Abstract
Protein degradation is a fundamental process in all living cells and is essential to remove both damaged proteins and intact proteins that are no longer needed by the cell. We are interested in creating synthetic genetic circuits that function in a cell-free expression system. This will require not only an efficient protein expression platform but also a robust protein degradation system in cell extract. Therefore, we purified and tested the activity of E. coli ClpXP protease in cell-free transcription-translation (TX-TL) systems that used E. coli S30 cell extract. Surprisingly, our studies showed that purified ClpXP added to the TX-TL system has very low proteolytic activity. The low activity of ClpXP was correlated with the rapid consumption of adenosine triphosphate (ATP) in cell extract. We improved the activity of ClpXP in cell extract by adding exogenous ATP and an energy regeneration system. We then established conditions for both protein synthesis, and protein degradation by ClpXP to occur simultaneously in the TX-TL systems. The optimized conditions for ClpXP activity will be useful for creating tunable synthetic genetic circuits and in vitro synthetic biology.
Highlights
Protein degradation is an essential process in all living cells that is used to remove damaged or misfolded proteins and to tune gene expression by temporally controlling the concentration of regulatory proteins
Our studies revealed that ClpXP activity is poor in the extract-based system because the concentration of adenosine triphosphate (ATP) is low in the standard S30 cell extract used for TX-TL
Our results suggest that TX-TL systems supplemented with known concentrations of ClpXP and an energy regeneration system could be used to create synthetic genetic circuits that function in vesicles, enabling robust and predictable protein degradation in artificial cells
Summary
Protein degradation is an essential process in all living cells that is used to remove damaged or misfolded proteins and to tune gene expression by temporally controlling the concentration of regulatory proteins. The amount of active ClpXP in cell extract is low, resulting in a low rate of protein degradation[4] To overcome this problem, one study added the genes coding for ClpX and ClpP to the TX-TL system to increase the concentration of the ClpXP protease in cell extract, which resulted in improved degradation of the target proteins[5]. A previous report showed that adding a purified, linked-hexameric version of ClpX to a TX-TL system increased the degradation of ssrA-tagged reporter proteins, indicating that this approach may be useful for creating dynamic gene circuits[13]. Our results suggest that TX-TL systems supplemented with known concentrations of ClpXP and an energy regeneration system could be used to create synthetic genetic circuits that function in vesicles, enabling robust and predictable protein degradation in artificial cells
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