Luo Xiao, Bathgate Ross A D, Liu Ya-Li, Shao Xiao-Xia, Wade John D, Guo Zhan-Yun
Institute of Protein Research, East Hospital, Tongji University, Shanghai, China.
FEBS J. 2009 Sep;276(18):5203-11. doi: 10.1111/j.1742-4658.2009.07216.x. Epub 2009 Aug 10.
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.
胰岛素样肽3（INSL3）主要在睾丸的间质细胞中表达，是肽类激素胰岛素超家族的成员之一。其主要功能之一是通过与G蛋白偶联受体RXFP2相互作用，启动并介导雄性胎儿睾丸的下降。对该肽的研究依赖于分别合成A链和B链并随后进行链重组。为了建立一种制备人INSL3的替代方法，我们设计并在大肠杆菌细胞中重组表达了一种单链INSL3前体。该前体从包涵体中溶解出来，通过固定化金属离子亲和色谱法几乎纯化纯化至几乎同质，并在体外有效复性。随后通过依次用内肽酶Lys-C和羧肽酶B处理，将复性的前体转化为成熟的人INSL3。圆二色光谱分析和肽图分析表明，复性的INSL3具有胰岛素样折叠结构和预期的二硫键连接。重组人INSL3在刺激表达RXFP2的细胞中的环磷酸腺苷（cAMP）活性方面表现出完全活性。有趣的是，单链前体的活性与成熟的双链INSL3相当，这表明B链中至C端的受体结合区域在前体中保持活性构象。本研究不仅为成熟INSL3的制备提供了一种有效方法，还获得了一个有用的单链模板，用于该肽的进一步结构和功能研究。
