Relative quantitation of protein-protein interaction strength within the yeast two-hybrid system via fluorescence β-galactosidase activity detection in a high-throughput and low-cost manner

K. Önder, P. Niedermayr, H. Hintner, K. Richter, L. Koller, A. Trost, J.W. Bauer, H. Hundsberger

Publikation: Beitrag in FachzeitschriftArtikelBegutachtung

Abstract

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 β2-interacting proteins in a human skin keratinocyte library. The identified clones were ranked by the amount of β-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 β-galactosidase assays, and still deliver accurate values for analysis of protein interaction data. Finally, we also achieved a better understanding of general aspects of β-galactosidase measurements in the Y2H system, such as protein normalization, the influence of yeast culture incubation time on optimal β-galactosidase detection, and the linearity of β-galactosidase detection in crude cell lysates. © Mary Ann Liebert, Inc.
OriginalspracheEnglisch
Seiten (von - bis)709-719
Seitenumfang11
FachzeitschriftAssay and Drug Development Technologies
Jahrgang4
Ausgabenummer6
DOIs
PublikationsstatusVeröffentlicht - 2 Jän. 2007

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