Biology Reference
In-Depth Information
AD
Bait
protein
Prey
protein
Transcription
DNA-BD
GAL UAS
Promoter
Reporter gene
Fig. 1
GAL4 yeast two-hybrid. The DNA-BD of the yeast GAL4 protein binds the
GAL UAS upstream of the reported genes. The AD of the GAL4 protein functions
as a transcriptional activator
Several varieties of the Y2H exist, commonly involving DNA-
binding domains that derive from the yeast Gal4 protein or the
Escherichia coli
LexA protein [
4
,
5
]
.
Transcriptional activation
domains are commonly derived from the Gal4 protein, the herpes
simplex virus VP16 protein, or a protein encoded by random
E. coli
sequence, the B42 “acid blob.” Reporter genes include the
E. coli lacZ
gene and selectable yeast genes such as HIS3, ADE2 (Gal4 sys-
tem), or LEU2 (LexA system).
Yeast two-hybrid systems provide a sensitive method for detect-
ing relatively weak and transient protein interactions. In addition,
because the two-hybrid assay is performed in vivo
,
the proteins are
more likely to be in their native conformations, which may lead to
increased sensitivity and accuracy of detection. In spite of its advan-
tages, the yeast two-hybrid system has also some limitations that
must be considered prior use.
Bait proteins may have DNA-binding and/or transcriptional
activating properties; hence, deletion of certain portions of
bait proteins will be required to eliminate the unwanted
activity.
●
Some fused proteins may not be able to entrance or be stably
expressed in the yeast nucleus. The GAL4 BD has its own
nuclear localization signal (NLS) within its N-terminal resi-
dues while LexA based system does not contain a NLS.
●
In some cases, the DNA-BD or AD fusion moiety may hinder
the interaction site, or interfere with the folding of the protein,
thus altering the ability of the proteins to interact.
●
Cellular yeast cell environments may not allow the proper fold-
ing or posttranslational modifi cations required for interaction
of some proteins.
●
Some protein interactions may not be detectable in a GAL4-
based two-hybrid system, but may be detectable using a LexA-
based system, and vice versa.
●
In this chapter we present protocols based on the yeast Gal4
protein where a bait gene is expressed as a fusion to the GAL4
