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PUREfrex® 1.0 / DsbC Set / disulfide bond formation / tissue plasminogen activator (tPA)/ acid phosphatase (AppA)/ alkaline phosphatase (AP) / Fab antibody
The PURE system is a reconstituted cell-free protein synthesis system, which consists of only purified factors necessary for transcription, translation and energy regeneration. The PURE system has the unique features that it contains less contaminants such as nucleases and proteases and that composition of the reagents can be easily adjusted in accordance to the purpose. Therefore, the PURE system is now used not only in preparation of the target proteins but also in in vitro display technology such as ribosome display (RD). But, we found that the original PURE system yet contained a large quantity of lipopolysaccharide (LPS) from incompletely purified components. We modified the preparation methods of all components that were purified from E. coli and developed the new PURE system (PUREfrex™). In PUREfrex, the amount of contaminants such as LPS and RNase is reduced and then both protein synthesis activity and selection efficiency in RD were improved.
However, because PUREfrex includes only translation factors under reduced condition, it is difficult to synthesize proteins containing disulfide bonds in active form. In this study, we examined the composition of reaction mixture and synthesis condition to synthesize functionally active proteins. We used 4 proteins, which form a different number of disulfide bridges, as model proteins. As a result, we succeeded to synthesize all 4 proteins with activities using PUREfrex supplemented with oxidized glutathione (GSSG) and DsbC protein (disulfide bond isomerase) and optimum concentration was different from each target protein.
This result shows that disulfide bonds-containing proteins can be also synthesized in active form using PUREfrex supplemented with optimized concentration of GSSG and DsbC.