Table 3. Possible modifications to enhance bait performance in specific applications

Bait problem:	Strongly activating	Weakly activating	Not transported to the nucleus, or low expression level	Continuous expression of LexA-fusion is toxic to yeast	Bait protein requires unblocked amino-terminal end for function	Bait protein expressed at high levels, unstable, or interacts promiscuously	Potential new problem^a
Response:
Truncate/modify bait	+	−	−	−	−	−	It may be necessary to subdivide bait into two or three overlapping constructs, each of which must be tested independently.
Use more stringent strain/reporter combination	+	+	−	−	+?	+?	Use of very stringent interaction strains may eliminate detection of biologically relevant interactions.
Fuse to nuclear localization sequence pJK202	−	−	+	−	−	−
Put LexA-fused protein under GAL1-inducible promoter pGilda	+?	−	−	+	+?	+?	Can no longer use GAL-dependence of reporter phenotype to indicate cDNA-dependent interaction
Fuse LexA to the carboxyl terminus of the bait pNLexA	−	−	−	−	+	−	Generally, LexA poorly tolerates attachment of the amino-terminal fusion domain; only ~60% of constructs are expressed correctly.
Integrate bait, reduce concentration pEG202I	+?	−	−	+?	+?	+	Reduced bait protein concentration may lead to reduced assay sensitivity.

(+) Would usually help; (+?) may help; (−) will not help.
^aAll of the alternative bait expression vectors remain on an Amp^R selection for bacteria. If using them as is, the investigator may need to use a KC8 bacterial passage to isolate the library plasmid after a library screen.