A trans-Golgi network retention signal YQRL fused to ricin A chain significantly enhances its cytotoxicity.
Ricin enters the cells by receptor-mediated endocytosis, followed by translocation across the membranes of intracellular organelles. A trans-Golgi retention peptide signal YQRL was fused to the C-terminus of ricin A chain (RTA) by polymerase chain reaction. The recombinant RTA and RTA-YQRL were expressed in Escherichia coli using plasmid pKK223.3 under the control of a tac promoter. The recombinant proteins were purified by affinity chromatography on a Blue-Sepharose 6B column. The cytotoxicities of RTA and the fusion toxin RTA-YQRL were measured by the MTT assay in HeLa, SKOV-3, and WISH cells following fluid-phase endocytosis. The rRTA-YQRL was 2-, 10-, and 40-fold more cytotoxic than rRTA itself in the three cell lines, respectively. The results indicate that addition of a TGN retention signal YQRL to the C-terminus of RTA can markedly increase its cytotoxicity, suggesting TGN may play an important role in the intracellular routing and translocation of RTA.
Zhan
Jinbiao
J
Department of Biochemistry, Zhejiang University Medical School, 353 Yan-An Road, Hangzhou 310006, PR China. jzhan2k@cmm.zju.edu.cn
Ge
Liping
L
Shen
Jiangen
J
Wang
Keyi
K
Zheng
Shu
S
D000595:Amino Acid Sequence; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D012276:Ricin / Q000235:genetics / Q000378:metabolism / Q000633:toxicity*; D021601:trans-Golgi Network / Q000378:metabolism*
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D012276:Ricin