ArticlePloS one2012
Optimisation of over-expression in E. coli and biophysical characterisation of human membrane protein synaptogyrin 1.
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 15 papers.
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Who cites it
15 citing papers in PubMed, 25 citations in OpenAlex.
- Soluble Expression of Recombinant Human Mitsugumin 53 in Escherichia coli NiCo21(DE3).The protein journal · 2026Article
- Cellugyrin (synaptogyrin-2) dependent pathways are used by bacterial cytolethal distending toxin and SARS-CoV-2 virus to gain cell entry.Frontiers in cellular and infection microbiology · 2024Article
- Phosphatidylserine-dependent structure of synaptogyrin remodels the synaptic vesicle membrane.Nature structural & molecular biology · 2023Article
- Cryo-EM Structure of an Atypical Proton-Coupled Peptide Transporter: Di- and Tripeptide Permease C.Frontiers in molecular biosciences · 2022Article
- Membrane Protein Production and Purification from Escherichia coli and Sf9 Insect Cells.Methods in molecular biology (Clifton, N.J.) · 2020Article
- High-throughput stability screening for detergent-solubilized membrane proteins.Scientific reports · 2019Article
- Smoothing membrane protein structure determination by initial upstream stage improvements.Applied microbiology and biotechnology · 2019Review
- Expression optimization, purification, and functional characterization of cholesterol oxidase from Chromobacterium sp. DS1.PloS one · 2019Article
- Expression optimization of recombinant cholesterol oxidase in Escherichia coli and its purification and characterization.AMB Express · 2018Article
- Lipid-like Peptides can Stabilize Integral Membrane Proteins for Biophysical and Structural Studies.Chembiochem : a European journal of chemical biology · 2017Article
- Uptake and Metabolism of Antibiotics Roseoflavin and 8-Demethyl-8-Aminoriboflavin in Riboflavin-Auxotrophic Listeria monocytogenes.Journal of bacteriology · 2016Article
- Production of functional human vitamin A transporter/RBP receptor (STRA6) for structure determination.PloS one · 2015Article
- A novel methodology for enhanced and consistent heterologous expression of unmodified human cytochrome P450 1B1 (CYP1B1).PloS one · 2014Article
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Authors and funding
5 authors at 2 institutions in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Progress in functional and structural studies of integral membrane proteins (IMPs) is lacking behind their soluble counterparts due to the great challenge in producing stable and homogeneous IMPs. Low natural abundance, toxicity when over-expressed and potential lipid requirements of IMPs are only a few reasons for the limited progress. Here, we describe an optimised workflow for the recombinant over-expression of the human tetraspan vesicle protein (TVP) synaptogyrin in Escherichia coli and its biophysical characterisation. TVPs are ubiquitous and abundant components of vesicles. They are believed to be involved in various aspects of the synaptic vesicle cycle, including vesicle biogenesis, exocytosis and endocytotic recycling. Even though TVPs are found in most cell types, high-resolution structural information for this class of membrane proteins is still missing. The optimisation of the N-terminal sequence of the gene together with the usage of the recently developed Lemo21(DE3) strain which allows the balancing of the translation with the membrane insertion rate led to a 50-fold increased expression rate compared to the classical BL21(DE3) strain. The protein was soluble and stable in a variety of mild detergents and multiple biophysical methods confirmed the folded state of the protein. Crosslinking experiments suggest an oligomeric architecture of at least four subunits. The protein stability is significantly improved in the presence of cholesteryl hemisuccinate as judged by differential light scattering. The approach described here can easily be adapted to other eukaryotic IMPs.
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