ( Bombyx mori ), has enabled us to produce proteins of interest to a higher level with both a satisfactory quantity and quality, where the resulting POIs hold most desirable posttranslational modifications, especially glycosylation, which is considered to be essential for glycoproteins [5,6,7,8,9]. Those advantages should contribute greatly to reducing the cost of the commercial proteins, such as enzymes and vaccines in the market, although several issues such as the development of purification methods and the safety risk (e.g., contaminations form either insect- or baculovirus-derived substances) remain to be clarified.…”