重组抗血红素ScFv包涵体蛋白的复性及纯化研究

Renaturation and Purification of Single Chain Fv Fragment of Monoclonal Antibody Against Haemachrome

抗血红素多二硫键ScFv在大肠杆菌中绝大多数表达产物为包涵体,为了获得可溶性的具有生物活性的ScFv,摸索了不同的复性条件,包括透析法、稀释和层析相结合的方法。研究发现,先对溶解的变性ScFv溶液稀释,进行初步的蛋白质复性,再利用Sephadex G-25凝胶层析进一步复性、降低变性剂浓度和纯化,至少可以得到95％纯度,产率为150mg／L的目标蛋白,通过一次凝胶过滤层析,达到了去除变性剂、复性及纯化ScFv蛋白三种目的,为多二硫键ScFv在大肠杆菌中的表达和纯化提供了一种经济可行的方法。

Most of single chain Fv fragment (ScFv) of monoclonal antibody deposited as insoluble inclusion bodies during expression in E. coli. In order to convert inactive and misfolded inclusion body ScFv into soluble products, the renaturation and purification procedure was developed, recombinant ScFv was redissolved, diluted, and was directly loaded onto a Sephadex G-25 column. The size-exclusion chromatography provided a favorable environment for the proteins to renature, it also spatially constrained partially refolded ScFv from aggregating and diffusing toward each other, promoted renaturation. In the size-exclusion chromatography, the ScFv protein flowed faster and was eluted earlier than the denaturant, this will allow the protein sample to pass through decreasing denaturant concentrations, which promotes ScFv refolding and removes denarurant. During this process, ScFv was simultaneously purified, and at least 150mg/L (with the purity more than 95 %) soluble ScFv was obtained. The strategy provides an economic and novel approach for the production of ScFv from inclusion body proteins.