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CFPS technology efficiently incorporates non‑natural amino acids, opening up new frontiers in scientific research!
Release date:
2024-08-20
Non‑natural amino acids (ncAAs) are a class of specialized amino acids that are not constrained by the genetic code. They possess unique spatial conformations and chemical properties, and by precisely incorporating them into proteins, we can endow these proteins with novel chemical, physical, and biological activities. In recent years, driven by rapid advances in biotechnology and medicine, non‑natural amino acids have demonstrated tremendous application potential in areas such as biological research, drug development, and medical diagnostics. Cell‑free protein synthesis (CFPS), as an innovative protein expression platform, has greatly facilitated the production of engineered proteins; by integrating ncAAs, it has enabled the development of versatile strategies that can markedly enhance the efficiency of ncAA incorporation.
Today, we would like to share with you a paper published in the journal Frontiers in Bioengineering and Biotechnology, titled “Emerging Methods for Efficient and Extensive Incorporation of Non-canonical Amino Acids Using Cell-Free Systems.”
I. Research Background
Cell-free protein synthesis (CFPS) has emerged as an efficient approach for producing recombinant proteins in specialized applications. By bypassing the cellular membrane barrier and the constraints imposed by cell viability, CFPS offers distinct advantages over in vivo protein expression: 1) Its open‑system nature allows researchers to precisely manipulate molecular components; 2) With no need to account for cell viability, toxic reagents and proteins that are difficult to express can be employed in CFPS, even under reaction conditions that are not biocompatible; 3) The absence of live cells ensures enhanced biosafety. These attributes also confer significant benefits for non‑canonical amino acid (ncAA) incorporation, including the ability to handle toxic ncAAs, streamline downstream processing, and circumvent the limitations associated with intracellular integration.
However, the incorporation of ncAAs into CFPS is subject to endogenous competition; to achieve precise ncAA incorporation, special codons must be reassigned. Although researchers have already re‑assigned stop and sense codons, codon competition between ncAA–tRNA complexes and endogenous biomolecules can still result in protein misfolding or truncation.
Figure 1: Schematic illustration of endogenous competition during CFPS system preparation and ncAA incorporation.
II. Strategies for Eliminating Competition
Codon competition can severely compromise the efficiency of ncAA incorporation. Currently, strategies to mitigate this competition include genome engineering, protein elimination, tRNA manipulation, and amino acid substitution.
Figure 2: Different strategies for eliminating ncAA competition
1. Genome Engineering
During protein synthesis, the amber codon (UAG) is typically recognized by release factor 1 (RF1) as a signal for translational termination. However, through genome engineering, researchers can repurpose UAG to encode non‑canonical amino acids (ncAAs). A key step in this strategy involves deleting the prfA gene, which encodes RF1; this deletion suppresses RF1 expression, thereby reducing its binding to UAG and allowing UAG to be recognized by the tRNA specific for the ncAA, enabling the incorporation of that ncAA into the growing polypeptide chain. This approach has been shown to be effective across diverse biological systems, facilitating the precise incorporation of ncAAs.
2. Protein Elimination Strategies
Because the open‑reaction environment can be directly controlled, the CFPS system is more flexible than in‑cellular translation systems. Various strategies for eliminating competition are feasible only within the CFPS framework. Among these, suppressing protein‑protein competition in CFPS represents a key approach: by modulating protein levels, one can maintain the activity of proteins essential for cell growth while simultaneously removing from the system those proteins that might otherwise compete.
Protein‑elimination strategies in CFPS systems are critical for enhancing protein synthesis efficiency and ensuring product purity. By selectively removing or inactivating specific proteins, such as RF1, the performance of CFPS systems can be optimized and the incorporation of nAAs promoted, thereby expanding the functional repertoire and application scope of proteins. The implementation of these strategies relies on advances in genetic and protein engineering, as well as the development of tailored elimination approaches targeting specific proteins.
3. tRNA manipulation
When ncAAs are incorporated into proteins via sense codons, endogenous aminoacyl‑tRNAs compete with the ncAA‑aminoacyl‑tRNAs at the ribosome during translation, resulting in partially native proteins. Directly manipulating cellular tRNAs is impractical; however, such manipulation is readily achievable in cell‑free protein synthesis (CFPS), where large numbers of sense codons can be employed to incorporate ncAAs by modulating tRNA pools. Given the one‑to‑one correspondence between tRNAs and codons, artificial codon tables—and even minimal codon sets—can be realized through tRNA engineering in CFPS.
4. Substitution of amino acids
During biosynthesis, the incorporation of non‑canonical amino acids (ncAAs) provides a means to introduce novel functionalities into proteins. However, because natural aminoacyl‑tRNA synthetases (aaRSs) exhibit relatively broad specificity for amino acids, a small subset of ncAAs that are structurally similar to canonical amino acids can be recognized by their cognate aaRSs and loaded onto native tRNAs. These structurally analogous ncAAs can then substitute for their corresponding canonical amino acids in protein function. A method known as “amino acid replacement CFPS,” as a cost‑effective and straightforward alternative to ncAA incorporation strategies, does not require engineering of either the aaRSs or the tRNAs.
III. Novel ncAAs in CFPS and an in vitro aminoacylation method
In the realm of novel ncAAs, researchers have designed and synthesized a large array of functionally specific ncAAs through chemical synthesis and genetic engineering. These ncAAs typically bear specialized chemical moieties, such as fluorescent tags or bioorthogonal reactive groups, enabling them to confer new functions or properties to proteins without disrupting endogenous biological processes.
Figure 3: The novel ncAA is used exclusively in CFPS.
In the realm of in vitro aminoacylation, optimizing reaction conditions, developing novel aminoacylating enzymes or aminoacylating reagents, and other strategies can enable the efficient and precise incorporation of ncAAs into proteins. The advancement of these approaches has not only enhanced the efficiency of ncAA incorporation in CFPS but has also broadened the diversity and scope of ncAAs that can be employed in this system.
Figure 4: In vitro aminoacylation coupled with CFPS
IV. Summary
CFPS, as a powerful platform for designing genetic codes—particularly thanks to its ability to efficiently incorporate non‑canonical amino acids—can optimize the efficiency and yield of synthetic protein production through a variety of strategies, including genome engineering, elimination of peptide release factors, tRNA manipulation, and amino acid substitution. These approaches enable multiple codons to be reassigned to distinct non‑canonical amino acids without endogenous competition, thereby providing greater flexibility in artificial protein synthesis. CFPS holds tremendous potential across several emerging fields, such as antibody therapeutics, protein labeling, and peptide‑based materials, with especially promising prospects for enhancing enzymatic activity—for instance, by integrating non‑canonical amino acids to mimic or augment critical post‑translational modifications like phosphorylation or methylation. By selecting appropriate methods, researchers can effectively integrate non‑canonical amino acids into CFPS systems, paving the way for future breakthroughs in both the technology and its applications.
In summary, CFPS holds great promise for applications in the field of non‑canonical amino acids. As the underlying technologies continue to advance and mature, the integration of CFPS with ncAAs will assume an increasingly pivotal role across drug discovery, materials science, biological research, and synthetic biology.
Currently, Perotin Bio has launched a site‑specific incorporation of non‑natural amino acids into proteins, leveraging its proprietary cell‑free protein synthesis (CFPS) platform. This milestone positions Perotin Bio as the first domestic provider to offer design, service, and manufacturing solutions based on its own proprietary CFPS system for site‑specific non‑natural amino acid incorporation.
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