Title
Insulin-like peptide 3 expressed in the silkworm possesses intrinsic disulfide bonds and full biological activity
Author
Takatsugu Miyazaki, Masaaki Ishizaki, Hideo Dohra, Sungjo Park, Andre Terzic, Tatsuya Kato, Tetsuya Kohsaka, Enoch Y. Park
Year
2017
Journal
Scientific Reports
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.
Full Article
Instrument
J-820
Keywords
Circular dichroism, Secondary structure, Biochemistry