Insulin-like peptide 3 expressed in the silkworm possesses intrinsic disulfide bonds and full biological activity.
Takatsugu MiyazakiMasaaki IshizakiHideo DohraSungjo ParkAndre TerzicTatsuya KatoTetsuya KohsakaEnoch Y ParkPublished in: Scientific reports (2017)
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.
Keyphrases
- type diabetes
- glycemic control
- induced apoptosis
- escherichia coli
- cell cycle arrest
- binding protein
- germ cell
- high throughput
- cell proliferation
- transcription factor
- cell free
- cell death
- metabolic syndrome
- staphylococcus aureus
- molecular dynamics simulations
- cystic fibrosis
- pi k akt
- long non coding rna
- genome wide identification
- crystal structure
- klebsiella pneumoniae