Expression screening yeast

For other production hosts (yeast, insect, and mammalian cells), standard promoter formats have been used in combination with FITP cloning methods to produce vectors for expression screening (see Section 2.3.2). A particularly interesting development is the use of multipromoter plasmids for expression in two or more hosts from a single vector. The construction of a dual E.coli (T7 promoter) and baculovirus transfer vector (polH promoter) for expression in insect cells has been described (Chambers et al., 2004). A three-promoter vector (T7, plO, and hCMV or CAG promoter) is available from Novagen (pTrlEX ) and its use reported for comparing protein expression in E. coli and insect cells (Xu and Jones, 2004). 

There are two yeast expression hosts that have an established track record for high-level production of heterologous proteins, namely Saccharomyces cerevisiae and Pichia pastoris. HTP expression screening using microplate formats has been reported for both these yeasts by Lang and coworkers (Holz et ah, 2002, 2003 Boettner et ah, 2002). In both cases standard protocols have been miniaturized with cells cultured in either 1.5 ml cultures in 96-deep-well plates for S. cerevisiae or 2 ml cultures in 24-deep-well plates for P. pastoris. Soluble... 

The yeast Saccharomyces cerevisae is well known as a host for the expression of heterologous proteins. It is also exceptionally useful for nutrient-dependent viability screening, and is even more versatile than E. coli. Examples of the myriad ways in which genes encoding heterologous proteins can be functionally expressed in yeast for HTS are readily available [13,36-39], However, few applications for antiparasitic drug discovery have been described. 

We have used isolated nucleoli to prepare monoclonal antibodies against nucleolar proteins (S. Chen, J. E. Dove, and J. P. Aris, unpublished results). Six monoclonal antibodies have been characterized by indirect immunofluorescence localization and Western blotting, and have been used to screen yeast expression libraries. At this stage, putative, novel nucleolar proteins are being expressed in yeast with an epitope tag for the purpose of confirming their subcellular localization. 

The two-hybrid system is a yeast-based genetic screen that is used to identify and characterize protein-protein interactions (/). In this system, two fusion proteins are expressed in yeast and their interaction is monitored in vivo using appropriate reporter-gene systems. One hybrid protein, termed the bait, contains the DNA-binding domain (DBD) of a transactivator fused to protein X. The second fusion protein, termed the prey or target, contains an activation domain (ACT) of the transactivator fused to protein Y. If DBD-X and ACT-Y are co-expressed in yeast, and if X and Y interact with one another, then the transactivator is reconstituted in a functionally relevant manner and expression of the reporter gene is enhanced. The power of the system resides in the ability to screen cDNA libraries constructed m the ACT vector to conduct global searches for novel proteins that interact with a protein of interest. 

Biological raw data are stored in public databanks (such as Genbank or EMBL for primary DNA sequences). The data can be submitted and accessed via the World Wide Web. Protein sequence databanks like trEMBL provide the most likely translation of all coding sequences in the EMBL databank. Sequence data are prominent, but also other data are stored, e.g.yeast two-hybrid screens, expression arrays, systematic gene-knock-out experiments, and metabolic pathways. 

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