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Methods Enzymol

Title:		A Novel Screening Approach for Optimal and Functional Fusion of T4 Lysozyme in GPCRs
Author(s):		Mathew E; Dumont ME;
Address:		"Department of Biochemistry and Biophysics, P.O. Box 712, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA. Department of Biochemistry and Biophysics, P.O. Box 712, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA. Electronic address: mark_dumont@urmc.rochester.edu"
Journal Title:		Methods Enzymol
Year:		2015
Volume:		20150324
Issue:
Page Number:		27 - 43
DOI:		10.1016/bs.mie.2014.12.031
ISSN/ISBN:		1557-7988 (Electronic) 0076-6879 (Print) 0076-6879 (Linking)
Abstract:		"Determination of high-resolution, three-dimensional structures of transmembrane proteins (TMPs) has, in many cases, only been accomplished through the use of stabilized variant forms of the proteins being studied. For the important G protein-coupled receptor superfamily, this has most often been achieved by inserting a stable soluble protein, such as T4 lysozyme (T4L), in an internal loop of a receptor. However, creation of such fusion proteins generally results in loss of the ability of receptors to activate their cognate cytoplasmic G proteins. Furthermore, the criteria for designing fusions that minimally perturb receptor structure are not well established. We describe here a method for creating a library of receptor variants containing T4L inserted into an internal loop at varying positions and as replacements for varying amounts of the original receptor sequence. We also describe methods for screening for variants displaying maximal expression levels, ligand binding capacity, and signaling function. When applied to the yeast alpha-factor receptor, Ste2p, this approach allowed recovery of well-expressed receptor variants containing internally fused T4L that retained nearly normal signaling function. The approach we describe can be readily adapted to creation of stabilized fusions of other TMPs expressed in yeast or other expression systems"
Keywords:		"Bacteriophage T4/chemistry/*genetics Gene Expression Muramidase/chemistry/*genetics Plasmids/genetics Protein Conformation Receptors, G-Protein-Coupled/chemistry/*genetics Receptors, Mating Factor/chemistry/*genetics Recombinant Fusion Proteins/chemistry/;"
Notes:		"MedlineMathew, Elizabeth Dumont, Mark E eng R01 GM084083/GM/NIGMS NIH HHS/ U54 GM094611/GM/NIGMS NIH HHS/ R01GM084083/GM/NIGMS NIH HHS/ U54GM09461/GM/NIGMS NIH HHS/ Research Support, N.I.H., Extramural 2015/05/08 Methods Enzymol. 2015; 557:27-43. doi: 10.1016/bs.mie.2014.12.031. Epub 2015 Mar 24"