8 Fungal Genetics Reports

A procedure is described for isolation and purification of four high-molecular mass stress-responsive proteins from the same starting material. Proteins are the major components determining cellular growth, metabolism, regulation, repair, homeostatic mechanisms, differentiation, organogenesis, response to environmental factors and ultimately survival. Extensive protein networks form the basis of interlocking metabolic pathways and their regulatory circuits in the eukaryotic and prokaryotic cells, involving transient interactions among a large, overwhelming array of actors. Availability of purified proteins in their native state is critical on account of the universal interest in unraveling how proteins function in their endogenous intracellular environment. With the rapid progress in genomics and availability of an ever increasing number of crystal structures, an understanding of the molecular basis of interactions of proteins with ligands, substrates and regulatory molecules is achievable. Structural studies, for instance X-ray crystallographic analysis, depend on homogeneous preparations of proteins. The use of heterologous expression systems with bacteria as host cells is a well established methodology. Other host systems include yeast, insect cells and plant cells. The technology for cloning and expression of proteins, vectors with selectable markers, enhancers, trafficking signals and a range of facile protocols for over-expression are also readily available. However, often it is feasible to express only individual domains of large multi-domain proteins efficiently in heterologous systems. Even when the entire polypeptide can be expressed it is not always possibl