表达于大肠杆菌中的人源性抗CTLA4单链抗体复性方法探索

探索表达于大肠杆菌中的人源性抗CTLA4单链抗体(Anti-CTLA4-scFv)的体外复性方法。采用稀释和透析两种复性方式,并分析影响复性得率的各种因素如复性时间、温度、适宜的氧化-还原体系对其的影响。通过非还原电泳确定复性效果,并测定蛋白含量。结果显示:含0.15mol·L-1NaCl、1μmol·L-1氧化型谷胱甘肽和3μmol·L-1还原型谷胱甘肽的50mmol·L-1Tris-HCl(pH8.0)缓冲液作为Anti-CTLA4-scFv的复性液,4℃下透析复性48~54h,可获得具有天然构象的蛋白质。该方法复性蛋白得率高,从3.9g菌体中可复性获得28mg蛋白。

The Study of In Vitro Refolding Conditions for Humanized Anti-CTLA4-scFv Expressed in E. coli

The purpose of the study is to explore the in vitro refolding protocol for humanized anti-CTLA4-scFv expressed in E. coli. The inclusion bodies are denatured and then diluted or dialyzed into a refolding buffer. We analyzed several factors affecting the refolding yield, including refolding time, temperature, and redox environment. The refolded target proteins are analyzed by non-reducing SDS-PAGE, and the concentration of refolded proteins are examined by Bio-Rad Dc Protein Assay kit. The result shows that a high yield of the protein with natural conformation can be acquired in the condition of 0.15 mol x L(-1) sodium chloride, 50 mmol x L(-1) Tirs-HCl, pH 8.0 buffer containing 1 micromol x L(-1) reduced glutathione and 3 micromol x L(-1) oxidized glutathione. The refolding time is 48 to 54 h at 4 degrees C. 28 mg refolded proteins are produced from 3.9g E. coli.