tailieunhanh - Báo cáo khoa học: Recombinant expression of an insulin-like peptide 3 (INSL3) precursor and its enzymatic conversion to mature human INSL3

Insulin-like peptide 3 (INSL3), which is primarily expressed in the Ley-dig cells of the testes, is a member of the insulin superfamily of peptide hormones. One of its primary functions is to initiate and mediate des-cent of the testes of the male fetus via interaction with its G protein-coupled receptor, RXFP2. Study of the peptide has relied upon chemical synthesis of the separate A- and B-chains and subsequent chain recombi-nation. | ỊFEBS Journal Recombinant expression of an insulin-like peptide 3 INSL3 precursor and its enzymatic conversion to mature human INSL3 Xiao Luo1 Ross A. D. Bathgate2 3 Ya-Li Liu4 Xiao-Xia Shao1 John D. Wade2 5 and Zhan-Yun Guo1 1 Institute of Protein Research Tongji University Shanghai China 2 Howard Florey Institute University of Melbourne Australia 3 Department of Biochemistry and Molecular Biology University of Melbourne Australia 4 East Hospital Tongji University Shanghai China 5 Schoolof Chemistry University of Melbourne Australia Keywords activity INSL3 recombinant expression refolding single-chain precursor Correspondence J. D. Wade Howard Florey Institute The University of Melbourne Vic 3010 Australia Fax 61 3 9348 1707 Tel 61 3 8344 7285 E-mail . Guo Institute of Protein Research Tongji University 1239 Siping Road Shanghai 200092 China Fax 86 21 658 98403 Tel 86 21 659 88634 E-mail Received 25 May 2009 accepted 16 July 2009 doi Insulin-like peptide 3 INSL3 which is primarily expressed in the Leydig cells of the testes is a member of the insulin superfamily of peptide hormones. One of its primary functions is to initiate and mediate descent of the testes of the male fetus via interaction with its G protein-coupled receptor RXFP2. Study of the peptide has relied upon chemical synthesis of the separate A- and B-chains and subsequent chain recombination. To establish an alternative approach to the preparation of human INSL3 we designed and recombinantly expressed a single-chain INSL3 precursor in Escherichia coli cells. The precursor was solubilized from the inclusion body purified almost to homogeneity by immobilized metal-ion affinity chromatography and refolded efficiently in vitro. The refolded precursor was subsequently converted to mature human INSL3 by sequential endoproteinase Lys-C and carboxypeptidase B treatment. CD spectroscopic analysis and peptide mapping showed