ArticleBiotechnology progress
A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell-free protein synthesis systems.
Article in Biotechnology progress. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 42 papers, 1 of them a synthesis that pooled it.
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Who cites it
42 citing papers in PubMed, 1 synthesis or guideline pooled it, 106 citations in OpenAlex.
- Cell-Free Approach for Non-canonical Amino Acids Incorporation Into Polypeptides.Frontiers in bioengineering and biotechnology · 2020Pooled it
- Egg-Derived Anti-SARS-CoV-2 Immunoglobulin Y (IgY) With Broad Variant Activity as Intranasal Prophylaxis Against COVID-19.Frontiers in immunology · 2022Trial
- Article
- Design-driven optimization of low-cost reagent formulations for reproducible and high-yielding cell-free gene expression.Nature communications · 2026Article
- The landscape of antibody production systems: recombinant expression for research, diagnostics and therapy.Frontiers in bioengineering and biotechnology · 2026Review
- On-demand insulin manufacturing using cell-free systems with an "on-column" conversion approach.New biotechnology · 2025Article
- Developing anACS synthetic biology · 2025Article
- Innovations, Challenges and Future Directions of T7RNA Polymerase in Microbial Cell Factories.ACS synthetic biology · 2025Review
- Cell-Free Systems to Mimic and Expand Metabolism.ACS synthetic biology · 2025Review
- Cell-free expression system: a promising platform for bacteriophage production and engineering.Microbial cell factories · 2025Review
- Cell-Free Gene Expression: Methods and Applications.Chemical reviews · 2025Review
- Chemiosmotic nutrient transport in synthetic cells powered by electrogenic antiport coupled to decarboxylation.Nature communications · 2024Article
- A cell-free strategy for host-specific profiling of intracellular antibiotic sensitivity and resistance.npj antimicrobials and resistance · 2023Article
- An approach to rapid distributed manufacturing of broad spectrum anti-viral griffithsin using cell-free systems to mitigate pandemics.New biotechnology · 2023Article
- A rapid cell-free expression and screening platform for antibody discovery.Nature communications · 2023Article
- Rapid biosynthesis of glycoprotein therapeutics and vaccines from freeze-dried bacterial cell lysates.Nature protocols · 2023Review
- Opportunities to accelerate extracellular vesicle research with cell-free synthetic biology.Journal of extracellular biology · 2023Article
- Automated design of protein-binding riboswitches for sensing human biomarkers in a cell-free expression system.Nature communications · 2023Article
- An Integrated In Vivo/In Vitro Protein Production Platform for Site-Specific Antibody Drug Conjugates.Bioengineering (Basel, Switzerland) · 2023Article
- A Low-Cost, Thermostable, Cell-Free Protein Synthesis Platform for On-Demand Production of Conjugate Vaccines.ACS synthetic biology · 2023Article
Corrections and comments
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Authors and funding
10 authors at 1 institution in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Cell-free protein synthesis (CFPS) systems allow for robust protein expression with easy manipulation of conditions to improve protein yield and folding. Recent technological developments have significantly increased the productivity and reduced the operating costs of CFPS systems, such that they can compete with conventional in vivo protein production platforms, while also offering new routes for the discovery and production of biotherapeutics. As cell-free systems have evolved, productivity increases have commonly been obtained by addition of components to previously designed reaction mixtures without careful re-examination of the essentiality of reagents from previous generations. Here we present a systematic sensitivity analysis of the components in a conventional Escherichia coli CFPS reaction mixture to evaluate their optimal concentrations for production of the immunoglobulin G trastuzumab. We identify eight changes to the system, which result in optimal expression of trastuzumab. We find that doubling the potassium glutamate concentration, while entirely eliminating pyruvate, coenzyme A, NAD, total tRNA, folinic acid, putrescine and ammonium glutamate, results in a highly productive cell-free system with a 95% reduction in reagent costs (excluding cell-extract, plasmid, and T7 RNA polymerase made in-house). A larger panel of other proteins was also tested and all show equivalent or improved yields with our simplified system. Furthermore, we demonstrate that all of the reagents for CFPS can be combined in a single freeze-thaw stable master mix to improve reliability and ease of use. These improvements are important for the application of the CFPS system in fields such as protein engineering, high-throughput screening, and biotherapeutics.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.