Expression and localization of plant membrane proteins in Saccharomyces.

Publisher Summary This chapter discusses the expression and localization of plant membrane proteins in Saccharomyces . The expression of cloned genes in heterologous systems has two major goals: (1) the production of antigen for antibody generation and (2) the expression of functional protein for study of its activity. Antigen production is usually accomplished by massive expression in Escherichia coli ( E. coli ) as insoluble, denatured fusion proteins with either P-galactosidase or anthranilate synthase. E. coli is also for most purposes the best host for the expression of soluble proteins in active form. In the case of membrane proteins, however, problems are usually found with the bacterial expression system. Hydrophobic membrane proteins are usually quite toxic to E. coli and are expressed at very low levels. This toxicity is probably due to the depolarization of the bacterial membrane by improperly assembled membrane proteins. The most crucial aspect in Western blot and immunofluorescence analysis is the specificity of the primary antibody. As animal sera are able to recognize yeast proteins, only monoclonal and affinity-purified polyclonal antibodies are adequate. Occasionally, crude antisera of high titer may be specific enough at high dilutions (more than 1/10,000).