人促肾上腺皮质激素释放因子Ⅰ型受体EC1片段在原核系统中的可溶性表达

Soluble Expression of EC1 Fragment of Human Corticotropin-Releasing Factor Receptor Ⅰ in Prokaryotic System

目的:研究人促肾上腺皮质激素释放因子Ⅰ型受体(hCRFR1)EC1区蛋白片段在原核表达系统中可溶性表达的影响因素。方法:以pcDNA3.1-hCRFR1全长质粒为模板,PCR扩增EC1区分别编码118和88个氨基酸残基(分别对应融合蛋白GST-EC1118和GST-EC188)的片段,将其插入pGEX-4T2载体,转化大肠杆菌BL21(DE3),用IPTG诱导目标蛋白表达;采用GSTrap FF亲和纯化柱对目标蛋白进行纯化,并用Western印迹证实。结果:与GST-EC1118相比,GST-EC188可溶性表达增加,为下一步hCRFR1相关研究的开展奠定了基础。结论:hCRFR1的EC1区M1～N19及N108～V118两个片段影响该区域在原核系统中表达的可溶性,推测可能与富含疏水性氨基酸有关。

Objective： To study the factors that affect soluble expression of human corticotropin-releasing factor receptorⅠ（hCRFR1） EC1 fragment in prokaryotic system.Methods： The sequences encoding 118-and 88-amino acid of hCRFR1 EC1 region（corresponding to fusion protein GST-EC1 118 and GST-EC1 88,respectively） were ampli-fied by PCR using pcDNA3.1-hCRFR1 as template and were then inserted into pGEX-4T2 vector.The expression of target proteins was induced by IPTG by employing Escherichia coli BL21（DE3） as host cells.After purified with GSTrap FF affinity chromatography column,the proteins were identified using anti-GST antibody.Results： Compared to GST-EC1 118,soluble expression of GST-EC1 88 was increased,which provides basis for further research of hCRFR1.Conclusion： The fragments M 1 ～N 19 and N 108 ～V 118 affect solubility of hCRFR1 EC1 region in prokaryotic expression system,which may be associated with the abundant hydrophobic amino acids in this region.