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简单且高效重组制备rxfp2激动剂的方法
首发时间：2024-06-25
摘要：胰岛素样肽3（insl3）是一种分泌的多肽类激素，主要在睾丸间质细胞中表达，其主要功能之一是通过激活g蛋白偶联受体rxfp2介导胎儿发育期间睾丸的下降。在本研究中，我们建立了一种简单且高效重组制备rxfp2激动剂的方法，可用于实验或治疗目的。首先设计含有6×his标签的人源pro-insl3前体dna序列，并在大肠杆菌中重组表达，接着从包涵体中溶解，用固定化金属离子亲和层析纯化前体，然后通过体外重折叠和使用氨肽酶去除标签，从而获得人源pro-insl3。该方法产量高，成熟人源pro-insl3的产率为每升培养液约5-6 mg，是人源双链insl3（命名为野生型insl3）的6~8倍。虽然人源pro-insl3的结合亲和力（pic50为8.53±0.03）略低于野生型insl3（pic50为9.48±0.02），但其激活受体的能力（pec50为10.09±0.08）与野生型insl3（pec50为10.38±0.09）相当，表明人源pro-insl3几乎具有完全的生物活性。该研究不仅为rxfp2激动剂的重组制备提供了简易且有效的方法，而且还获得了一个有用的pro-insl3单链模板，可用于进一步的其结构和功能研究。
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a simple and efficient method for recombinant preparation of rxfp2 agonist
abstract：insulin-like factor 3 (insl3) is a secreted peptide hormone, which is primarily expressed in the leydig cells of the testes. one of its primary functions is to mediate testes descent during fetal development via activating its g protein coupled receptor, rxfp2. in the present study, we established an efficient approach for preparing human pro-insl3 that may be used for experimental or therapeutic purposes. the designed pro-insl3 precursor carried a 6×his tag was recombinantly expressed in escherichia coli. solubilized from the inclusion body, the precursor was purified using immobilized metal?ion affinity chromatography. subsequent to in vitro refolding and the removal of the tag by aminopeptidase cleavage, mature pro-insl3 was obtained with a considerable yield. the yield of mature human pro-insl3 was 5-6 mg per liter of culture broth, which is 6 to 8 times that of human double-stranded insl3 (named wild type insl3). although the binding affinity of human pro-insl3 (pic50 of 8.53 ± 0.03) was slightly lower than human wild-type insl3 (pic50 of 9.48 ± 0.02), the activation potency of pro-insl3 (pec50 of 10.09 ± 0.08) was comparable to wild-type insl3 (pec50 of 10.38 ± 0.09), suggesting human pro-insl3 was almost full biological activity. this study not only provides a simple and efficient approach for rxfp2 agonist preparation, but alsoyields a valuable pro-insl3 single-chain template for further structural and functional investigations.
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