Protein Extraction And Purification

Standard analytical methods for protein purification and characterization are routinely used in the study of Saccharomyces proteins. It is important to have a

Figure 2.6 Immuno-gold localization. a-Factor receptor Ste2p is localized to the plasma membrane of a mating type cells. In response to the binding of a-factor, Ste2p is internalized in the form of vesicles by a process called endocytosis. Immunogold labeling methods are used here to visualize the subcellular location of Ste2p during this internalization process. The cells are fixed, sectioned, stained with osmium to visualize subcellular structures, and treated with anti-Ste2p antibody followed by the gold-labeled secondary antibody. One can see a series of black spheres (the electron dense gold beads) concentrated over a tubular-vesicular membrane compartment that appears to span the region from the plasma membrane to the vacuole (labeled V). Taken from Mulholland et al. (1999). Reproduced by permission of the American Society for Cell Biology

Figure 2.6 Immuno-gold localization. a-Factor receptor Ste2p is localized to the plasma membrane of a mating type cells. In response to the binding of a-factor, Ste2p is internalized in the form of vesicles by a process called endocytosis. Immunogold labeling methods are used here to visualize the subcellular location of Ste2p during this internalization process. The cells are fixed, sectioned, stained with osmium to visualize subcellular structures, and treated with anti-Ste2p antibody followed by the gold-labeled secondary antibody. One can see a series of black spheres (the electron dense gold beads) concentrated over a tubular-vesicular membrane compartment that appears to span the region from the plasma membrane to the vacuole (labeled V). Taken from Mulholland et al. (1999). Reproduced by permission of the American Society for Cell Biology

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1C 2C DNA Content

1C 2C DNA Content

Figure 2.7 FACS analysis of DNA content. Strains carrying the temperature-sensitive mutation mcm2-\, encoding a protein required for the initiation of DNA replication at chromosomal origins of replication, and the mutation dbf4-6, a component of a regulatory kinase, and the wild-type strain are analyzed for DNA content by FACS analysis. Cells were grown at the permissive temperature and exposed to the nonpermissive temperature for at least one cell cycle. The DNA was stained with propidium iodide and the cells subjected to FACS analysis to determine the number of cells containing different amounts of DNA. The results are presented graphically, as seen in the figure. A 1 C DNA content is the amount found in a haploid cell during G1 of the cell cycle and a 2C DNA content is the amount found in a haploid cell in G2 following the completion of DNA replication. Cells having DNA levels between 1 C and 2 C are in the S phase and in the process of replicating their DNA. As can be seen, the effect of the mcm2-\ mutation is to arrest cells in G2 but the dbf4-6 mutation suppresses (bypasses) the mcm2-l alteration. Taken from Lei et al. (1997). Reproduced by permission of Cold Spring Harbor Laboratory Press basic understanding of these methods and be familiar with the terminology since one will undoubtedly encounter many of these while reading the literature whether on Saccharomyces or other systems.

Saccharomyces can be cultured in liquid in large volume sufficient to provide milligram quantities of many proteins that then can be purified by standard methods such as gel filtration, affinity chromatography, and ion exchange chromatography. A thorough description of the variety of protein purification techniques available to the researcher is beyond the scope of this book. Instead the reader is referred to the several reference texts on protein purification, particularly Marshak et al. (1996), Ausubel et al. (2001), Spector et al. (1998a,b,c) and certain volumes of Methods in Enzymology edited by Abelson and Simons and others.

In Saccharomyces these protein purification techniques may be used in conjunction with overexpression vectors that place the ORF of any cloned gene under the control of high-level constitutive promoters in multicopy plasmids (see the discussion of expression vectors in Chapter 1). In this way, one can achieve sufficiently abundant levels of expression to allow the purification of almost any desired protein. Overexpression of a particular protein makes possible its purification but may not be appropriate for the in vivo analysis of function. Artifacts such as mislocalization to unusual subcellular compartments may occur. The protein may have toxic effects when produced in abundance. For example, overproduction of an integral membrane protein may have serious consequences for the transit of other proteins through the secretory pathway. In fact, overproduction of any protein that relies on a saturable process for its synthesis and localization may lead to unforeseen effects. Examples of saturable processes are nuclear import via import proteins and the nuclear pore complex or protein modification and processing events. Additionally, many proteins are components of larger functional complexes or interact in a highly regulated manner with several different competing regulatory components. Overproduction of one member of a complex may impact the level of expression of the other components of the complex or may alter their interactions so as to cause aberrant regulatory patterns. Caution must be used in the interpretation of any result obtained by the overproduction of a single protein.

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