PUREfrex® is a reconstituted cell free protein synthesis kit developed based on the PURE system. Now the productivity of PUREfrex® is up to 1 mg/mL. However, depending on the protein, it may be difficult to synthesize as functional and soluble form by using only PUREfrex®.

In such cases, we can apply chaperones (DnaK and GroE) for correct folding and isomerases (DsbC and PDI) for disulfide bond formation as additives for PUREfrex®. Here, we introduce examples of protein synthesis that could not be solved by these additives alone, but could be solved by adding other additives or synthesizing at lower temperature.

We mainly show examples of human acetylcholinesterase (hAChE) synthesis. hAChE has three intramolecular and one intermolecular disulfide bonds. When hAChE was synthesized by using PUREfrex® with PDI, active hAChE was obtained but there was a problem in solubility. The synthesis under the presence of surfactant Brij 58 increased the solubility of hAChE but decreased its activity. Next, we examined whether the difference in the solubility of hAChE is depending on the synthesis temperature. The solubility of the product was less than 10% at 30ºC although the amount of the product was high. In contrast, the product synthesized at 25 ºC was almost soluble although the amount was low. It was also found that DnaK/DnaJ/GrpE are essential for the synthesis of soluble hAChE even at lower temperature.

This result indicates that it is possible to synthesize difficult-to-express proteins by using PUREfrex® with optimization of conditions. In this presentation, we also discuss the results from other types of proteins and additives.