Relative quantitation of protein-protein interaction strength within the yeast two-hybrid system via fluorescence beta-galactosidase activity detection in a high-throughput and low-cost manner.
The yeast two-hybrid (Y2H) method is capable of delivering vast amounts of interacting positive yeast colonies from a single library screen, particularly if a multifunctional protein is used as bait. However, the selection of definitive colonies for further molecular analysis is limited by both technical practicality and high costs. Here we demonstrate a cost-effective and simple method for the rapid selection and ranking of those Y2H-positive interaction clones that are suitable for further analysis. We performed a Y2H screen for the identification of human transforming growth factor beta2- interacting proteins in a human skin keratinocyte library. The identified clones were ranked by the amount of beta-galactosidase enzyme produced, as well as by the interaction strength of the positive colonies. The combination of high-throughput microplate fluorescence readers and specific fluorescence assays can be utilized for relative quantitation of protein-protein interaction strength of Y2H-positive colonies in crude yeast-cell lysates. We demonstrate here that the high sensitivity of the fluorescence approach can bypass cumbersome conventional methods of cell lysis used in beta-galactosidase assays, and still deliver accurate values for analysis of protein interaction data. Finally, we also achieved a better understanding of general aspects of beta-galactosidase measurements in the Y2H system, such as protein normalization, the influence of yeast culture incubation time on optimal beta-galactosidase detection, and the linearity of beta-galactosidase detection in crude cell lysates.
Oender
K
K
Division of Molecular Dermatology, Department of Dermatology, Paracelsus Private Medical University Salzburg, Salzburg, Austria. k.oender@salk.at
Niedermayr
P
P
Hintner
H
H
Richter
K
K
Koller
L
L
Trost
A
A
Bauer
J W
JW
Hundsberger
H
H
D003362:Cost-Benefit Analysis; D005189:False Positive Reactions; D005453:Fluorescence; D015723:Gene Library; D006801:Humans; D015603:Keratinocytes / Q000201:enzymology; D009930:Organic Chemicals; D025941:Protein Interaction Mapping / Q000379:methods*; D012680:Sensitivity and Specificity; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics*; D001616:beta-Galactosidase / Q000378:metabolism*
C433144:NanoOrange; D009930:Organic Chemicals; D001616:beta-Galactosidase