Yeast interaction trap assay

Details Yeast interaction trap assay Yeast interaction trap assay

2000-03-16

2001-12-04

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007100, C435S320100, C435S254200, C435S254210

Reexamination Certificate

active

06326150

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the field of molecular biology. More specifically, the invention provides novel compositions and methods to facilitate the isolation and characterization of novel, protein-protein interactions involved in the regulation of cell growth and metabolism.
BACKGROUND OF THE INVENTION
Several publications are referenced in this application by numerals in parenthesis in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
Biological regulatory systems require the specific organization of proteins into multi-component complexes. Two-hybrid systems have been used to identify novel components of signaling networks based on interactions with defined partner proteins (1-5). An important consideration in use of two-hybrid systems has been the degree to which interacting proteins distinguish their biological partner from evolutionarily conserved related proteins, and the degree to which observed interactions are specific.
In the basic version of the Yeast Interaction Trap Assay (20;
FIG. 1A
herein), the plasmid pEG202 or a related vector is used to express the probe or “bait” protein as a fusion to the heterologous DNA binding-protein LexA. Many proteins, including transcription factors, kinases and phosphatases, have been successfully used as bait proteins. The essential requirements for the bait protein are that it should not be actively excluded from the yeast nucleus, and it should not possess an intrinsic ability to strongly activate transcription. The plasmid expressing the LexA fusion bait protein is used to transform the yeast possessing a dual reporter system responsive to transcriptional activation through the Lex A operator. In one such example, the yeast strain EGY48 contains the reporter plasmid pSH18-34. In this case, binding sites for LexA are located upstream of two reporter genes. In the EGY48 strain, the upstream activating sequences of the chromosomal LEU2 gene, required for the biosynthesis of leucine, are replaced with LexA operator DNA binding sites. pSH18-34 contains a LexA operator-lacZ fusion gene. These two reporters allow selection for transcriptional activation by permitting selection for viability when cells are plated on medium lacking Leu, and discrimination based on color when the yeast is grown on medium containing X-gal.
In the basic protocol, EGY48/pSH18-34 transformed with a bait is characterized for its ability to express the fusion protein, growth on medium lacking leu, and for the level of transcriptional activation of lacZ.
In an interactor hunt, the strain EGY48/pSH18-34 containing the bait expression plasmid is transformed with a conditionally expressed library made in the vector pJG-5. This library uses the inducible yeast Gall promoter to express proteins as fusions to an acidic domain (“acid blob”) that functions as a portable transcriptional activation motif. Expression of library-encoded proteins is induced by plating transformants on medium containing galactose (Gal), and the yeast cells are subsequently plated in gal medium lacking leucine. Yeast cells containing library proteins that do not interact specifically with the bait protein will fail to grow in the absence of Leu. Yeast cells containing library proteins that interact with the bait protein will form colonies within 2-5 days, and the colonies will turn blue when the cells are streaked on gal medium containing Xgal. The cells will not grow or turn blue on glucose medium—leucine+Xgal. The plasmids are isolated and characterized by a series of tests to confirm specificity of the interaction with the initial bait protein. Those found to be specific are ready for further analysis. Thus, in summary, existing reagents assay the interaction of an activation-domain-fused protein A with a DNA-binding-domain fused protein B by their ability to activate transcription of two DNA-binding domain responsive reporters using a single bait moiety.
Kits or systems for practicing the methods described above are commercially available. Typically such a kit includes several components, i.e., a bait expression plasmid, and activation domain fusion plasmid, and a lexA operator-LacZ reporter plasmid. The lexA operator-LEU-2 gene is present in the host yeast strain. The first vector or plasmid contains a promoter and may include a transcription termination signal functionally associated with the first chimeric gene in order to direct the transcription of the first chimeric gene. The first chimeric gene includes a DNA sequence that encodes a DNA-binding domain and a unique restriction site(s) for inserting a DNA sequence encoding a first test protein or protein fragment in such a manner that the first test protein is expressed as part of a hybrid protein with the means for replicating itself in the host cell and in bacteria. Also included in the first vector is a first marker gene, the expression of which in the host cell permits selection of cells containing the first marker gene from cells that do not contain the first marker gene. The kit also includes a second vector which encodes a second chimeric gene. The second chimeric gene also includes a promoter and a transcription termination signal to direct transcription. The second chimeric gene also includes a DNA sequence that encodes a transcriptional activation domain and a unique restriction site(s) to insert a DNA sequence encoding the second test protein or protein fragment into the vector, in such a manner that the second test protein is capable of being expressed as part of a hybrid protein with the transcriptional activation domain. In one embodiment, the DNA-binding domain of the first hybrid protein and the transcriptional activation domain of the second hybrid protein are derived from transcriptional activators having separate DNA-binding and transcriptional activation domains. However, the DNA binding domain can be from any protein that binds DNA. The second vector also includes a means for replicating itself in the host cell and in bacteria. The second vector also includes a second marker gene, the expression of which, in the host cell permits selection of cells containing the second marker gene from cells that do not contain the second marker gene. The kit also includes a host yeast strain. The host yeast strain contains the detectable gene having a binding site for the DNA-binding domain of the first hybrid protein. The binding site is positioned so that the detectable gene expresses a detectable protein when the detectable gene is activated by the transcriptional activation domain encoded by the second vector. Activation of the detectable gene is possible when the transcriptional activation domain is in sufficient proximity to the detectable gene as when it associates with the protein encoded by the first vector. The host yeast strain, by itself, is incapable of expressing a protein having a function of the first marker gene, the second marker gene, the DNA-binding domain, or the transcriptional activation domain. In using the kit of the prior art, the interaction of the first test protein and the second test protein in the host cell causes a measurably greater expression of the detectable gene than when the DNA-binding domain and the transcriptional activation domain are present, in the absence of an interaction between the first test protein and the second test protein. The detectable gene may encode an enzyme or other product that can be readily measured. Such measurable activity may include the ability of the cell to grow only when the marker gene is transcribed, or the presence of detectable enzyme activity only when the marker gene is transcribed. U.S. Pat. Nos. 5,283,173 and 5,580,736 disclose two variations of the original interaction trap assay. The disclosures of these two patents are incorporated by reference herein.
Certain difficulties have been experienced in implementing the systems des

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