Mammalian recombinant production of mature peptide hormone insulin-like 3 (INSL3)

Abstract Insulin-like 3 (INSL3) is a peptide hormone important for regulating male reproductive physiology. INSL3 belongs to the insulin/relaxin family of peptide hormones, which are synthesized in cells as prohormones containing B-C-A chains. These hormones mature following proteolytic processing by prohormone convertases to remove C chain and yield mature hormones containing B-A chains that are stabilized by two inter-chain and one intra-chain disulfide bonds. It has been a challenge in the field to produce recombinant active mature INSL3 in large quantities because the enzymes that process INSL3 are unclear. Here we report prohormone convertases PCSK1 and PCSK3, and carboxypeptidase E together process INSL3 prohormone into mature INSL3 and established a simple method to produce recombinant active mature INSL3 in a mammalian expression system using an engineered Fc-fusion approach with co-expression of processing enzymes. Furthermore, we confirmed the molecular weight of the recombinant INSL3 matches the native hormone by intact mass spectrometry, and the recombinant mature INSL3 display full activity (EC 50 = 360 pM) in signaling assays of its cognate receptor RXFP2. Notably, the Fc-fusion INSL3 is also fully active (EC 50 = 609 pM) and has a much higher yield and a simpler production method. Lastly, peptide hormones of this family are conserved prohormone convertase substrates, therefore this method can potentially be applied to the expression and purification of other peptide hormones within this family with some optimization.