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Screening in Yeast With a Bacterial/Yeast Two-Hybrid System 285be assayed by Western blot, see Subheading 3.2.1.). The reasons for this are notclear; however, it appears to be a bait-specific phenomenon, and may be linked tosome degree of toxicity associated with continued expression of particular proteinsin yeast. It is usually necessary to modify such bait(s), as use of the unmodifiedbaits is associated with high backgrounds of false-positives and artifactualresults. In case of problematic (toxic, autoactivating nonnuclear) baits, a numberof bait modification strategies have been described for LexA-based (but not forcI-based) fusions (23), which may provide useful models for subsequent steps.10. In addition to the simplified technique described in this chapter, a number of moreelaborate (and time-consuming) protocols exist (e.g., see Clontech’s Yeast ProtocolsHandbook, available at http://www.clontech.com.11. Many fusion proteins exhibit sharp decreases in detectable levels of protein withthe onset of stationary phase. Therefore, use of the saturated cultures is not recommendedfor this assay.12. Frozen samples will be stable for at least 4–6 mo, and will need to be boiled for5 min at 100°C before loading on an sodium dodecyl sulfate-polyacrylamide gel.13. The lysates prepared from yeast cultures containing pGLS22-Ras and pEG202-Krev1 allows comparison of expression levels of new baits with two well-expressedbait proteins that have worked well under library screening conditions. Some proteinsmay be synthesized at lower levels, or be posttranslationally cleaved by proteases(resulting in anomalously small baits). This can be because of the size ofthe fused domain derived from the protein of interest (proteins of 60–80 kDa andlarger often have problems). In case of proteolytically clipped proteins, screensmight inadvertently be performed with DBD fused only to the amino(N)-terminalend of the larger intended bait. It is also possible for the proteins expressed at lowlevels, and seemingly inactive in transcriptional activation assays to be upregulatedto much higher levels under the auxotrophic selection, and suddenly demonstratea high background of transcriptional activation. Hence, it is often a goodidea to remake baits showing these properties as smaller derivatives of the proteinsof interest. A high percentage of the colonies not appropriately expressing the baitprotein, although containing the bait plasmid, may be indicative that the bait istoxic in the yeast. Finally, the best way to find out if a bait protein is correctlyexpressed is to coexpress it in yeast with a known interaction partner as a prey (i.e.,expressed as an AD-fusion), and scoring for transcriptional activation on appropriatedropout media.14. A “traditional” but more laborious alternative to the mating, directly transforming thelibrary into yeast containing the bait (21), is not really practical for dual bait system.Such direct transformation in the bait strain requires media not only selective forlibrary plasmid, but also maintaining selective pressure to keep both baits and reporter.15. A good library transformation efficiency should yield approx 10 5 transformants/µgof library DNA (for transformation with a single plasmid). Transformation ofyeast in multiple small aliquots in parallel helps reduce the likelihood of contamination;furthermore, it frequently results in significantly better transformationefficiency than that obtained by using larger volumes of yeast in a smaller number