Tagging yeast proteins

Figure 1.2 Schematic outlining the purification protocol. The principal steps of purifying FLAG-tagged eIF2B proteins from yeast whole cell extracts are outlined in the diagram for details, refer to the main text.

Incorporation of a stable isotopic tag into proteins/peptides in metabolically active cells was first described to quantify protein abundance in yeast (43). Wild-type and mutant cell populations were grown in media containing the naturally abundant isotopes of nitrogen and enriched in 15N, respectively, followed by trypsin digestion and LC-ESI-MS/MS analysis to identify and quantify relative phosphopeptide levels in both populations (43). 

Schneider, B. L., Seufert, W., Steiner, B., Yang, Q. H., and Futcher, A. B. 1995. Use of polymerase chain reaction epitope tagging for protein tagging in Saccharomyces cerevisiae. Yeast, 11, 1265-1274. 

Eukaryotic expression vectors can be used to express cloned genes in yeast or mammalian cells (see Figure 9-29). An Important application of these methods is the tagging of proteins with an epitope for antibody detection. 

The second method also relies on site-specific chemical modification ofphosphoproteins (Oda et al., 2001). It involves the chemical replacement of phosphates on serine and threonine residues with a biotin affinity tag (Fig. 2.7B). The replacement reaction takes advantage of the fact that the phosphate moiety on phosphoserine and phosphothreonine undergoes -elimination under alkaline conditions to form a group that reacts with nucleophiles such as ethanedithiol. The resulting free sulfydryls can then be coupled to biotin to create the affinity tag (Oda et al., 2001). The biotin tag is used to purify the proteins subsequent to proteolytic digestion. The biotinylated peptides are isolated by an additional affinity purification step and are then analyzed by mass spectrometry (Oda et al., 2001). This method was also tested with phosphorylated (Teasein and shown to efficiently enrich phosphopeptides. In addition, the method was used on a crude protein lysate from yeast and phosphorylated ovalbumin was detected. Thus, as with the method of Zhou et al. (2001), additional fractionation steps will be required to detect low abundance phosphoproteins. 

The discovery of Green Fluorescent Protein (GFP) and the development of technology that allows specific proteins to be tagged with GFP has fundamentally altered the types of question that can be asked using cell biological methods. It is now possible not only to study where a protein is within a cell, but also feasible to study the precise dynamics of protein movement within living cells. We have exploited these technical developments and applied them to the study of translation initiation factors in yeast, focusing particularly on the... 

Janke, C., Magiera, M. M., Rathfelder, N., Taxis, C., Reber, S., Maekawa, H., Moreno-Borchart, A., Doenges, G., Schwob, E., Schiebel, E., and Knop, M. (2004). A versatile toolbox for PCR-based tagging of yeast genes New fluorescent proteins, more markers, and promoter substitution cassettes. Yeast 21, 947-962. 

Li, J., Steen, H., and Gygi, S.P. (2003) Protein profiling with cleavable isotope-coded affinity tag (cICAT) reagents. The yeast salinity stress response. Mol. Cell. Proteomics 2, 1198-1204. 

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