Question

High throughput technologies have been underway for several years now and include both yeast two-hybrid screening and tandem affinity protein (TAP) or co-IPs for each predicted open reading frame (ORF) in an organism’s proteome. These strategies have been successful in identifying a large number of predicted protein-protein interactions.

a. In general, what is a primary limitation on these two techniques in their ability to identify all biologically relevant binding partners for any given protein?

b. Although both these methods have been employed to analyze some of the same targets in proteomic studies designed to identify all interacting partners, they often do not identify the same exact set of partners. Drawing on your knowledge of the two methods, propose a reason for the difference in partners identified by the two methods.

Answer 1

a) All biotechnology techniques have certain advantages and certain limitations. The major limitations of yeast two hybrid (y2h) assays is that the bait and prey proteins must be able to express properly in the host yeast. Some proteins do not fold correctly in the yeast system and some are not stable. Some proteins also tend to be toxic leading to the death of the host yeasts. Moreover, this techniques has been known to give a large number of false positives.

The major limitations of coimmunoprecipitation (co-IP) are that the transient interactions are often not detected. Co-IP cannot tell whether two proteins interact directly or indirectly via some other proteins.

b) The protein-protein interactions detected by both these techniques are not exactly the same. This is so because both the methods have a very different basic principle. Y2H depends on the introduction of the bait and prey proteins into the yeast vector and then mating between these yeast. Certain reporter genes are transcribed in the yeast having the proteins which interact, which allows those yeast to grow and be detected.

Co-IP is based on the immunological interaction between the bait and the prey proteins. The bait protein ia captured by a specific antibody and if a prey protein interacts with this bait it can be detected because it will precipitate along with the bait.

Since certain proteins may not be stably express in the yeast, they may not be detected by y2h, but they can be detected by co-IP.