Relative Quantitation of Protein–Protein Interaction Strength Within the Yeast Two-Hybrid System via Fluorescenceβ-Galactosidase Activity Detection in a High-Throughput and Low-Cost Manner

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 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.

H2O2-dependent translocation of TCTP into the nucleus enables its interaction with VDR in human keratinocytes: TCTP as a further module in calcitriol signalling

“…A cDNA library derived from human keratinocytes was cloned in the GAL4 AD vector pGADT7 (4,128 × 10 6 independent transformants) as described before [25]. For the Y2H screening that was conducted with the MATCHMAKER GAL4 Two-hybrid System 3, bait and prey plasmids were co-transformed into the haploid yeast strain AH109 and plated on SD-trp-leu-ade-his synthetic medium where tryptophane and leucine represent auxotrophic selection markers and ADE2/HIS3 select for actual reporter activity.…”

A Saccharomyces cerevisiae cell‐based quantitative β‐galactosidase assay compatible with robotic handling and high‐throughput screening

“…luciferase or green fluorescent protein) have become increasingly popular tools (Bovee et al , 2004). Luminescent or fluorescent β‐galactosidase substrates are now available and offer a route to increase the sensitivity of detection and range of applications for monitoring gene expression with a range of strains containing lacZ fusions (Oender et al , 2006; Serebriiskii and Golemis, 2000). At the same time, advances in microplate technology have allowed both assay miniaturization and the possibility to save time, reagents and increase dramatically the number of samples analysed (Berg et al , 2000; Brouchon‐Macari et al , 2003).…”

PRIMOS: An Integrated Database of Reassessed Protein–Protein Interactions Providing Web-Based Access toIn SilicoValidation of Experimentally Derived Data

“…This example details part of a standard Y2H screening that we have performed to identify novel TGF-b2 interactors within a human keratinocyte cDNA library. Out of 32 reproducible hits, 116 PRIMOS positively validates two previously unknown PPIs: the interaction of TGF-b2 with secretory leukocyte protease inhibitor (SLPI, PRIMOS score: high), and with dual specificity mitogen-activated protein kinase kinase 2 (MAPKK2, PRIMOS score: high). These results complement a plethora of earlier, and, to a large extent, methodically divergent studies that were performed to systematically decipher the C. elegans abnormal dauer formation (DAF)-7/ TGF-b signaling network, 117 the human/mammalian TGF-b superfamily-regulated Smad signaling cascade, 118,119 and the TGF-b receptor type I interactome.…”

“…These results complement a plethora of earlier, and, to a large extent, methodically divergent studies that were performed to systematically decipher the C. elegans abnormal dauer formation (DAF)-7/ TGF-b signaling network, 117 the human/mammalian TGF-b superfamily-regulated Smad signaling cascade, 118,119 and the TGF-b receptor type I interactome. 120 In doing so, PRIMOS surpasses the strategy of measuring b-galactosidase activity within protein content-normalized samples in which false-positive PPIs can be readily distinguished, 116 although numerous additional yeast co-transformants similarly range between the internal negative and the exceptionally strong p53-SV40 positive controls. SLPI is a cationic, lowmolecular weight protein, which, in addition to its inhibitory activity against serine proteases that are thought to be primarily involved in protection against detrimental neutrophil protease-induced tissue damage at sites of inflammation, 121 also exerts antibacterial, antifungal, antiretroviral, and immunomodulatory activities.…”