Unraveling time-inhibition mechanisms in mammalian cell-free protein synthesis

Abstract

We present a highly efficient human HEK293-based cell-free in vitro protein synthesis (CFPS) system that produces 300 µg/ml of reporter protein. The main challenge with the CFPS assay is its limited activity for a few hours, during which all protein is synthesized. If the activity of the CFPS system can be extended, more protein can be produced. The time-dependent inhibition has been studied in the yeast system, but not much is known in the mammalian system. We used the HEK293 CFPS assay to investigate the reasons for time inhibition. We observed that the main culprit is the energy regeneration system, which is depleted quickly. We also demonstrate that the CFPS assay can be used with other mammalian cells or tissues, as evidenced by the active human neuroblastoma SH-SY5Y-based CFPS assay. We observe differences between the yeast and mammalian systems; for example, there is no need to add creatine kinase (CK) as the native CK is functional. This knowledge helps to reduce the costs of CFPS-based systems for biotechnological purposes.