Expression, purification, and characterization of an immunotoxin containing a humanized anti-CD25 single-chain fragment variable antibody fused to a modified truncated Pseudomonas exotoxin A.
Vascular leak syndrome (VLS) is the major dose-limiting toxicity of immunotoxin therapy. In our previous study, a modified PE38KDEL, denoted PE38KDELKQK, was engineered to eliminate VLS. The PE38KDELKQK-based immunotoxin has been proved to retain potent anti-tumor activity but with a remarkable attenuation in VLS. In the present study, we have constructed and expressed a recombinant immunotoxin CD25-PE38KDELKQK containing humanized anti-CD25 single-chain antibody (scFv) genetically fused to PE38KDELKQK in Escherichia coli. After washing with buffer containing 2 M urea, the purity of inclusion body was about 82%. The denatured inclusion bodies were refolded on-column in Tris buffer (pH 8.0) containing 4mM of GSH and 1 mM of GSSG using a gradient of decreasing urea. We found that the presence of GSH/GSSG (4:1) in the on-column refolding buffer was important for efficient refolding. In addition, slow flow rate was another important factor could increase refolding. Under these conditions, the activity of the refolded protein could reach about 90% of that of the native protein. The refolded proteins were purified to homogeneity ( approximately 95% purity) by a combination of His-Ni(2+) metal affinity chromatography and gel filtration chromatography. The in vitro cytotoxicity assay indicated the purified immunotoxin CD25-PE38KDELKQK had specific cytotoxicity to CD25-positive leukemic cells comparable to wild-type CD25-PE38KDEL (wt). In contrast, CD25-PE38KDELKQK was shown to be much weaker in inducing VLS in mice than wt. The protein expression, purification, and refolding system established in this paper is important for further study on immunotoxin CD25-PE38KDELKQK.
Wang
Huajing
H
International Joint Cancer Institute, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People's Republic of China.
Dai
Jianxin
J
Li
Bohua
B
Fan
Kexing
K
Peng
Lin
L
Zhang
Dapeng
D
Cao
Zhiguo
Z
Qian
Weizhu
W
Wang
Hao
H
Zhao
Jian
J
Guo
Yajun
Y
D036002:ADP Ribose Transferases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism; D019559:Capillary Leak Syndrome / Q000209:etiology; D045744:Cell Line, Tumor; D005098:Exotoxins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000633:toxicity; D000922:Immunotoxins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity; D053645:Interleukin-2 Receptor alpha Subunit / Q000276:immunology*; D019169:Jurkat Cells; D008168:Lung / Q000166:cytology / Q000276:immunology; D051379:Mice; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D005098:Exotoxins; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D053645:Interleukin-2 Receptor alpha Subunit; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A