ArticlePloS one2012
Fluorescent in situ folding control for rapid optimization of cell-free membrane protein synthesis.
Article in PloS one, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 10 papers.
What it found
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Who cites it
10 citing papers in PubMed, 31 citations in OpenAlex.
- Functional mapping and engineering of the Sec translocon unlocked by a cell-free system.Science advances · 2026Article
- Direct observation of fluorescent proteins in gels: A rapid, cost-efficient, and quantitative alternative to immunoblotting.Biology of the cell · 2025Article
- Article
- A synthetic method to assay polycystin channel biophysics.bioRxiv : the preprint server for biology · 2024Article
- Preparation of cationic proteoliposomes using cell-free membrane protein synthesis: the chaperoning effect of cationic liposomes.RSC advances · 2020Article
- Functional Analysis of Aquaporin Water Permeability Using anFrontiers in bioengineering and biotechnology · 2020Article
- Formate-nitrite transporters carrying nonprotonatable amide amino acids instead of a central histidine maintain pH-dependent transport.The Journal of biological chemistry · 2019Article
- Cell-Free and Yeast-Based Production of the Malarial Lactate Transporter, PfFNT, Delivers Comparable Yield and Protein Quality.Frontiers in pharmacology · 2019Article
- Cell-free methods to produce structurally intact mammalian membrane proteins.Scientific reports · 2016Article
- Article
Corrections and comments
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Cell-free synthesis is an open and powerful tool for high-yield protein production in small reaction volumes predestined for high-throughput structural and functional analysis. Membrane proteins require addition of detergents for solubilization, liposomes, or nanodiscs. Hence, the number of parameters to be tested is significantly higher than with soluble proteins. Optimization is commonly done with respect to protein yield, yet without knowledge of the protein folding status. This approach contains a large inherent risk of ending up with non-functional protein. We show that fluorophore formation in C-terminal fusions with green fluorescent protein (GFP) indicates the folding state of a membrane protein in situ, i.e. within the cell-free reaction mixture, as confirmed by circular dichroism (CD), proteoliposome reconstitution and functional assays. Quantification of protein yield and in-gel fluorescence intensity imply suitability of the method for membrane proteins of bacterial, protozoan, plant, and mammalian origin, representing vacuolar and plasma membrane localization, as well as intra- and extracellular positioning of the C-terminus. We conclude that GFP-fusions provide an extension to cell-free protein synthesis systems eliminating the need for experimental folding control and, thus, enabling rapid optimization towards membrane protein quality.
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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.