The budding yeast Saccharomyces cerevisiae is a viable system for the overexpression and functional analysis of eukaryotic integral membrane proteins (IMPs). In this chapter we describe a general protocol for the initial cloning, transformation, overexpression, and subsequent purification of a putative IMP and discuss critical optimization steps and approaches. Since expression and purification are often the two predominant hurdles one will face in studying this difficult class of biological macromolecules the intent is to outline the general workflow while providing insights based upon our collective experience. These insights should facilitate tailoring of the outlined protocol to individual IMPs and expression or purification routines.CI - Copyright (c) 2010 Elsevier Inc. All rights reserved.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|