A simple approach for the preparation of mature human relaxin-3

Relaxin-3 (also known as INSL7) is the most recently identified member of the insulin-like family. It is predominantly expressed in the nucleus incertus of the brain and involved in the control of stress response, food intake, and reproduction. In the present work, we have established a simple approach for the preparation of the mature human relaxin-3 peptide. We first designed and recombinantly expressed a single-chain relaxin-3 precursor in E. coli cells. After purification by immobilized metal ion affinity chromatography, refolding in vitro through disulfide reshuffling, and digestion by endoproteinase Asp-N, mature human relaxin-3 was obtained in high yield and at low cost. Peptide mapping and circular dichroism spectroscopy studies suggested that the recombinant relaxin-3 adopted an insulin-like fold with the expected disulfide linkages. The recombinant mature relaxin-3 was fully active in both RXFP3 binding and activation assays. The activity of the single-chain precursor was very low, suggesting that a free C-terminus of the B-chain is necessary for receptor-binding and activation of relaxin-3. Our present work provides a highly efficient approach for the preparation of relaxin-3 as well as its analogues for functional and structural analyses.