A simplified and highly efficient cell-free protein synthesis system for prokaryotes

Reviewer #1 (Public review):

Summary:

The authors presented a simplified E. coli cell-free protein synthesis (eCFPS) system that reduces core reaction components from 35 to 7, improving protein expression levels. They also presented a "fast lysate" protocol that simplifies extract preparation, enhancing accessibility and robustness for diverse applications.

Strengths:

The authors present a valuable new protocol for eCFPS, which simplifies its application.

Weaknesses:

The authors only provided the data for optimization, leaving the underlying mechanism that explains the phenomena unexplained.

Reviewer #2 (Public review):

Summary:

The authors have made a convincing argument that the current system of in vitro translation using E. coli extracts can be significantly optimized to work with much lesser components, while maintaining activity. They have showcased their improved activity using not only physical but also functional readouts.

Strengths:

The experiments are designed in a very logical and easy-to-understand manner, which makes it easier not only to follow the paper but also to reproduce the results. Functional assays with the synthesized proteins are a good way to demonstrate functionality and applicability of the system.

Weaknesses:

The production of the lysate requires special instrumentation, limiting accessibility. While the strengths of the study are well-emphasized, the limitations are not mentioned. Representation of some experiments could be done in a more complete manner.

Reviewer #3 (Public review):

Summary:

The authors aimed to overcome the challenges associated with complex, conventional prokaryotic cell-free protein synthesis (CFPS) systems, which require up to thirty-five components, by developing a streamlined and efficient E. coli CFPS platform to encourage broader adoption. The main objective was to reduce the number of reaction components from thirty-five to seven, while also developing an accessible 'fast lysate' preparation protocol that eliminates time-consuming runoff and dialysis steps. The authors also sought to demonstrate the robustness and translational quality of this streamlined system by efficiently synthesising challenging functional proteins, including the cytotoxic restriction endonuclease BsaI and the self-assembling intermediate filament protein vimentin.

Strengths:

This study presents several key strengths of the optimised E. coli cell-free protein synthesis system in terms of its design, performance and accessibility.

(1) The reaction mixture has been dramatically simplified, with the number of essential core components successfully reduced from up to thirty-five in conventional systems to just seven.

(2) The "fast lysate" protocol is a significant advance in terms of procedure.

(3) The system's ability to synthesise challenging, functional proteins is evidence of its robustness.

Weaknesses:

(1) Title: "A simplified and highly efficient cell-free protein synthesis system for prokaryotes".

(a) This title is misleading since one would expect a simplified and highly efficient cell-free protein synthesis system to yield similar protein levels compared to current cell-free protein synthesis systems. What this study shows is that the composition of cell-free protein synthesis systems can be simplified while maintaining a certain level of protein synthesis. Here, optimisation does not involve maintaining protein synthesis yield while simplifying the cell-free protein synthesis system; rather, it involves developing a simplified cell-free protein synthesis system. As mentioned in my comments below, this study lacks a comparison of protein levels with a typical cell-free protein synthesis system.

(b) What do the authors mean by "highly efficient"? Highly efficient compared to what experimental conditions? If one is interested in the yield of protein synthesis, is this simplified system highly efficient compared to current systems?

(2) Figures 1, 3-5 :

(a) What do relative luciferase units represent? How are these units calculated?

(b) In this system, the level of expression depends mainly on the level of NLuc transcripts and the efficiency of NLuc translation. How did the authors ensure that the chemical composition of the different eCFPS buffers only affected protein translation and not transcript levels? In other words, are luciferase units solely an indicator of protein synthesis efficiency, or do they also depend on transcription efficiency, which could vary depending on the experimental conditions?

(c) How long were the eCFPS reactions allowed to proceed before performing the luciferase activity measurement? Depending on the reaction time, the absence or presence of certain compounds may or may not impact NLuc expression. For example, it can be assumed that tRNA does not significantly affect NLuc levels over a short period of time, and that endogenous tRNA in the lysate is present at sufficient concentrations. However, over a longer period of time, the addition of tRNA could be essential to achieve optimal NLuc levels.

(d) The authors show that tRNA and amino acids are not strictly essential for the expression of NLuc, likely due to residual amounts within the cell lysate. However, are the protein levels achieved without added amino acids and tRNA sufficient for biochemical assays that require a certain amount of protein? It is important to note that the focus here is on optimising the simplicity of the buffer rather than the level of protein expression. In fact, the simplicity of the buffer is prioritised over the amount of protein produced. This should be made clear.

(e) How would the NLuc level compare if all the components were optimised individually and present in an optimised buffer, compared to a buffer optimised for simplicity as described by the authors?

(3) Line 71, Streamlining eCFPS: removal of dispensable components. This title is misleading because it creates the false impression that proteins can be produced in vitro without the addition of certain compounds. While this is true, the level of protein produced may not be sufficient for subsequent biochemical analyses. This should be made clear.

(4) Figure 2: In the legend, "(A) Protein expression levels of the eCFPS system measured at varying concentrations of KGlu and MgGlu2" would be more accurate if changed to "(A) Protein expression levels of the eCFPS system using an Nanoluciferase (NLuc) reporter DNA measured at varying concentrations of KGlu and MgGlu2".

(5) Lanes 302-303: "The thorough optimization of the seven core components was a critical step in achieving high protein expression levels". What are "high expression levels"? Compared to what?

Author response:

Thank you for overseeing the review of our manuscript and for providing the eLife Assessment and Public Reviews. We are highly appreciative of the detailed, constructive feedback from the editors and reviewers.

We acknowledge the core issues raised and we are committed to undertaking the necessary experiments and textual revisions to address every critique.

Here is a summary of the key revisions we plan to undertake to address the major points raised:

(1) Absolute yield comparison and efficiency clarification (eLife Assessment, R#3)

We will perform new quantitative experiments to provide the absolute protein yield of our optimized eCFPS system and benchmark it against a published, widely recognized high-yield CFPS protocol. This will directly address the central requirement for industry comparison and strengthen the claim of "high efficiency." Furthermore, we will revise the manuscript's terminology, especially in the title and abstract, to accurately reflect the system's success in "streamlining" and "robustness" in addition to performance.

(2) Mechanistic rationale for simplification (eLife Assessment, R#1)

We will substantially expand the Discussion to provide a mechanistic explanation for why activity is maintained after removing up to 28 components. This analysis will focus on the retention of endogenous metabolic enzymes and residual factors within the "Fast Lysate," citing relevant literature (e.g., Yokoyama et al., 2010, as suggested by R#1) to support the role of metabolic pathways in compensating for the lack of exogenous tRNA, CTP/UTP, and specific amino acids.

(3) Transcription-translation coupling (R#3)

To address the concern that expression changes might be due to transcription rather than translation efficiency, we will perform control experiments to monitor mRNA levels under key optimized conditions. This will help confirm that the observed efficiency changes are primarily attributable to translation.

(4) Data presentation and completeness (R#2)

We will revise the presentation of data in figures (e.g., Figure 2) to use appropriate graph types for discrete data and ensure all units, incubation times, and conditions are clearly and consistently specified. Furthermore, we will add a paragraph to the Discussion addressing the study's limitations, specifically the potential implications of DTT removal for certain protein types.

We are confident that these planned revisions will address the reviewers' recommendations and result in a stronger manuscript.