Ricin A chain reaches the endoplasmic reticulum after endocytosis.
Ricin is a potent ribosome inactivating protein and now has been widely used for synthesis of immunotoxins. To target ribosome in the mammalian cytosol, ricin must firstly retrograde transport from the endomembrane system to reach the endoplasmic reticulum (ER) where the ricin A chain (RTA) is recognized by ER components that facilitate its membrane translocation to the cytosol. In the study, the fusion gene of enhanced green fluorescent protein (EGFP)-RTA was expressed with the pET-28a (+) system in Escherichia coli under the control of a T7 promoter. The fusion protein showed a green fluorescence. The recombinant protein can be purified by metal chelated affinity chromatography on a column of NTA. The rabbit anti-GFP antibody can recognize the fusion protein of EGFP-RTA just like the EGFP protein. The cytotoxicity of EGFP-RTA and RTA was evaluated by the MTT assay in HeLa and HEP-G2 cells following fluid-phase endocytosis. The fusion protein had a similar cytotoxicity of RTA. After endocytosis, the subcellular location of the fusion protein can be observed with the laser scanning confocal microscopy and the immuno-gold labeling Electro Microscopy. This study provided important evidence by a visualized way to prove that RTA does reach the endoplasmic reticulum.
Department of Biochemistry and Molecular Biology, Zhejiang University Medical School, Hangzhou 310006, PR China.
Qiong
Q
Liu
Jinbiao
J
Zhan
Xinhong
X
Chen
Shu
S
Zheng
D015153:Blotting, Western; D004705:Endocytosis; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D010957:Plasmids; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism / Q000633:toxicity; D012276:Ricin / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity
D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012276:Ricin