Expression and purification of recombinant immunotoxin--a fusion protein stabilizes a single-chain Fv (scFv) in denaturing condition.
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 6M guanidine hydrochloride (GuHCl). It was found that the immunotoxin maintains a binding activity in denaturing condition of 6M GuHCl and the fused PE contributes to the stability of immunotoxin in such condition. Dialysis against PBS buffer after purification under 6M GuHCl keeps the binding activity of immunotoxin.
Kim
Se-Ho
SH
Antibody Engineering Lab., Central Research Center of the Korea Green Cross Corp., Gugal-Ri, Giheung-Eup, Yongin City, Kyunggi-Do 449-903, South Korea. sehokim@greencross.com
D036002:ADP Ribose Transferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000276:immunology; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002272:Carcinoembryonic Antigen / Q000276:immunology; D003001:Cloning, Molecular; D015179:Colorectal Neoplasms / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005098:Exotoxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015870:Gene Expression; D019791:Guanidine / Q000494:pharmacology; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000922:Immunotoxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D037521:Virulence Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D002272:Carcinoembryonic Antigen; D005098:Exotoxins; D019791:Guanidine; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A