免疫毒素ScFv(anti HER2)-PE38的复性与纯化

目的建立免疫毒素ScFv(anti HER2)-PE38的分离纯化工艺。方法免疫毒素ScFv(anti HER2)-PE38变性后,进行金属螯合柱层析复性和纯化,再用分子筛层析进行精纯,SDS-PAGE分析纯化产物。结果包涵体形式存在的免疫毒素ScFv(anti HER2)-PE38以8 mol/L尿素溶解效果较好;当复性梯度为稀释液从0到100%,共用时150 min,流速为2 ml/min时,复性效果较好,复性后的目的蛋白纯度可达95%以上;以凝胶柱Superdex 200进行分子筛层析效果较好,纯化产物的纯度可达98%以上。结论已建立了免疫毒素ScFv(anti HER2)-PE38的分离纯化工艺,为进一步的研究奠定了基础。

Renaturation and Purification of Immunotoxin ScFv(anti HER2)-PE38

Objective To develop a procedure for purification of immunotoxin ScFv(anti HER2)-PE38.Methods Immunotoxin ScFv(anti HER2)-PE38 was de-naturalized,then re-naturalized and purified by metal chelate column chromatography,and further purified by molecular sieve chromatography.The purified product was analyzed by SDS-PAGE.Results The immunotoxin ScFv(anti HER2)-PE38 in a form of inclusion body was effectively dissolved with 8 mol / L urea.The optimal gradient of diluent,time and flow rate for renaturation were 0 ～ 100%,150 min and 2 ml / min respectively.The purity of target protein after renaturation reached more than 95%.The molecular sieve chromatography with Superdex 200 showed satisfactory effect,by which the purity of purified product reached more than 98%.Conclusion A procedure for purification of immunotoxin ScFv(anti HER2)-PE38 was developed,which laid a foundation of further study.