Optimizing Human Cell-Free System for Efficient Protein Production

We present a highly efficient human HEK293-based cell-free protein synthesis (CFPS) system capable of producing up to 300 μg/ml reporter protein. One of the limitations of the CFPS systems with respect to protein yield has been the decline of the protein-synthesizing activity of the system upon prolonged incubation. Though factors contributing to this decline in activity have been investigated in yeast, little is known about the factors in mammalian systems. We find that a rapid depletion of the components of the energy-regeneration system is a major factor behind the decreasing protein-synthesis activity in the HEK293-derived system. In addition, we demonstrate that a functional CFPS system can be prepared from other mammalian cell lines as evidenced by our use of a human neuroblastoma SH-SY5Y-derived CFPS system. We also find that exogenous creatine kinase (CK) is dispensable for the functionality of the energy-regeneration system in HEK293 due to the presence of a sufficiently high endogenous CK activity in an HEK293 cell-free extract.