Possible modifications to enhance LexA-fused bait performance in specific applications:

Bait Problem

Response

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

truncate / modify bait

Use more stringent strain/reporter combination

fuse to nuclear localization sequence
pJK202

Put LexA-fused protein under GAL1-inducible promoter
pGilda

Fuse LexA to the carboxy terminus of the bait
pNLexA

Integrate bait, reduce concentration
pEG202I

Potential New Problem*

It may be necessary to subdivide bait into two or three overlapping constructs, each of which must be tested independently

Use of very stringent interaction strains may eliminate detection of biologically relevant interactions

Can no longer use GAL-dependence of reporter phenotype to indicate cDNA-dependent interaction

Generally, LexA poorly tolerates attachment of the N-terminal fusion domain. Only ~60% of constructs are expressed correctly.

Reduced Bait protein concentration may lead to reduced assay sensitivity

+: would usually help; +?: may help; - : will not help

*: all 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 bacteria to isolate the library plasmid after a library screen.