eEF1A1基因克隆、原核分泌表达及融合蛋白纯化

Cloing,Secretion Expression and Purification of eEF1A1 in Escherichia coli

eEF1A1作为蛋白合成中的重要翻译延伸因子,可与多种功能性蛋白如F-actin、BPOZ-2结合,并在细胞凋亡、蛋白降解方面起重要作用。以往原核基因工程蛋白表达系统大多为包涵体表达的变性分子,需要复性。为了获得eEF1A1原核分泌性可溶性蛋白分子,克隆了人eEF1A1蛋白编码序列(约1 300 bp),并成功构建pET22b-A原核分泌表达重组质粒,转化到大肠杆菌BL21(DE3)菌株,0.4 mmol/L终浓度IPTG诱导,经不同温度下包涵体与胞浆蛋白组分分析,快速明确蛋白表达情况,即诱导4 h后,37℃表达于包涵体组分,在30℃分泌表达至胞浆组分。通过His-Trap亲和层析纯化柱进行线性洗脱,Bradford法测定蛋白浓度高达620 mg/mL,SDS-PAGE分析纯度约为95%,蛋白大小符合50 kD,Western blotting显示目的蛋白能被eEF1A1抗体识别;质谱分析证实重组蛋白为人eEF1A1蛋白分子。为进一步研究其与重要功能性蛋白的相互作用及在细胞凋亡和蛋白降解中的作用奠定基础。

As a central molecular in protein biosynthesis,eEF1A1 had been reported more possible roles in apoptosis and protein degradation because of its interaction with paramount functional protein such as F-actin and BPOZ-2. Most reported that protein formed as inclusion body by genetic engineering. To get soluble protein, we cloned eEF1 A1 gene（ about 1 300 bp）and constructed the secretion expression plasmid pET22b-A. In the study of expression form under different condition ,we quickly and clearly knew that E. coli culti- vated at 30℃ about 4 h can prevent the formation of inclusion bodies. Then ＇after enriched in cytoplasm at 30℃, the protein was extracted and purified by His-Trap Crude column chromatography. SDS-PAGE analyze that the protein was about 50 kD and was up to 620 mg/L,which can be detected by eEF1AI antibody in Western blotting. Moreover, mass spectrum result showed that it＇ s correctly eukaryotic translation elongation factor 1 alpha 1 [ Homo sapiens ]. This work has provided the foundation for research on eEF1 A1 ＇ s inter- action with paramount functional protein and its function in cell apoptosis and protein degradation.