Abstract: In the process of troubleshooting the activity of our PURE system, we stumbled upon a surprising discovery: folinic acid — a key ingredient in the Energy Mix involved in translation initiation — doesn’t work as expected unless isomerized, either chemically beforehand, as an additional step, or in situ by an enzyme (methenyltetrahydrofolate synthetase, MTHFS) in the PURE reaction. This enzyme isn’t formally one of the 36 proteins in Protein Mix. We hypothesize it co-purifies with the other proteins during OnePot protein purification and not in commercial (36-Pot PURE) preparations. If MTHFS is absent and folinic acid is not prepared correctly, translation efficiency drops dramatically. Here’s the story of how we found this hidden helper and what it means for future PURE systems.
Folinic acid (5-formyl-THF) is one of the components in Energy Mix, the small molecule components of PURE reactions, and is crucial to the production of N-formylmethionine (fMet) by the enzyme Methionyl-tRNA formyltransferase (MTF). fMet is a modified amino acid required for translation initiation in bacterial systems like PURE. However, folinic acid cannot be used directly by MTF and first has to be converted into 10-formyltetrahydrofolate (10-formyl-THF). This process happens in two steps: (1) folinic acid is converted (chemically or enzymatically) to an intermediate product 5,10-methenyl-THF, which then (2) spontaneously hydrolyzes into 10-formyl-THF.
Reaction pathway for the production of 10-formyl-THF from folinic acid by MTHFS. The formyl group used in fMet synthesis is highlighted in yellow. We highlight an unchanging portion of the molecule in blue for visual convenience.
There are two ways to prepare folinic acid for use in PURE described in the literature:
Implied protocol (from Lavickova et al. 2019):
When we used folinic acid resuspended in water for our PURE reactions, we observed a consistent reduction in activity. In fact, our Energy Mix had higher activity when we omitted folinic acid altogether. One explanation is that omission of folinic acid reduces the total amount of salt in the PURE reaction. It is well known that the performance of PURE is sensitive to ions, and folinic acid solutions introduce additional calcium; however other mechanisms might be at play.
In contrast, folinic acid chemically converted to 5,10-methenyl-THF, yielded high activity and fast translation [see Figures 1 and 2]. We also observe that, when 5,10-methenyl-THF is included in the Energy Mix, its function diminishes significantly after one freeze-thaw cycle, indicating it may be unstable in solution.
These discrepancies led us to acknowledge that folinic acid, the more stable THF derivative, must be present in the Energy Mix and hypothesize that an additional enzyme may be allowing OnePot PURE users to convert folinic acid to the usable formyl donor—explaining how these users are able to simply dissolve folinic acid in water and still achieve high performance, unlike our experience with multi-pot preparations.
Methenyltetrahydrofolate synthetase (MTHFS, EC 6.3.3.2]), also known as 5-formyltetrahydrofolate cyclo-ligase, is the enzyme responsible for converting folinic acid to 5,10-methenyl-THF. MTHFS is a metalloprotein, binding magnesium, and utilizes ATP as a cofactor, making it well-suited as an additive for PURE.
OnePot purification workflows — where all 36 PURE proteins are co-purified — increases the likelihood that MTHFS might copurify due to protein-protein interactions. In contrast, 36-Pot workflows purify each individual component separately. 36-Pot Protein Mix may have less MTHFS carryover and thus be unable to process folinic acid. It has been reported that the distribution of contaminants in OnePot and 36-Pot PURE are distinct (Fig. S6 Lavickova et al. 2019).
To test this hypothesis, we supplemented commercial MTHFS (Prospec Bio [link]) into PURE reactions with energy mix containing folinic acid dissolved in water. The addition of MTHFS resulted in a dramatic improvement in PURE system performance, confirming that folinic acid processing is required.
Figure 1: Translation kinetics of PURE system reactions under different folinic acid conditions and MTHFS supplementation. The graph shows plamGFP fluorescence measurements over time.
Figure 2: Steady state fluorescence measurements comparing PURE system performance across different folinic acid preparations and MTHFS enzyme concentrations.