Assay methods

Details Assay methods Assay methods

1998-03-27

2001-12-25

Schwartzman, Robert A. (Department: 1636)

Measuring and testing

C435S091400

Reexamination Certificate

active

06332897

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to methods useful for detecting protein-protein interactions. Protein-protein interactions enable the association of two or more proteins through the formation of non-covalent bonds when two protein surfaces are precisely matched. These bonds account for the specificity of recognition. Protein-protein interactions are involved, for example, in the assembly of enzyme subunits; in antigen-antibody reactions; in forming the supramolecular structures of ribosomes, filaments, and viruses; in transport; and in the interaction of receptors on a cell with growth factors and hormones. Products of oncogenes can give rise to neoplastic transformation through protein-protein interactions.
BACKGROUND OF THE INVENTION
The yeast two-hybrid (Y2H) assay is a method for detecting protein-protein interactions using a genetic system. The technique may be used for charting protein interactions, and hence, identifying potential partners in genetic pathways. The assay is sensitive and yields the DNA sequences encoding proteins that interact. In a typical two-hybrid assay, a known protein that forms part of a DNA-binding domain hybrid is assayed against a library of all possible proteins present as transcriptional activation domain hybrids. Some two hybrid approaches rely on interaction mating. In this method, the protein fused to the DNA-binding domain and the protein fused to the activation domain are expressed in two different haploid yeast strains of opposite mating type and the strains are mated to determine if the two proteins interact. When haploid yeast strains of opposite mating type come into contact, mating occurs and results in fusion of the two haploids to form a diploid yeast strain. An interaction can thus be determined by measuring activation of a two-hybrid reporter gene in the dipoid strain.
WO 94/10300 and U.S. Pat. No. 5,283,173 describe methods for detecting the interaction between proteins using reconstitution of the activity of a transcriptional activator. This reconstitution makes use of chimeric genes which express hybrid proteins. The first hybrid contains the DNA-binding domain of a transcriptional activator fused to a known protein (the “bait”), with the DNA binding domain DNA binding element placed upstream of a reporter gene. “Prey” proteins are cloned as either random sequences or cDNAs and are fused to the amino- or carboxy-terminus of a transcription activation domain. If the bait and prey proteins are able to interact, they bring into close proximity the two domains of the transcriptional activator. This proximity is sufficient to cause transcription, which can be detected by the activity of a reporter gene that contains a binding site for the DNA-binding domain.
The disadvantages of these techniques is that irrelevant interactions with yeast proteins are generated. These include false-positive interactions that are unlikely to be found in living cells, and false-negative interactions, that is, those interactions that would otherwise be detected but are not. The techniques as disclosed in WO 94/10300 and U.S. Pat. No. 5,283,173 require the use of mating in solid medium which is cumbersome, labor-intensive, and does not preserve diploid cells for further analysis.
We have developed the mating strategy of the yeast two-hybrid assay into an automated format which allows many bait proteins to be processed. The format uses an arraying means, for example, microtiter plates and liquid mass-mating of a subset of a large, complex library. By tracking positive interactions in the library, we have also developed a method to create a functionally-subtracted library, that is, one that can be made devoid of a scorable phenotype. For example, our method allows for the determination of detection of hybrids that react promiscuously with many targets, such as heat shock proteins, and their elimination from any future considerations.
SUMMARY OF THE INVENTION
In accordance with the present invention there is provided a method for detecting protein-protein interactions comprising liquid mass-mating of subsets of a large, complex library. The method provides a means for subtracting irrelevant protein-protein interactions to yield a “functionally-subtracted” assay.
DETAILED DESCRIPTION OF THE INVENTION
According to an aspect of the present invention, there is provided a method for detecting an interaction between a first test protein and a second test protein, comprising:
(a) providing a host cell containing a reporter gene wherein the reporter gene expresses a detectable protein when the reporter gene is activated by an amino acid sequence including a transcriptional activation domain when the transcriptional activation domain is in sufficient proximity to the reporter gene;
(b) providing a first chimeric gene that is capable of being expressed in the host cell, the first chimeric gene comprising a DNA sequence that encodes a first hybrid protein, the first hybrid protein comprising:
(i) a DNA-binding domain that recognizes a binding site on the reporter gene in the host cell; and
(ii) a first test protein or fragment thereof that is to be tested for interaction with at least one second test protein or fragment thereof;
(c) providing a second chimeric gene that is capable of being expressed in the host cell, the second chimeric gene comprising a DNA sequence that encodes a second hybrid protein, the second hybrid protein comprising:
(i) the transcriptional activation domain; and
(ii) a second test protein or fragment thereof that is to be tested for interaction between the first test protein or fragment thereof; wherein interaction between the first test protein and the second test protein in the host cell causes the transcriptional activation domain to activate transcription of the reporter gene;
(d) introducing the second chimeric gene into the host cell and subsequently introducing said cells into an arraying means thereby creating a master library plate;
(e) introducing cells from the master library plate into a second arraying means thereby creating a mating set;
(f) introducing the first chimeric gene into the host cell and subsequently introducing said cell into the mating set;
(g) selecting for outgrowth of the interaction of the first and second genes;
(h) removing a portion of the mating set to a third arraying means thereby creating a rescue set;
(i) determining whether the reporter gene has been expressed in the mating set; and
(j) analyzing the cells from the rescue plate.
The term “reporter gene” or “marker gene” as used herein means any gene whose expression may be assayed. More than one reporter gene may be encoded by the host cell in step (a) above.
The term “arraying means” as used herein means any method for holding clones in liquid media, suspension, or solid media, for example, microtiter plates or test tubes.
The term “selecting for outgrowth” as used herein means any method using a selectable means to either amplify or isolate a set of interacting proteins. This selectable means may include outgrowth in a nutritionally-deficient growth medium wherein the interacting proteins cause transcription of a biosynthetic gene or pathway. Examples of other useful selectable means include amino acid, metabolic, catabolic and nucleic acid biosynthetic genes, such as yeast HIS3, URA3, and LYS2, GAL1,
E. coli
galK, and CAT, GUS, antibiotic resistance, and any gene encoding a cell surface antigen for which antibodies are available. Outgrowth may be allowed to proceed for 5-10 days prior to selecting for outgrowth.
The term “analyzing” as used herein means any method for obtaining information regarding protein-protein interactions, for example, selecting positive clones, performing PCR, DNA sequence analysis, and comparison with databases such as LifeSeq® (Incyte Pharmaceuticals) or Genbank.
The term “functionally substracted” means devoid of a detectable phenotype that represents an irrelevant protein-protein interaction.
In a further aspect of the invention, determination of reporter gene expression and analysis of cells may

Affiliated with

Also associated with

Rating

Assay methods does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Assay methods, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Assay methods will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2570240