117 results found
Bill R, Byrne B, 2013, Foreword for EDICT Special Edition, volume 2, MOLECULAR MEMBRANE BIOLOGY, Vol: 30, Pages: 113-113, ISSN: 0968-7688
Rana RR, Zhang M, Spear AM, et al., 2013, Bacterial TIR-containing proteins and host innate immune system evasion, MEDICAL MICROBIOLOGY AND IMMUNOLOGY, Vol: 202, Pages: 1-10, ISSN: 0300-8584
Bill R, Byrne B, 2013, Introduction to EDICT volume 1, MOLECULAR MEMBRANE BIOLOGY, Vol: 30, Pages: 2-2, ISSN: 0968-7688
Leung J, Cameron AD, Diallinas G, et al., 2013, Stabilizing the heterologously expressed uric acid-xanthine transporter UapA from the lower eukaryote Aspergillus nidulans, MOLECULAR MEMBRANE BIOLOGY, Vol: 30, Pages: 32-42, ISSN: 0968-7688
Chae PS, Rana RR, Gotfryd K, et al., 2013, Glucose-Neopentyl Glycol (GNG) amphiphiles for membrane protein study, CHEMICAL COMMUNICATIONS, Vol: 49, Pages: 2287-2289, ISSN: 1359-7345
Glassford SE, Govada L, Chayen NE, et al., 2012, Micro ATR FTIR imaging of hanging drop protein crystallisation, VIBRATIONAL SPECTROSCOPY, Vol: 63, Pages: 492-498, ISSN: 0924-2031
Chae PS, Rasmussen SGF, Rana RR, et al., 2012, A New Class of Amphiphiles Bearing Rigid Hydrophobic Groups for Solubilization and Stabilization of Membrane Proteins, CHEMISTRY-A EUROPEAN JOURNAL, Vol: 18, Pages: 9485-9490, ISSN: 0947-6539
Spear AM, Rana RR, Oyston PCF, et al., 2012, A TIR domain protein from Yersinia pestis interacts with mammalian IL-1/TLR pathways but does not play a central role in the virulence of Y. pestis in a mouse model of bubonic plague, Microbiology, Vol: 158, Pages: 1593-1606
The Toll/interleukin (IL)-1 receptor (TIR) domain is an essential component of eukaryotic innate immune signalling pathways. Interaction between TIR domains present in Toll-like receptors and associated adaptors initiates and propagates an immune signalling cascade. Proteins containing TIR domains have also been discovered in bacteria. Studies have subsequently shown that these proteins are able to modulate mammalian immune signalling pathways dependent on TIR interactions and that this may represent an evasion strategy for bacterial pathogens. Here, we investigate a TIR domain protein from the highly virulent bacterium Yersinia pestis, the causative agent of plague. When overexpressed in vitro this protein is able to downregulate IL-1β- and LPS-dependent signalling to NFκB and to interact with the TIR adaptor protein MyD88. This interaction is dependent on a single proline residue. However, a Y. pestis knockout mutant lacking the TIR domain protein was not attenuated in virulence in a mouse model of bubonic plague. Minor alterations in the host cytokine response to the mutant were indicated, suggesting a potential subtle role in pathogenesis. The Y. pestis mutant also showed increased auto-aggregation and reduced survival in high-salinity conditions, phenotypes which may contribute to pathogenesis or survival.
Logez C, Alkhalfioui F, Byrne B, et al., 2012, Preparation of Pichia pastoris expression plasmids, Methods in Molecular Biology, Vol: 866, Pages: 25-40
Glassford S, Chan KLA, Byrne B, et al., 2012, Chemical Imaging of Protein Adsorption and Crystallization on a Wettability Gradient Surface, LANGMUIR, Vol: 28, Pages: 3174-3179, ISSN: 0743-7463
Singh S, Zhang M, Bertheleme N, et al., 2012, Purification of the human G protein-coupled receptor adenosine A(2a)R in a stable and functional form expressed in Pichia pastoris., Curr Protoc Protein Sci, Vol: Chapter 29, Pages: Unit-29.4
The isolation of membrane proteins with the aim of producing highly pure, homogeneous, stable, and functional material remains challenging, and it is often necessary to develop protein-specific purification protocols by trial and error. One key tool that is required in the development of a suitable protocol is a functional assay. This unit describes a range of different protocols for isolation of the human adenosine A2a receptor (A(2a)R). These protocols show the importance of developing a robust method for comparing the quality of protein obtained by a combination of biophysical analyses including SDS-PAGE, analytical size-exclusion chromatography, and functional analysis. One of the keys to isolating and maintaining a functional receptor, found not only in the optimal protocol described here but in other published examples, is that there should be no more than two chromatographic steps.
Singh S, Zhang M, Bertheleme N, et al., 2012, Radioligand binding analysis as a tool for quality control of GPCR production for structural characterization: adenosine A(2a)R as a template for study., Curr Protoc Protein Sci, Vol: Chapter 29, Pages: Unit-29.3
Functional characterization of G protein-coupled receptors is essential to ascertain the suitability of a protein target for downstream studies and to help develop optimal expression and isolation procedures. Radioligand binding analysis is a well-established technique, which allows direct measurement of the amount of functional receptor in a sample. It can be readily applied to both membrane-bound and soluble receptor samples and is an ideal method for monitoring the amount of functional protein at each stage in the expression and isolation process. This unit presents protocols for the radioligand binding analysis of the human adenosine A(2a) receptor and provides examples of how these assays can be used at several stages to help optimize expression, solubilization, and isolation procedures.
Byrne B, 2012, Producing stable membrane protein for structural studies, Publisher: INT UNION CRYSTALLOGRAPHY, Pages: S10-S10, ISSN: 2053-2733
Singh S, Gras A, Fiez-Vandal C, et al., 2012, Screening for high-yielding Pichia pastoris clones: the production of G protein-coupled receptors as a case study., Methods Mol Biol, Vol: 866, Pages: 65-73
Pichia pastoris is an established host for the production of a wide range of recombinant proteins including membrane proteins. The system has a particularly good track record for the production of G protein-coupled receptors (GPCRs). Generation and screening of expression clones with this system use standard molecular biology techniques. Multiple clones can be generated and screened in a matter of a few weeks making this similar to Escherichia coli in terms of speed. In addition, basic buffer components and the lack of expensive equipment make small-scale expression screening in P. pastoris very cost-effective. Here we describe the procedures used for small-scale GPCR production screening.
Singh S, Gras A, Fiez-Vandal C, et al., 2012, Large-scale production of membrane proteins in Pichia pastoris: the production of G protein-coupled receptors as a case study., Methods Mol Biol, Vol: 866, Pages: 197-207, ISSN: 1064-3745
One of the major advantages of using Pichia pastoris is that it is readily adapted to large-scale culture in bioreactors. Bioreactors allow precise regulation of cell growth parameters increasing both yields and reproducibility of the culture. P. pastoris cultures grow to very high cell densities which helps minimise culture volume and facilitates downstream processing of the sample. Here, we provide protocols for the large-scale production of the human adenosine A(2A) receptor (A(2A)R) and provide some details of how bioreactor cultures can be used for optimisation of expression of the human dopamine D2 receptor (D2DR).
Rana RR, Simpson P, Zhang M, et al., 2011, Yersinia pestis TIR-domain protein forms dimers that interact with the human adaptor protein MyD88, MICROBIAL PATHOGENESIS, Vol: 51, Pages: 89-95, ISSN: 0882-4010
Sonoda Y, Newstead S, Hu N-J, et al., 2011, Benchmarking Membrane Protein Detergent Stability for Improving Throughput of High-Resolution X-ray Structures, STRUCTURE, Vol: 19, Pages: 17-25, ISSN: 0969-2126
Chae PS, Gotfryd K, Pacyna J, et al., 2010, Tandem Facial Amphiphiles for Membrane Protein Stabilization, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Vol: 132, Pages: 16750-16752, ISSN: 0002-7863
Chae PS, Rasmussen SGF, Rana RR, et al., 2010, Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins, NATURE METHODS, Vol: 7, Pages: 1003-U90, ISSN: 1548-7091
Singh S, Hedley D, Kara E, et al., 2010, A purified C-terminally truncated human adenosine A(2A) receptor construct is functionally stable and degradation resistant, PROTEIN EXPRESSION AND PURIFICATION, Vol: 74, Pages: 80-87, ISSN: 1046-5928
Leung J, Karachaliou M, Alves C, et al., 2010, Expression and purification of a functional uric acid-xanthine transporter (UapA), PROTEIN EXPRESSION AND PURIFICATION, Vol: 72, Pages: 139-146, ISSN: 1046-5928
Tanabe M, Szakonyi G, Brown KA, et al., 2009, The multidrug resistance efflux complex, EmrAB from Escherichia coli forms a dimer in vitro, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol: 380, Pages: 338-342, ISSN: 0006-291X
Singh S, Gras A, Fiez-Vandal C, et al., 2008, Large-scale functional expression of WT and truncated human adenosine A(2A) receptor in Pichia pastoris bioreactor cultures, MICROBIAL CELL FACTORIES, Vol: 7, ISSN: 1475-2859
Gutmann DAP, Mizohata E, Newstead S, et al., 2007, A high-throughput method for membrane protein solubility screening: The ultracentrifugation dispersity sedimentation assay (vol 16, pg 1422, 2007), PROTEIN SCIENCE, Vol: 16, Pages: 2775-2775, ISSN: 0961-8368
Tanabe M, Mirza O, Bertrand T, et al., 2007, Structures of OppA and PstS from Yersinia pestis indicate variability of interactions with transmembrane domains, ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, Vol: 63, Pages: 1185-1193, ISSN: 2059-7983
Martin CA, Longman E, Wooding C, et al., 2007, Cd36, a class B scavenger receptor, functions as a monomer to bind acetylated and oxidized low-density lipoproteins, PROTEIN SCIENCE, Vol: 16, Pages: 2531-2541, ISSN: 0961-8368
Gutmann DAP, Mizohata E, Newstead S, et al., 2007, A high-throughput method for membrane protein solubility screening: The ultracentrifugation dispersity sedimentation assay, PROTEIN SCIENCE, Vol: 16, Pages: 1422-1428, ISSN: 0961-8368
Byrne B, 2007, Expression, purification, and crystallisation of membrane proteins, Conference of the NATO Advanced Study Institute on Evolving Methods for Macromolecular Cystallography, Publisher: SPRINGER, Pages: 11-23
Tanabe M, Atkins HS, Harland DN, et al., 2006, The ABC transporter protein OppA provides protection against experimental Yersinia pestis infection, INFECTION AND IMMUNITY, Vol: 74, Pages: 3687-3691, ISSN: 0019-9567
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