BAPJ69-4A: a yeast two-hybrid strain for both positive and negative genetic selection.
Genetic selection systems, such as the yeast two-hybrid system, are efficient methods to detect protein-protein and protein-ligand interactions. These systems have been further developed to assess negative interactions, such as inhibition, using the URA3 genetic selection marker. Previously, chemical complementation was used to assess positive selection in Saccharomyces cerevisiae. In this work, a new S. cerevisiae strain, called BAPJ69-4A, containing three selective markers ADE2, HIS3, and URA3 as well as the lacZ gene controlled by Gal4 response elements, was developed and characterized using the retinoid X receptor (RXR) and its ligand 9-cis retinoic acid (9cRA). Further characterization was performed using RXR variants and the synthetic ligand LG335. To assess the functionality of the strain, RXR was compared to the parent strain PJ69-4A in adenine, histidine, and uracil selective media. In positive selection, associating partners that lead to cell growth were observed in all media in the presence of ligand, whereas partners that did not associate due to the absence of ligand displayed no growth. Conversely, in negative selection, partners that did not associate in 5-FOA medium did not display cell death due to the lack of expression of the URA3 gene. The creation of the BAPJ69-4A yeast strain provides a high-throughput selection system, called negative chemical complementation, which can be used for both positive and negative selection, providing a fast, powerful tool for discovering novel ligand receptor pairs for applications in drug discovery and protein engineering.
Department of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, United States.
Hally Anne
HA
Shaffer
Michael Kenneth
MK
Rood
Badar
B
Kashlan
Eileen I-ling
EI
Chang
Donald Francis
DF
Doyle
Bahareh
B
Azizi
D003470:Culture Media / Q000737:chemistry; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D012641:Selection, Genetic; D020798:Two-Hybrid System Techniques
D003470:Culture Media; D029701:Saccharomyces cerevisiae Proteins