Design and recombinant expression of insulin-like peptide 5 precursors and the preparation of mature human INSL5
Design and recombinant expression of insulin-like peptide 5 precursors and the preparation of mature human INSL5
复制标题
胰岛素样肽5前体的设计、重组表达及成熟人INSL5的制备
DOI：
10.1007/s00726-010-0586-3
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发表时间：
2010-11-01
期刊：
影响因子：
3.5
通讯作者：
Guo, Zhan-Yun
中科院分区：
文献类型：
--
作者：
Luo, Xiao;Bathgate, Ross A. D.;Guo, Zhan-Yun
胰岛素样肽5(INSL5)是新近发现的胰岛素超家族成员。尽管它与G蛋白偶联受体RXFP4结合并激活，但其确切的生物学功能仍不清楚。为了帮助确定其功能，需要大量的INSL5。本工作设计了三个单链INSL5前体，其中两个在INSL5中成功表达。结肠癌细胞。表达的前体从包涵体中溶解，通过固定化金属离子亲和层析纯化到几乎均一，然后在体外复性。一种前体可以通过Lys-C内蛋白酶和羧基肽酶B的顺序处理而转化为带有A链延长N末端的双链人INSL5(命名为Long-INSL5)。Long-INSL5的6个残基A链N端延伸随后被气单胞菌氨基肽酶去除，产生天然的INSL5，命名为Short-INSL5。圆二色谱分析和肽图分析表明，重组INSL5以类胰岛素构象存在，并具有预期的类胰岛素二硫键特征。活性分析表明，与化学合成的人INSL5相比，长的和短的INSL5都具有完整的RXFP4受体活性。这表明，Long-INSL5 A链N-末端的延长不会对RXFP4受体的结合或激活产生不利影响。然而，单链INSL5前体没有活性，这表明B链的自由C末端对INSL5的活性至关重要。我们的工作为通过重组表达来制备INSL5及其类似物提供了一条有效的途径。结肠癌细胞。
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 inE. colicells. 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 byAeromonasaminopeptidase 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 inE. colicells.