0%

​

Share this page with:

High-throughput: yeast two-hybrid

One of the most frequently used laboratory methods for experimentally determining molecular interactions is yeast two-hybrid (Y2H) screening.7

Y2H is a complementation assay. The readout mechanism is based on a transcription factor, which is split into two independent parts, the DNA-binding domain (BD) and the DNA-activation domain (AD). The BD and AD domains are fused to two proteins of interest, the bait (X) and the prey (Y). This ensures that the readout can only take place when the two halves are brought into close proximity. If the bait and prey proteins bind to each other when expressed in a yeast cell, the transcription machinery becomes activated and a reporter gene is turned on (Figure 3).

Figure 3 The yeast two hybrid (Y2H) concept and a typical readout. [1] The BD domain fused to the bait protein (X) and the AD domain fused to prey protein (Y) are expressed in yeast cells. [2] If proteins X and Y interact, BD binds DNA and AD activates RNA polymerase. An example readout [3]  of a Y2H assay with two bait proteins (Bait 1 and Bait 2) and five prey proteins (1 to 5) (Figure from Koh et. al.3). 

In the above example, positive interactions are shown by colony growth. Readouts for a Y2H assay can also be detected by DNA sequencing or colorimetric methods such as the beta-galactosidase assay (Figure 3).

Advantages:

  • Fast
  • Inexpensive
  • Scalable
  • An in vivo system in which binding sites can be accurately mapped
  • Provides binary relationships (despite limitations, see below)
  • Clone libraries readily available are common

Disadvantages:

  • Interactions occur between proteins that would not normally be present in the same cellular compartment, in the same cell type, or at the same time
  • False positives occur when a yeast protein acts as a bridge for the interaction
  • Limitations inherent to heterologous expression: Both bait and prey proteins can fail to be expressed or might be toxic to the cell, big proteins might need to be cut down into manageable fragments, etc
  • Needs a clone library!