ArticleProtein expression and purification2010
Purification of transmembrane proteins from Saccharomyces cerevisiae for X-ray crystallography.
Article in Protein expression and purification, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 21 papers.
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Who cites it
21 citing papers in PubMed, 37 citations in OpenAlex.
- Comparison of Experimental Approaches Used to Determine the Structure and Function of the Class D G Protein-Coupled Yeast α-Factor Receptor.Biomolecules · 2022Review
- First purified recombinant CYP75B including transmembrane helix with unexpected high substrate specificity to (2R)-naringenin.Scientific reports · 2022Article
- Phosphoramidon inhibits the integral membrane protein zinc metalloprotease ZMPSTE24.Acta crystallographica. Section D, Structural biology · 2018Article
- Acquisition of accurate data from intramolecular quenched fluorescence protease assays.Analytical biochemistry · 2017Article
- Human CaaX protease ZMPSTE24 expressed in yeast: Structure and inhibition by HIV protease inhibitors.Protein science : a publication of the Protein Society · 2017Article
- Structure of the SLC4 transporter Bor1p in an inward-facing conformation.Protein science : a publication of the Protein Society · 2017Article
- Current strategies for protein production and purification enabling membrane protein structural biology.Biochemistry and cell biology = Biochimie et biologie cellulaire · 2016Review
- Deducing the symmetry of helical assemblies: Applications to membrane proteins.Journal of structural biology · 2016Article
- The Crystal Structure of an Integral Membrane Fatty Acid α-Hydroxylase.The Journal of biological chemistry · 2015Article
- A Novel Screening Approach for Optimal and Functional Fusion of T4 Lysozyme in GPCRs.Methods in enzymology · 2015Article
- DNASU plasmid and PSI:Biology-Materials repositories: resources to accelerate biological research.Nucleic acids research · 2014Article
- Membrane protein structure determination - the next generation.Biochimica et biophysica acta · 2014Review
- Structure of the integral membrane protein CAAX protease Ste24p.Science (New York, N.Y.) · 2013Article
- Article
- Recombinant production of human Aquaporin-1 to an exceptional high membrane density in Saccharomyces cerevisiae.PloS one · 2013Article
- Functional fusions of T4 lysozyme in the third intracellular loop of a G protein-coupled receptor identified by a random screening approach in yeast.Protein engineering, design & selection : PEDS · 2013Article
- Oligomeric state of the oxalate transporter, OxlT.Biochemistry · 2011Article
- High throughput platforms for structural genomics of integral membrane proteins.Current opinion in structural biology · 2011Review
- PSI:Biology-materials repository: a biologist's resource for protein expression plasmids.Journal of structural and functional genomics · 2011Article
- New tools in membrane protein determination.F1000 biology reports · 2011Article
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Authors and funding
8 authors at 2 institutions in 1 country.
Funding
Abstract
To enhance the quantity and quality of eukaryotic transmembrane proteins (TMPs) available for structure determination by X-ray crystallography, we have optimized protocols for purification of TMPs expressed in the yeast Saccharomyces cerevisiae. We focused on a set of the highest-expressing endogenous yeast TMPs for which there are established biochemical assays. Genes encoding the target TMPs are transferred via ligation-independent cloning to a series of vectors that allow expression of reading frames fused to C-terminal His10 and ZZ (IgG-binding) domains that are separated from the reading frame by a cleavage site for rhinovirus 3C protease. Several TMP targets expressed from these vectors have been purified via affinity chromatography and gel filtration chromatography at levels and purities sufficient for ongoing crystallization trials. Initial purifications were based on expression of the genes under control of a galactose-inducible promoter, but higher cell densities and improved expression have been obtained through use of the yeast ADH2 promoter. Wide variations have been observed in the behavior of different TMP targets during purification; some can be readily purified, while others do not bind efficiently to affinity matrices, are not efficiently cleaved from the matrices, or remain tightly associated with the matrices even after cleavage of the affinity tags. The size, oligomeric state, and composition of purified protein-detergent complexes purified under different conditions were analyzed using a colorimetric assay of detergent concentrations and by analytical size-exclusion chromatography using static light scattering, refractive index, and UV absorption detection to monitor the elution profiles. Effective procedures were developed for obtaining high concentrations of purified TMPs without excessively concentrating detergents.
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