Expression of ricin A chain and ricin A chain-KDEL in Escherichia coli.
Ricin and its A chains can be used to conjugate with monoclonal antibodies to prepare immunotoxins. Ricin A chain (RTA) and its modification RTA-KDEL (ER-retrieval signal) were expressed with the pKK223.3 system in Escherichia coli under control of a tac promoter. The recombinant proteins can be purified by one-step affinity chromatography on a column of Blue-Sepharose 6B. The toxicities of RTA and its mutant RTA-KDEL were evaluated by the MTT assay in HeLa, MCF, and ECV-304 cells following fluid-phase endocytosis. RTA-KDEL was somewhat more cytotoxic than RTA itself in the different cell lines. The results suggest that rRTA-KDEL may be useful for the synthesis of more potent immunotoxins.
Department of Biochemistry, Zhejiang University Medical School, 353 Yan An Road, Hangzhou 310006, PR China. jzhan2k@cmm.zju.edu.cn
Jinbiao
J
Zhan
Yongdui
Y
Chen
Keyi
K
Wang
Shu
S
Zheng
D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004705:Endocytosis / Q000502:physiology*; D042783:Endothelial Cells / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012276:Ricin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000922:Immunotoxins; D011994:Recombinant Proteins; D012276:Ricin