人β-淀粉样肽Aβ42的融合表达与纯化

Fusion expression and purification of human β-amyloid peptide Aβ_(42)

[目的]建立一种快速可靠、获取足量和高纯度的人β-淀粉样肽(Aβ_(42))的方法。[方法]首先利用重叠PCR技术扩增获得Aβ_(42)基因全长。随后将基因连入p GEX-4T-1载体,利用GST系统表达融合蛋白。分别在16℃、25℃、30℃和37℃诱导表达,SDS-PAGE检测融合蛋白的表达情况,确定表达的最佳温度。根据优化条件进行目的蛋白的大量表达,利用Gstrap FF柱亲和纯化GST-Aβ_(42)融合蛋白。[结果]成功构建p GEX/Aβ_(42)表达载体,确定30℃为诱导表达的最佳温度。大量表达并经过纯化可获得分子量为30.7 k Da的融合蛋白。[结论]利用GST融合系统表达纯化可得到纯度超过90%的GST-Aβ_(42)融合蛋白,重组蛋白的产率约为1.2 mg/L培养基。当用凝血酶切除GST融合标签后,Aβ_(42)易聚集沉淀。

[Objective]This study is performed to establish a simple and reliable method to obtain ample and high- purity human β- amyloid peptide Aβ_（42）. [Methods]Overlap PCR was used to amplify Aβ_（42）gene,and then the gene was cloned into p GEX- 4T- 1 vector. GST expression system was used to express Aβ_（42）peptide. The recombinant protein was induced under16℃,25℃,30℃,and 37℃,respectively. SDS- PAGE was performed to examine the expression of the fusion protein and determine the best temperature for expression. The interested protein was expressed efficiently under the optimized condition,after which the GST- Aβ_（42）fusion protein was purified using Gstrap FF affinity chromatography. [Results]The expressing vector p GEX / Aβ_（42）was established successfully. The best temperature for soluble expression was determined at 30℃. Soluble GST-Aβ_（42）fusion protein could be expressed with 30. 7 k Da of molecular mass using prokaryotic expression system. [Conclusion]GST fusion expression system was used to obtain GST- Aβ_（42）fusion protein with over 90% of purity,and the productivity of the recombinant protein was estimated to 1. 2 mg / L medium. When GST fusion tag was removed by thrombin cleavage,Aβ_（42）was apt to aggregate.