XynⅢ包涵体的纯化与变复性研究

Study of Purification, Denaturation and Renaturation of xynⅢ's Inclusion Body

常规诱导表达后收集的粗包涵体经 3mol/L尿素洗涤加上表面活性剂TritonX -10 0作用 ,可将包涵体中xynⅢ的含量从 3 7%提高到 92 .4% ;8mol/L尿素可将包涵体完全溶解 ;蛋白浓度在 0 .2～ 0 .2 5mg/mL时 ,pH4.5 5 0mmol/LNaOAc与pH 7.5 2 0mmol/LTris HCl缓冲液复性效果相当 ;降低蛋白浓度 ,则是pH 7.5 2 0mmol/LTris HCl缓冲液复性效果相对较好 ;复性过程中加入DTT、EDTA、尿素、木糖等处理复性效果没有明显提高 ;pH7.5 2 0mmol/LTris HCl、0 .0 1mg/mL蛋白浓度条件下 。

XynⅢ was cloned and expressed for the first time in our lab, and the purification, denaturation and renaturation of its inclusion body were studied in the present paper. XynⅢ inclusion body would be produced in great quantity under 37℃, 220?rpm in LB medium added with IPTG to the final concentration of 1?mmol/L and expressed for 3h. Cells were harvested, sonicated and centrifuged after expression under this condition, and the pellet (crude xynⅢ inclusion body) was washed by 4 successive steps as follows: pH 7 5,20?mmol/L Tris-HCl, Triton X-100, pH 7 5,20?mmol/L Tris-HCl combined with 3?mol/L Urea and at last pH 7 5,20?mmol/L Tris-HCl. Xyn Ⅲ content in the pellet increased from 37% to 92 4%. This pellet could be dissolved completely in 8?mol/L Urea. As to the renaturation effects, they were similar in 50?mmol/L NaOAc buffer system at pH 4 5 and in 20?mmol/L Tris-HCl buffer system at pH 7 5 when the inclusion body protein content was from 0 2 to 0 25?mg/mL. This was reported for the first time that inclusion body could be renaturated in an acid condition. The renaturation effect of 20?mmol/L Tris-HCl buffer system at pH 7 5 would be better when the inclusion body protein content decreased, and it did not increased when this renaturation system containing 1?mmol/L DTT, or 1?mmol/L EDTA, or 2?mol/L Urea, or 1?mmol/L xylose etc. Renaturation effect would be the best when in 20 mmol/L Tris-HCl buffer system at pH 7 5 with the inclusion body protein content was 0 01?mg/mL.