title: Design and recombinant expression of insulin-like peptide 5 precursors and the preparation of mature human INSL5.
abstract: 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 its function, significant quantities of INSL5 are required. In the present work, three single-chain INSL5 precursors were designed, two of which were successfully expressed in E. coli cells. The expressed precursors were solubilized from inclusion bodies, purified almost to homogeneity by immobilized metal-ion affinity chromatography, and then refolded in vitro. One precursor could be converted to two-chain human INSL5 bearing an extended N-terminus of the A-chain (designated long-INSL5) by sequential Lys-C endoproteinase and carboxypeptidase B treatment. The 6 residue A-chain N-terminal extension of long-INSL5 was subsequently removed by Aeromonas aminopeptidase to yield native INSL5 that was designated short-INSL5. Circular dichroism spectroscopic analysis and peptide mapping showed that the recombinant INSL5s adopted an insulin-like conformation and possessed the expected characteristic insulin-like disulfide linkages. Activity assay showed that both long- and short-INSL5 had full RXFP4 receptor activity compared with chemically synthesized human INSL5. This suggested that extension of the N-terminus of the A-chain of long-INSL5 did not adversely impact upon the binding to or activation of the RXFP4 receptor. However, the single-chain INSL5 precursor was inactive which indicated that a free C-terminus of the B-chain is critical for the activity of INSL5. Our present work thus provides an efficient approach for preparation of INSL5 and its analogs through recombinant expression in E. coli cells.
authors.affiliation: Institute of Protein Research, Tongji University, School of Medicine, and Central Laboratory, Shanghai East Hospital, 1239 Siping Road, Shanghai, 200092, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Luo
authors.forename: Ross A D
authors.initials: RA
authors.lastname: Bathgate
authors.forename: Wei-Jie
authors.initials: WJ
authors.lastname: Zhang
authors.forename: Ya-Li
authors.initials: YL
authors.lastname: Liu
authors.forename: Xiao-Xia
authors.initials: XX
authors.lastname: Shao
authors.forename: John D
authors.initials: JD
authors.lastname: Wade
authors.forename: Zhan-Yun
authors.initials: ZY
authors.lastname: Guo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D006801:Humans; D007328:Insulin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D017510:Protein Folding; D011498:Protein Precursors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011506:Proteins / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D007328:Insulin; C084797:Leydig insulin-like protein; D011498:Protein Precursors; D011506:Proteins; D011994:Recombinant Proteins