Recombinant expression of an insulin‐like peptide 3 (INSL3) precursor and its enzymatic conversion to mature human INSL3
Recombinant expression of an insulin‐like peptide 3 (INSL3) precursor and its enzymatic conversion to mature human INSL3
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DOI：
10.1111/j.1742-4658.2009.07216.x
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发表时间：
2009-09
期刊：
影响因子：
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通讯作者：
Xiao Luo;R. Bathgate;Ya‐Li Liu;X. Shao;J. Wade;Zhan-Yun Guo
中科院分区：
文献类型：
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作者：
Xiao Luo;R. Bathgate;Ya‐Li Liu;X. Shao;J. Wade;Zhan-Yun Guo
胰岛素样肽3 （Insulin‐like peptide 3, INSL3）主要表达于睾丸间质细胞，是胰岛素超家族的一员。其主要功能之一是通过与其G蛋白偶联受体RXFP2的相互作用启动和介导男性胎儿睾丸的下降。该肽的研究依赖于分离的A链和B链的化学合成以及随后的链重组。为了建立一种制备人类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个单链INSL3前体。采用固定化金属离子亲和层析法将前驱体从包裹体中溶解，纯化至几乎均匀，并在体外有效地折叠。通过内源性蛋白酶Lys - C和羧基肽酶B处理，将重组的前体转化为成熟的人INSL3。CD光谱分析和肽图谱分析表明，重新折叠的INSL3具有胰岛素样折叠，具有预期的二硫键。重组人INSL3在RXFP2‐表达的细胞中显示出充分刺激cAMP活性的活性。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端的受体结合区在前体中保持活跃构象。这项研究不仅为成熟的INSL3制备提供了有效的方法，而且还获得了一个有用的单链模板，用于肽的进一步结构和功能研究。
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.