ArticleJournal of molecular microbiology and biotechnology2010
An Escherichia coli-based cell-free system for large-scale production of functional mammalian membrane proteins suitable for X-ray crystallography.
Article in Journal of molecular microbiology and biotechnology, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
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Who cites it
4 citing papers in PubMed, 15 citations in OpenAlex.
- Selecting an appropriate method for expressingBiotechnology reports (Amsterdam, Netherlands) · 2017Article
- A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis.PloS one · 2016Article
- Detergent-solubilized Patched purified from Sf9 cells fails to interact strongly with cognate Hedgehog or Ihog homologs.Protein expression and purification · 2014Article
- Optimisation of over-expression in E. coli and biophysical characterisation of human membrane protein synaptogyrin 1.PloS one · 2012Article
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Authors and funding
3 authors at 1 institution in 1 country.
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Abstract
A cell-free expression system using an Escherichia coli extract was adapted for large-scale expression and purification of mammalian membrane proteins. The system was tested with a set of human membrane proteins of different sizes, numbers of transmembrane domains, oligomeric arrangements, and native membrane locations. Tens of milligrams of protein were readily expressed and purified from an overnight cell-free reaction. Both reaction 'mode A' (proteins were expressed as precipitant) and 'mode B' (proteins were expressed in the presence of mild detergents to keep them soluble) were investigated. The combination of 'mode B' and the right detergents, used in the subsequent extraction and purification steps, is critical for obtaining properly folded proteins (CX32 and VDAC1) that can be crystallized and diffracted (VDAC1). The E. coli cell-free system is capable of efficient expression of many mammalian membrane proteins. However, fine-tuning of the system, especially to facilitate proper protein folding, will be required for each specific target.
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Registered trials
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