Figure 2. Bait Expression from a combined bacterial/yeast expression plasmid.

A. Plasmid pGLS20, pGLS22 and pGLS23 (pGLS22 and pGLS23 only differ in the presence of an extra EcoRI site in the Cm^R gene of pGLS22) use a combined TEF1/uvLac promoter to express λ cI fused baits in yeast or bacteria. Plasmids are selected in yeast by selection for G418 resistance (pGLS20) or HIS5 complementation (pGLS23), and in bacteria by selection for kanamycin resistance (pGLS20) or chloramphenicol resistance (pGLS23). Relative expression of cI baits from these plasmids, versus the previously described pGBS10 (yeast two-hybrid, (11)) or pBT (bacterial two-hybrid, Stratagene) vectors is shown in bacteria (center panel). B. To demonstrate relative bait levels, equal total protein concentration was confirmed by Coomassie staining of a PAGE gel loaded with equivalent amounts of cell lysate for bacteria expressing each plasmid (not shown). Then, equal volumes of 1:40 (for pGLS20) or 1:100 (for pBT) dilutions of extracts in sample buffer were loaded in parallel with the same volume of undiluted extract from pGBS10-bearing cells. Western blots using anti-cI antibodies are shown. C. pGBS10 and pGLS20 express comparable levels of λ cI baits in yeast, based on Western analysis with antibodies to λ cI. 1, 2 denotes two independent transformants in bacteria or yeast; -, denotes yeast containing no bait plasmid.