Overexpression and purification of integral membrane proteins in yeast.
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.
Hays
Franklin A
FA
Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California, USA.
Roe-Zurz
Zygy
Z
Stroud
Robert M
RM
D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
D008565:Membrane Proteins