大肠杆菌表达的单链抗体柱复性的研究

In Situ Refolding of Bacterially Expressed ScFv on Column

单链抗体(single chain Fv SeFv)是由抗体重链可变区V_H和轻链可变区V_L通过一段连接肽连接而成的重组蛋白.由于其分子量小穿透力强,免疫原性低,利于基因工程操作等特点,而具有良好的应用前景.近年来国内外学者对单链抗体进行了大量研究.本室构建了抗乙肝病毒表面抗原HBsAg的单链抗体ScFv,并在大肠杆菌表达体系中以包含体形式获得高效表达,由于包含体为没有活性的变性蛋白,其纯化和复性是大量制备SeFv的重要环节.传统的稀释透析复性方法对包含体蛋白的复性效率较低,操作复杂,限制了生物技术的开发应用.为此我们对包含体表达的抗乙肝病毒表面抗原(HBsAg)SeFv的柱上在位复性进行了探索,为探讨最佳纯化过程及柱上复性的可行性,我们设计了几种纯化复性方案.A:包含体在变性条件下经铜金属整合亲合层析柱(为应用此介质纯化,在构建ScFv时加了一聚组氨酸片段)纯化后,所得产品以尿素进行梯度透析.B:在铜金属螯合亲合层析柱进行在位复性.C:溶解的包含体直接上Sephaeyl S-200进行柱复性.对几种方法的回收率,复性效果进行了比较,发现柱上复性效果良好.铜金属螯合亲合层析柱可进行在位复性,其相对复性率大于透析法,但蛋白丢失较多.原因可能是分子中的His.被固定于柱上限制了分子的自由折叠,国外有人用铜金属螯合亲和?

Purification and refolding of bacterially expressed anti HBsAg ScFv by means ofsitu refolding on column was investigated. Three refolding approaches were adopted: dialysis, insitu refolding via metal chelated chromatography and via gel filtration chromatography. The rela-tive refolding index and relative recovery of the ScFv inclusion body were compared using the threeprocedures. The results showed that the refolding on gel filtration column (sephacyl s-200) wasfar more efficient that the other two approaches, with relative refolding index of 98% and relativerecovery of 81%, illustrating the potential of this technique in production of recombinant ScFv.