A Single Polyphosphate Kinase-Based NTP Regeneration System Driving Cell-Free Protein Synthesis

20 August 2019, Version 2
This content is a preprint and has not undergone peer review at the time of posting.


Current reconstituted cell-free protein synthesis systems regenerate the ATP and GTP required for peptide bond formation using phosphocreatine and three coupled kinase reactions. Here, we replace the three-kinase system with a single polyphosphate kinase, resulting in a comparable protein yield and enabling functional expression of a heat-sensitive enzyme at 37°C.


cell-free protein synthesis
polyphosphate kinase
NTP regeneration
functional protein expression

Supplementary materials

Supporting Information V2


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