ArticleAnalytical biochemistry2012
Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system.
Article in Analytical biochemistry, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 31 papers.
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Who cites it
31 citing papers in PubMed, 59 citations in OpenAlex.
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- Online monitoring of the mitochondrial respiration activity and protein formation in the Almost Living Cell-free Expression (ALiCE) system.BMC biotechnology · 2025Article
- Real-Time Affinity Measurements of Proteins Synthesized in Cell-Free Lysate Using Fluorescence Correlation Spectroscopy.Analytical chemistry · 2025Article
- High temperature delays and low temperature accelerates evolution of a new protein phenotype.Nature communications · 2024Article
- Paracatalytic induction: Subverting specificity in hedgehog protein autoprocessing with small molecules.Methods in enzymology · 2023Article
- Intrinsically Unstructured Sequences in the mRNA 3' UTR Reduce the Ability of Poly(A) Tail to Enhance Translation.Journal of molecular biology · 2022Article
- Reliable online measurement of population dynamics for filamentous co-cultures.Microbial biotechnology · 2022Article
- Immediate protein expression from exogenous mRNAs in embryonic brain.Scientific reports · 2022Article
- Actinosomes: Condensate-Templated Containers for Engineering Synthetic Cells.ACS synthetic biology · 2022Article
- Improving the Robustness of Engineered Bacteria to Nutrient Stress Using Programmed Proteolysis.ACS synthetic biology · 2022Article
- SystematicACS synthetic biology · 2022Article
- A CHO-Based Cell-Free Dual Fluorescence Reporter System for the Straightforward Assessment of Amber Suppression and scFv Functionality.Frontiers in bioengineering and biotechnology · 2022Article
- Sensitive Detection of Protein Binding to the Plasma Membrane with Dual-Color Z-Scan Fluorescence.Biophysical journal · 2020Article
- Nanoparticle-based local translation reveals mRNA as a translation-coupled scaffold with anchoring function.Proceedings of the National Academy of Sciences of the United States of America · 2019Article
- Cell-free protein synthesis from non-growing, stressed Escherichia coli.Scientific reports · 2017Article
- A new mechanism of voltage-dependent gating exposed by KThe Journal of general physiology · 2017Article
- Investigating State Restriction in Fluorescent Protein FRET Using Time-Resolved Fluorescence and Anisotropy.The journal of physical chemistry. C, Nanomaterials and interfaces · 2017Article
- Boudin trafficking reveals the dynamic internalisation of specific septate junction components in Drosophila.PloS one · 2017Article
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
Abstract
Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturation of fluorescent proteins, which is of crucial importance for fluorescence studies. A droplet sample protocol was developed that ensured sufficient oxygenation for chromophore maturation and ease of manipulation for titration studies. The kinetics of chromophore maturation of EGFP, EYFP, and mCherry were analyzed as a function of temperature. A strong increase in the rate from room temperature to 37°C was observed. We further demonstrate that all EGFP proteins fully mature in the cell-free solution and that brightness is a robust parameter specifying stoichiometry. Finally, FFS is applied to study the stoichiometry of the nuclear transport factor 2 in a cell-free system over a broad concentration range. We conclude that combining cell-free expression and FFS provides a powerful technique for quick, quantitative study of chromophore maturation and protein-protein interaction.
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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.