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.
Background: What does folinic acid do in PURE?
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.
There are two ways to prepare folinic acid for use in PURE described in the literature:
- Dissolve folinic acid in 50 mM β-mercaptoethanol (50 mM) and 1M HCl,
- Incubate at room temperature for 3 hr until reaction is visibly yellow (
Make Energy Mix).
The Problem: Implied folinic acid preparation results in lower PURE activity
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.
The Twist: The difference between OnePot vs. 36-Pot Purification
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).
Experimental Confirmation: Supplementing MTHFS Restores Activity
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.
Table 1: Kinetic parameters
While the performance did not fully match that observed using pre-converted 5,10-methenyl-THF, the improvement was substantial (~3x), demonstrating proof-of-concept that MTHFS activity is significant. This finding supports our hypothesis that MTHFS must be a "shadow component" unintentionally contributing to the success of some OnePot preparations.
Future directions
In this developer note, we want to emphasize a key finding: simply dissolving folinic acid in water without MTHFS does not support high-performing PURE reactions. This explains why some labs, particularly those using 36-Pot purification methods, may struggle with folinic acid-based energy mixes.
Our next step is to express bacterial MTHFS and integrate it into our PURE mixture. Based on our initial results, we encourage adding MTHFS to PURE systems to ensure a more stable energy mix, as folinic acid is a more stable THF derivative compared to 5,10-methenyl-THF. This could improve long-term reproducibility and performance.
Finally, we want to highlight a tradeoff in PURE system design. One-pot purification is a cheaper and more convenient approach that many researchers rely on, but it may come at the cost of a less-defined system, where unexpected enzyme carryover (like MTHFS) influences performance. Understanding these hidden factors is crucial as the PURE community continues refining and optimizing the system.
References
Research notebook
- Shimizu, Y. & Ueda, T. PURE Technology. Methods in Molecular Biology 11–21 (2009) [link]
- Lavickova, B. & Maerkl, S. J. A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System. ACS Synthetic Biology vol. 8 455–462 (2019). [link]
- Ecocyc (6.3.3.2)
ecocyc.org
Credits
- Yemo Ku (b.next)