ArticleACS synthetic biology2014
Cell-free protein synthesis from a release factor 1 deficient Escherichia coli activates efficient and multiple site-specific nonstandard amino acid incorporation.
Article in ACS synthetic biology, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 69 papers, 1 of them a synthesis that pooled it.
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Who cites it
69 citing papers in PubMed, 1 synthesis or guideline pooled it.
- Cell-Free Approach for Non-canonical Amino Acids Incorporation Into Polypeptides.Frontiers in bioengineering and biotechnology · 2020Pooled it
- Purification of post-transcriptionally modified tRNAs for enhanced cell-free translation systems.Nucleic acids research · 2026Article
- High throughput screening of eukaryotic release factor 1 variants to enhance noncanonical amino acid incorporation.bioRxiv : the preprint server for biology · 2025Article
- Enhancing thermostability of Moloney murine leukemia virus reverse transcriptase through greedy combination of multiple mutant residues.Bioresources and bioprocessing · 2025Article
- Cell-Free Gene Expression: Methods and Applications.Chemical reviews · 2025Review
- Cell-free protein synthesis platforms for accelerating drug discovery.Biotechnology notes (Amsterdam, Netherlands) · 2025Review
- Promoting the production of challenging proteins via induced expression in CHO cells and modified cell-free lysates harboring T7 RNA polymerase and mutant eIF2α.Synthetic and systems biotechnology · 2024Article
- Xenobiology for the Biocontainment of Synthetic Organisms: Opportunities and Challenges.Life (Basel, Switzerland) · 2024Review
- Enabling technology and core theory of synthetic biology.Science China. Life sciences · 2023Review
- Cell-Free Expression of a Therapeutic Protein Serratiopeptidase.Molecules (Basel, Switzerland) · 2023Article
- Crystal Structure of Pyrrolysyl-tRNA Synthetase from a Methanogenic Archaeon ISO4-G1 and Its Structure-Based Engineering for Highly-Productive Cell-Free Genetic Code Expansion with Non-Canonical Amino Acids.International journal of molecular sciences · 2023Article
- The Use of Cell-free Protein Synthesis to Push the Boundaries of Synthetic Biology.Biotechnology and bioprocess engineering : BBE · 2023Review
- Cell-Free Display Techniques for Protein Evolution.Advances in biochemical engineering/biotechnology · 2023Article
- Translation initiation consistency betweenFrontiers in bioengineering and biotechnology · 2023Article
- Advancing synthetic biology through cell-free protein synthesis.Computational and structural biotechnology journal · 2023Review
- Incorporation of Non-Canonical Amino Acids into Antimicrobial Peptides: Advances, Challenges, and Perspectives.Applied and environmental microbiology · 2022Review
- Ribosome-mediated biosynthesis of pyridazinone oligomers in vitro.Nature communications · 2022Article
- An efficient cell-free protein synthesis platform for producing proteins with pyrrolysine-based noncanonical amino acids.Biotechnology journal · 2022Article
- Toward efficient multiple-site incorporation of unnatural amino acids using cell-free translation system.Synthetic and systems biotechnology · 2022Article
- Best Practices for DNA Template Preparation Toward Improved Reproducibility in Cell-Free Protein Production.Methods in molecular biology (Clifton, N.J.) · 2022Article
9 more citing papers are in PubMed but not listed here.
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6 authors.
Funding
Abstract
Site-specific incorporation of nonstandard amino acids (NSAAs) into proteins enables the creation of biopolymers, proteins, and enzymes with new chemical properties, new structures, and new functions. To achieve this, amber (TAG codon) suppression has been widely applied. However, the suppression efficiency is limited due to the competition with translation termination by release factor 1 (RF1), which leads to truncated products. Recently, we constructed a genomically recoded Escherichia coli strain lacking RF1 where 13 occurrences of the amber stop codon have been reassigned to the synonymous TAA codon (rEc.E13.ΔprfA). Here, we assessed and characterized cell-free protein synthesis (CFPS) in crude S30 cell lysates derived from this strain. We observed the synthesis of 190±20 μg/mL of modified soluble superfolder green fluorescent protein (sfGFP) containing a single p-propargyloxy-L-phenylalanine (pPaF) or p-acetyl-L-phenylalanine. As compared to the parent rEc.E13 strain with RF1, this results in a modified sfGFP synthesis improvement of more than 250%. Beyond introducing a single NSAA, we further demonstrated benefits of CFPS from the RF1-deficient strains for incorporating pPaF at two- and five-sites per sfGFP protein. Finally, we compared our crude S30 extract system to the PURE translation system lacking RF1. We observed that our S30 extract based approach is more cost-effective and high yielding than the PURE translation system lacking RF1, ∼1000 times on a milligram protein produced/$ basis. Looking forward, using RF1-deficient strains for extract-based CFPS will aid in the synthesis of proteins and biopolymers with site-specifically incorporated NSAAs.
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