A simple and efficient method for recombinant preparation of RXFP2 agonist

• 1、Cyrus Tang Hematology Center, Soochow University, Suzhou, P. R. China

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.

Keywords： biochemistry INSL3 RXFP2 recombinant expression activity circular dichroism