Soluble Expression And Purification Strategy Research Of Anti-VEGF Fab Antibody In E.Coli

As a kind of micromolecule antibodies,Fab fragments have distinct advantages over IgG in clinical application.Escherichia coli(E.coli)is the most important expression system for Fab fragments,however,heterologous proteins are often formed as inactive inclusion bodies in E.coli.Protein refolding may be time-and resources-consuming step during purification.So people always focus on the soluble expression of target protein in E.coli.In this research,we take anti-VEGF Fab fragments for example,and study strategies of soluble expression and purification of recombinant Fab in E.coli.Then we analyze the qualities of the purified production so that can provide experience and reference to soluble expression of other recombinant Fab fragments and lay the foundation for industrial manufacture.Anti-VEGF Fab fragments can inhibit the angiogenesis promoted by VEGF,which results in the application in the treatment of eye diseases such as diabetic macular edema.So we used expression vectors carried the anti-VEGF Fab fragments genes which has been designed to secret the expressed product into the pariplamic space of E.coli and acquired products with high purity by purification.It was validated that the soluble expression could be improved by using the phoA expression system,coexpression with molecular chaperones,adding osmolytes and reducing induced temperature.And the target protein with purity >99% was obtained after purification by Capto L affinity chromatograghy.The purified production were analysed by SEC-HPLC,MALDI-TOF MS,BIAcore SRP assay and vascular endothelial cell activity assay.The results indicated that the purity of the product was 99.34%,the molecular weight of the product was 48.35 kDa which was closed to the theoretical value,the affinity of the prodcut to VEGF was 10.2 pmol/L,higher than that of the commercial ranibizumab,and the EC50 of the product was 24.07mg/mL,so the activity of it was 94.35% of that of reference standard which was considered as the same level.