Design and recombinant expression of insulin-like peptide 5 precursors and the preparation of mature human INSL5

摘要: Insulin-like peptide 5 (INSL5) is a recently identified insulin superfamily member. Although it binds to and activates the G-protein coupled receptor, RXFP4, its precise biological function remains unknown. To help determine function, significant quantities of INSL5 are required. In present work, three single-chain precursors were designed, two which successfully expressed in E. coli cells. The solubilized from inclusion bodies, purified almost homogeneity by immobilized metal-ion affinity chromatography, then refolded vitro. One precursor could be converted two-chain human bearing an extended N-terminus A-chain (designated long-INSL5) sequential Lys-C endoproteinase carboxypeptidase B treatment. 6 residue N-terminal extension long-INSL5 was subsequently removed Aeromonas aminopeptidase yield native that designated short-INSL5. Circular dichroism spectroscopic analysis mapping showed recombinant INSL5s adopted insulin-like conformation possessed expected characteristic disulfide linkages. Activity assay both long- short-INSL5 had full RXFP4 receptor activity compared with chemically synthesized INSL5. This suggested did not adversely impact upon binding or activation receptor. However, inactive indicated free C-terminus B-chain critical for Our work thus provides efficient approach preparation analogs through expression