【Poster_MBSJ 2015】 In vitro production of Fab, Toxin, and Immunotoxin using PUREfrex® 2.0 with increased productivity
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PUREfrex® 2.0 / DsbC Set / DnaK Mix / scFv / Fab antibody / protein toxin / Gelonin / PE38 / antibody toxin conjugate / Immunotoxin
The PURE system is a cell-free protein synthesis system reconstituted from only purified factors involved in transcription and translation in E. coli. The PURE system has features not found in other expression systems, such as almost no contamination of RNase or protease, and the composition of the reaction can be easily changed according to the purpose. Based on that concept, we have improved the preparation method of the components and developed new PURE system (PUREfrex® 1.0) in which the residual amount of lipopolysaccharide (LPS) is reduced to 1/1000 compared to the conventional method. It was also shown that, by adding molecular chaperones and other proteins, proteins with disulfide bonds and proteins that are easily insolubilized can be synthesized as active form. Furthermore, by optimizing the composition of the reaction solution, we succeeded in dramatically improving the protein synthesis ability of PUREfrex (PUREfrex® 2.0).
Here, as an example of application of PUREfrex® 2.0, anti-HER2 Fab, protein toxin (Gelonin, PE38, etc.) and Immunotoxin linking them by a linker were synthesized and their functions were evaluated. The amount of Fab synthesized by PUREfrex® 2.0 was about 0.5 mg/mL, which showed the same binding activity as Fab produced in E. coli (EC50 = 0.5 to 0.6 nM). Protein toxins were synthesized as soluble proteins, and, they showed inhibiting effect of protein synthesis in the assay using eukaryotic cell-free protein synthesis system. The Immunotoxins showed both function of Fab and protein toxins. When the unpurified products from PUREfrex® 2.0 reaction was added to the HER2-highly expressed cell line SK-BR-3, Fab and the protein toxin alone had no cell proliferation inhibitory effect. However, it was confirmed that Immunotoxin had an inhibitory effect on cell proliferation depending on the amount added.
These results indicate that PUREfrex® 2.0 can be used to synthesize complex proteins easily, such as Immunotoxin, in sufficient amounts for functional evaluation.