Yeast fusion proteins

A representative set of a- and -keto esters was also tested as substrates (total 11) for each purified fusion protein (Figure 8.13b,c) [9bj. The stereoselectivities of -keto ester reductions depended both on the identity of the enzyme and the substrate stmcture, and some reductases yielded both l- and o-alcohols with high stereoselectivities. While a-keto esters were generally reduced with lower enantioselec-tivities, it was possible to identify pairs of yeast reductases that delivered both alcohol antipodes in optically pure form. These results demonstrate the power of genomic fusion protein libraries to identify appropriate biocatalysts rapidly and expedite process development. 

Figure 5.1. Yeast two-hybrid system. Interaction of proteins X and Y upstream of a reporter gene leads to transcriptional activation. Protein X is part of a fusion protein that binds to a site on DNA upstream of the reporter gene by means of a DNA binding domain. Protein Y is part of a fusion protein that contains a transcriptional activation domain. Interaction of proteins X and Y places the activation domain in the vicinity of the reporter gene and stimulates its transcription.

Figure 7.6. Purification of protein from pooled yeast strains. Each yeast ORF was cloned as a fusion to glutathione-S-transferase in a protein expression vector to create 6144 yeast strains. The individual strains were pooled in groups of 96 to create a set of 64 pools. Each pool was grown and the 96 fusion proteins are purified in batch. Each pool was then assayed for a biochemical function (Martzen et al., 1999). Pools positive for function were then deconvoluted using smaller pools consisting of strains from rows and columns of a 96-well plate.

Schlumpberger, M., Wille, H., Baldwin, M. A., Buder, D. A., Herskowitz, I., and Prusiner, S. B. (2000). The prion domain of yeast Ure2p induces autocatalytic formation of amyloid fibers by a recombinant fusion protein. Protein Sci. 9, 440-451. 

Schematic diagram outlining the principle of the CytoTrap, Sos recruitment, yeast two-hybrid system. The figure shows the bait and fish fusion constructs. The bait vector encodes a Sos fusion protein. The fish vector encodes a myristylation sequence that targets the fusion protein to the cell membrane. Interaction between the fish and bait proteins targets the Sos fusion protein to the intracellular face of the cell membrane where it can interact with Ras and rescue cell growth at 37°C...

In addition to identifying protein partners, yeast two-hybrid technology can be used to identify and study in detail the interaction domains between two proteins. Here, bait and/or fish truncation or deletion constructs of the parent proteins are engineered and characterized as described earlier (see 3.1 Selection and characterization of bait constructs). These are then investigated for association in a yeast two-hybrid interaction assay. Once the BD has been identified, it can be further refined by mutagenesis. The same caveat applies to these studies as for the identification of associating proteins, i.e., it is assumed that the respective fusion proteins fold and adopt the same or a similar three-dimensional conformation to the native protein. This is not always the case and results should be interpreted with caution and if possible, always validated by an alternative experimental approach. O Table 19-1 shows an example of mapping the... 

Sherman MY, Goldberg AL. (1991) Eorma-tion in vitro of complexes between an abnormal fusion protein and the heat shock proteins from Escherichia coli and yeast mitochondria. JBacteriol 173, 7249-56. 

Yeast cells can exist as haploids of opposite mating types (either a or a). When an a and an a cell are allowed to mate, they form a diploid cell (a/a). To study interactions between two proteins, cDNA sequences of a protein of interest (PT1) are expressed as a fusion protein, linked to a DNA-binding domain (DBD) of a yeast gene-transcript activator in a haploid cell (e.g., a). cDNA sequences corresponding to another test protein (PT2) are linked to the Continued on next page)... 

Figure 16.1. Schematic representation of the yeast two-hybrid system for evaluation of protein-protein interactions. Haploid yeast of a and a cells can mate to form (a/a) diploid cells. (A) If two test proteins, PT1 and PT2—expressed in (a/a) diploid cells as fusion proteins of DNA binding domains (DAB) and activation domains (AD) of yeast gene-transcript activator proteins—bind to each other, the binding interaction allows the diploid cells to grow in histidine selection media. Histidine selection media is permissive for diploid cells that express the HISS reporter gene only if PT1 and PT2 interact. (B) If PT1 and PT2 do not interact, no HISS gene product is expressed and the hybrid cell cannot grow in histidine media.

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