Expression and purification of recombinant immunotoxin - a fusion protein stabilizes a single-chain Fv (scFv) in denaturing condition

摘要

Carcinoembryonic antigen (CEA) is expressed at greatly increased levels in nearly all human colorectal carcinomas. Anti-CEA antibodies have been proved to be useful for targeting several cancer types known to express CEA. A recombinant immunotoxin was constructed, in which the cell-binding domain of Pseudomonas exotoxin (PE) was replaced with the single-chain Fv (scFv) of anti-CEA monoclonal antibody for targeting to colorectal carcinomas. This single-chain immunotoxin was expressed in E. coli and purified under denaturing condition of 6 M guanidine hydrochloride (GuHCl). It was found that the immunotoxin maintains a binding activity in denaturing condition of 6 M GuHCl and the fused PE contributes to the stability of immunotoxin in such condition. Dialysis against PBS buffer after purification under 6 M GuHCl keeps the binding activity of immunotoxin. (C) 2002 Elsevier Science (USA). All rights reserved. [References: 24]