Gholami Samaneh, Goodarzvand Chegini Koroush, Gheibi Nematollah, Mokhtarian Kobra, Mohamadi Mohsen, Falak Reza
Immunology Research Center, Iran University of Medical Sciences, Tehran, Iran.
Department of Biotechnology, Qazvin University of Medical Sciences, Qazvin, Iran.
Med J Islam Repub Iran. 2017 Dec 18;31:102. doi: 10.14196/mjiri.31.102. eCollection 2017.
Betatrophin, a novel secretory protein from liver and fatty tissues, is believed to be involved in lipid and glucose metabolism. However, its precise physiological role remains unclear. Here, we report the cloning, expression, and purification steps of mouse betatrophin in a prokaryotic system, followed by its structural analysis. Specific cloning primers were used to amplify the coding sequence of mouse liver betatrophin. The product was cloned into pET28 and expressed in E.coli BL21 (DE3) cells. The suitability of the refolding procedure was assessed by determining secondary structures of the initial and refolded proteins using circular dichroism spectroscopy. The polymerase chain reaction resulted in a 549 bp nucleotide sequence, encoding a 183 amino acid polypeptide, with an apparent molecular weight of 21 kDa, which was expressed in an inclusion body. Following an optimization and refolding procedure, the recombinant protein was purified by anion exchange and metal affinity chromatography. CD spectra revealed that the refolded protein has suitable configuration. We believe that the produced betatrophin is suitable for further biochemical studies on glucose and lipid metabolism.
β-促胰岛素分泌素是一种源自肝脏和脂肪组织的新型分泌蛋白，被认为参与脂质和葡萄糖代谢。然而，其确切的生理作用仍不清楚。在此，我们报告了小鼠β-促胰岛素分泌素在原核系统中的克隆、表达和纯化步骤，随后对其进行了结构分析。使用特异性克隆引物扩增小鼠肝脏β-促胰岛素分泌素的编码序列。将产物克隆到pET28中，并在大肠杆菌BL21(DE3)细胞中表达。通过使用圆二色光谱法测定初始蛋白和重折叠蛋白的二级结构，评估重折叠程序的适用性。聚合酶链反应产生了一个549 bp的核苷酸序列，编码一个183个氨基酸的多肽，表观分子量为21 kDa，该多肽以包涵体形式表达。经过优化和重折叠程序后，重组蛋白通过阴离子交换和金属亲和色谱法进行纯化。圆二色光谱显示重折叠蛋白具有合适的构象。我们认为所产生的β-促胰岛素分泌素适用于进一步关于葡萄糖和脂质代谢的生化研究。
