Counteracting ros in extract
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Background: The intracellular environment is maintained in a reduced state and cells spend energy to combat the effects of reactive oxygen species (ROS) such as hydrogen peroxide by reducing these radicals (among others) to water. ROS can cause irreversible damage to DNA and proteins such as polymerases and ribosomes, both of which are in finite supply in the context of TX TL. Thus since metabolism is activated in a TX TL reaction, it would not be unreasonable to presume that ROS species are generated in a standard TX TL reaction and thus may be contributing to the 'death' of TX TL reactions by causing damage to polymerases and or ribosomes and thus affecting the protein expression ability of a TX TL reaction.
Glutathione (GSH) is the main antioxidant found in cells and serves to neutralize radicals (ROS) found inside the cell by reducing them to water. At the same time as reducing the radicals, GSH itself becomes oxidized into its disulfide form (GSSG) and is no longer active. To become active again, it must be reduced itself, which is done by the enzyme glutathione reductase (GR). GR transfers electrons from NADPH to GSSG to convert it back into the active form GSH.
Thus the mechanism requires the enzyme GR, the antioxidant GSH, the presence and a continued source of the co-factor NADPH to function continuously. Since GR is a ubiquitous enzyme, it should be contained / found in most if not all cell extracts generated for TX TL reactions and thus does not to be added.
Lastly, the biosynthesis of GSH is limited by the presence of the amino acid L-cysteine. Thus it was hypothesized that the addition of L-cysteine in the presence of GSH would be beneficial. Furthermore the attached graph (publication info included) showed that the addition of L-cysteine increased protein production, unlikely to be solely due to the fact that L-cysteine is a rare amino acid, as the addition of other rare amino acids had no effect.
Additional Background: A highly efficient and economical cell-free protein synthesis system using the S12 extract of Escherichia coli
Note: Although the co-factor NADPH is required for continued replenishment of GSH, it was not added in these experiments!!!!!
Results: Day 1: High concentrations of glutathione outright killed the reaction with no positive effect seen. Concentrations of glutathione tested = [1.25, 2.5, 5.0, 10.0, 20mM] High concentrations of L-cysteine impaired the reaction, but did not have as a dramatic effect as glutathione. Concentrations of L-cysteine tested = [0.5, 1.0, 2.0, 4.0, 8.0mM].
Day 2: given the results from day 1, three different glutathione concentrations were tested across the range of L-cysteine concentrations. Shown are the best results.
Three glutathione concentrations tested = [0.25, 0.5, 1.25mM]. Concentrations of L-cysteine tested = [0.5, 1.0, 2.0, 4.0, 8.0mM] in combination with the three glutathione concentrations tested.
Overall: A small increase in output is seen relative to the control.
- Again NADPH was not added in these results.***
