Purification of transmembrane proteins from Saccharomyces cerevisiae for X-ray crystallography. 2010

Kathleen M Clark, and Nadia Fedoriw, and Katrina Robinson, and Sara M Connelly, and Joan Randles, and Michael G Malkowski, and George T DeTitta, and Mark E Dumont
Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.

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.

UI MeSH Term Description Entries
D008565 Membrane Proteins Proteins which are found in membranes including cellular and intracellular membranes. They consist of two types, peripheral and integral proteins. They include most membrane-associated enzymes, antigenic proteins, transport proteins, and drug, hormone, and lectin receptors. Cell Membrane Protein,Cell Membrane Proteins,Cell Surface Protein,Cell Surface Proteins,Integral Membrane Proteins,Membrane-Associated Protein,Surface Protein,Surface Proteins,Integral Membrane Protein,Membrane Protein,Membrane-Associated Proteins,Membrane Associated Protein,Membrane Associated Proteins,Membrane Protein, Cell,Membrane Protein, Integral,Membrane Proteins, Integral,Protein, Cell Membrane,Protein, Cell Surface,Protein, Integral Membrane,Protein, Membrane,Protein, Membrane-Associated,Protein, Surface,Proteins, Cell Membrane,Proteins, Cell Surface,Proteins, Integral Membrane,Proteins, Membrane,Proteins, Membrane-Associated,Proteins, Surface,Surface Protein, Cell
D011485 Protein Binding The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments. Plasma Protein Binding Capacity,Binding, Protein
D002846 Chromatography, Affinity A chromatographic technique that utilizes the ability of biological molecules, often ANTIBODIES, to bind to certain ligands specifically and reversibly. It is used in protein biochemistry. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Chromatography, Bioaffinity,Immunochromatography,Affinity Chromatography,Bioaffinity Chromatography
D002850 Chromatography, Gel Chromatography on non-ionic gels without regard to the mechanism of solute discrimination. Chromatography, Exclusion,Chromatography, Gel Permeation,Chromatography, Molecular Sieve,Gel Filtration,Gel Filtration Chromatography,Chromatography, Size Exclusion,Exclusion Chromatography,Gel Chromatography,Gel Permeation Chromatography,Molecular Sieve Chromatography,Chromatography, Gel Filtration,Exclusion Chromatography, Size,Filtration Chromatography, Gel,Filtration, Gel,Sieve Chromatography, Molecular,Size Exclusion Chromatography
D005822 Genetic Vectors DNA molecules capable of autonomous replication within a host cell and into which other DNA sequences can be inserted and thus amplified. Many are derived from PLASMIDS; BACTERIOPHAGES; or VIRUSES. They are used for transporting foreign genes into recipient cells. Genetic vectors possess a functional replicator site and contain GENETIC MARKERS to facilitate their selective recognition. Cloning Vectors,Shuttle Vectors,Vectors, Genetic,Cloning Vector,Genetic Vector,Shuttle Vector,Vector, Cloning,Vector, Genetic,Vector, Shuttle,Vectors, Cloning,Vectors, Shuttle
D012441 Saccharomyces cerevisiae A species of the genus SACCHAROMYCES, family Saccharomycetaceae, order Saccharomycetales, known as "baker's" or "brewer's" yeast. The dried form is used as a dietary supplement. Baker's Yeast,Brewer's Yeast,Candida robusta,S. cerevisiae,Saccharomyces capensis,Saccharomyces italicus,Saccharomyces oviformis,Saccharomyces uvarum var. melibiosus,Yeast, Baker's,Yeast, Brewer's,Baker Yeast,S cerevisiae,Baker's Yeasts,Yeast, Baker
D014965 X-Rays Penetrating electromagnetic radiation emitted when the inner orbital electrons of an atom are excited and release radiant energy. X-ray wavelengths range from 1 pm to 10 nm. Hard X-rays are the higher energy, shorter wavelength X-rays. Soft x-rays or Grenz rays are less energetic and longer in wavelength. The short wavelength end of the X-ray spectrum overlaps the GAMMA RAYS wavelength range. The distinction between gamma rays and X-rays is based on their radiation source. Grenz Ray,Grenz Rays,Roentgen Ray,Roentgen Rays,X Ray,X-Ray,Xray,Radiation, X,X-Radiation,Xrays,Ray, Grenz,Ray, Roentgen,Ray, X,Rays, Grenz,Rays, Roentgen,Rays, X,X Radiation,X Rays,X-Radiations
D056890 Eukaryota One of the three domains of life (the others being BACTERIA and ARCHAEA), also called Eukarya. These are organisms whose cells are enclosed in membranes and possess a nucleus. They comprise almost all multicellular and many unicellular organisms, and are traditionally divided into groups (sometimes called kingdoms) including ANIMALS; PLANTS; FUNGI; and various algae and other taxa that were previously part of the old kingdom Protista. Eukaryotes,Eucarya,Eukarya,Eukaryotas,Eukaryote
D018360 Crystallography, X-Ray The study of crystal structure using X-RAY DIFFRACTION techniques. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) X-Ray Crystallography,Crystallography, X Ray,Crystallography, Xray,X Ray Crystallography,Xray Crystallography,Crystallographies, X Ray,X Ray Crystallographies
D029701 Saccharomyces cerevisiae Proteins Proteins obtained from the species SACCHAROMYCES CEREVISIAE. The function of specific proteins from this organism are the subject of intense scientific interest and have been used to derive basic understanding of the functioning similar proteins in higher eukaryotes. Baker's Yeast Proteins,S cerevisiae Proteins

Related Publications

Kathleen M Clark, and Nadia Fedoriw, and Katrina Robinson, and Sara M Connelly, and Joan Randles, and Michael G Malkowski, and George T DeTitta, and Mark E Dumont
July 1992, Biochimica et biophysica acta,
Kathleen M Clark, and Nadia Fedoriw, and Katrina Robinson, and Sara M Connelly, and Joan Randles, and Michael G Malkowski, and George T DeTitta, and Mark E Dumont
February 2007, Acta crystallographica. Section F, Structural biology and crystallization communications,
Kathleen M Clark, and Nadia Fedoriw, and Katrina Robinson, and Sara M Connelly, and Joan Randles, and Michael G Malkowski, and George T DeTitta, and Mark E Dumont
January 1994, Methods of biochemical analysis,
Kathleen M Clark, and Nadia Fedoriw, and Katrina Robinson, and Sara M Connelly, and Joan Randles, and Michael G Malkowski, and George T DeTitta, and Mark E Dumont
January 2014, Acta crystallographica. Section F, Structural biology communications,
Kathleen M Clark, and Nadia Fedoriw, and Katrina Robinson, and Sara M Connelly, and Joan Randles, and Michael G Malkowski, and George T DeTitta, and Mark E Dumont
June 2006, Acta crystallographica. Section F, Structural biology and crystallization communications,
Kathleen M Clark, and Nadia Fedoriw, and Katrina Robinson, and Sara M Connelly, and Joan Randles, and Michael G Malkowski, and George T DeTitta, and Mark E Dumont
April 2008, Acta crystallographica. Section F, Structural biology and crystallization communications,
Kathleen M Clark, and Nadia Fedoriw, and Katrina Robinson, and Sara M Connelly, and Joan Randles, and Michael G Malkowski, and George T DeTitta, and Mark E Dumont
November 2006, Acta crystallographica. Section F, Structural biology and crystallization communications,
Kathleen M Clark, and Nadia Fedoriw, and Katrina Robinson, and Sara M Connelly, and Joan Randles, and Michael G Malkowski, and George T DeTitta, and Mark E Dumont
August 2021, Journal of molecular biology,
Kathleen M Clark, and Nadia Fedoriw, and Katrina Robinson, and Sara M Connelly, and Joan Randles, and Michael G Malkowski, and George T DeTitta, and Mark E Dumont
January 2005, Methods in enzymology,
Kathleen M Clark, and Nadia Fedoriw, and Katrina Robinson, and Sara M Connelly, and Joan Randles, and Michael G Malkowski, and George T DeTitta, and Mark E Dumont
January 2008, Methods in enzymology,
Copied contents to your clipboard!