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 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 that the refolded INSL3 possessed an insulin-like fold with the expected disulfide linkages. Recombinant human INSL3 demonstrated full activity in stimulating cAMP activity in RXFP2-expressing cells. Interestingly, the activity of the single-chain precursor was comparable with that of the mature two-chain INSL3, suggesting that the receptor-binding region within the mid- to C-terminal of B-chain is maintained in an active conformation in the precursor. This study not only provides an efficient approach for mature INSL3 preparation, but also resulted in the acquisition of a useful single-chain template for additional structural and functional studies of the peptide.
Luo
Xiao
X
Institute of Protein Research, East Hospital, Tongji University, Shanghai, China.
Bathgate
Ross A D
RA
Liu
Ya-Li
YL
Shao
Xiao-Xia
XX
Wade
John D
JD
Guo
Zhan-Yun
ZY
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000096:biosynthesis* / Q000737:chemistry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D017510:Protein Folding; D011498:Protein Precursors / Q000096:biosynthesis*; D011506:Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis*
D007328:Insulin; C084797:Leydig insulin-like protein; D011498:Protein Precursors; D011506:Proteins; D011994:Recombinant Proteins; D000242:Cyclic AMP