Targeting and isolation of tagged membrane proteins

Membrane proteins heterologously expressed in yeast are rather Instable, if correctly folded and inserted into lipid bilayers. They are resistant against proteolytic attacks by soluble proteases. Nevertheless, one often experiences difficulties expressing such proteins, especially when a transmembrane spanning segment of the protein is part of a desired fusion site. This is because few techniques exist for obtaining information about putative transmembrane spanning segments, making it difficult to precisely localize extra-membrane regions safe for the insertion of tags or targeting sequences. Computer-supported models are limited in their practical application.1 For example, predicted membrane spanning segments based on hydropathy plots very seldomly have charged amino acid residues in the hydrophobic phase. However, charged amino acid residues are absolutely necessary here for solute transport and for stabilization of the tertiary protein structure by salt bridges.2