Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1998-08-25
2000-06-13
Schwartzman, Robert A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
4353201, C12Q 168, C12N 1563
Patent
active
06074829&
DESCRIPTION:
BRIEF SUMMARY
The invention is concerned with improvements in methods for detecting protein-protein interactions, more specifically with the reduction or elimination of false positives. The invention relates in particular to improvements to two-hybrid or interaction trap systems for selecting for interacting proteins in living cells. The invention also includes modifications to hybrid constructs and to vectors and yeast strains expressing such constructs.
BACKGROUND OF THE INVENTION
In the two-hybrid system of Fields and Song(1) described in U.S. Pat. No. 5,283,173 two chimeric genes which encode hybrid proteins are used to test the interaction between a known protein and protein of interest. The first chimeric gene codes for a known protein, often called the bait protein, fused to a DNA-binding domain. The second chimeric gene codes for a protein of interest fused to the transcriptional activation domain. Additionally, the protein of interest may not be known and could be derived for example from a cDNA library. In a suitable host cell such as yeast, if the protein of interest and the bait protein do interact they bring into proximity the DNA-binding and transcriptional activation domains. This proximity is sufficient to cause transcription of a marker gene placed under the control of a promoter containing a binding site for the DNA-binding domain.
Yeast genetic systems have also been used in methods for defining DNA-binding domains of proteins. One such method (2) uses a chimeric protein containing a transcriptional activation domain together with two DNA-binding domains, each capable of binding to a different reporter gene. One of the DNA-binding domains is mutated to analyse its DNA-binding properties.
In the two-hybrid system, once a specific cDNA-encoded protein or known protein of interest has been shown to give rise to activation of the marker gene, it is important to show that this is indeed due to an interaction between the bait protein and the protein of interest and not due to a "false positive" interaction (3). At least four classes of false positives may occur in two-hybrid systems where a protein of interest, which may be encoded by a cDNA library, is fused to the activation domain: would activate transcription independent of the DNA-binding hybrid, by binding: the bait hybrid; between the protein and an epitope tag or tags encoded by the bait hybrid vector, or to the epitope tag(s) itself; between the protein of interest and an eptiope tag or tags encoded by the activator hybrid vector.
A number of strategies have been previously described which remove some of the above classes of false positives (3). Three such strategies are as follows: selectable marker (eg. HIS3) and the other one a measurable marker activity (eg. lacZ) and the reporter gene promoters are usually different. This allows the removal of those proteins in the first class of false positive where a non-specific interaction occurs between the protein and the promoter of the marker gene, since non-specific interaction with both of the marker gene promoters is less likely to occur. removes the bait hybrid plasmid, so that only the activator hybrid expressing the protein of interest is present in the cell. If the marker gene expression remains high, then this shows that the activation is due to spurious promoter activation by the protein of interest hybrid protein, rather than via binding through the bait protein hybrid. protein interacts with the bait protein of interest and no other part of the bait hybrid protein.
A review by Allen et al 1995 (5) describes strategies for the removal of false positives. One of these, elaborated on in Harper et al 1993 (6), involves curing out the bait hybrid and using unrelated bait proteins fused to the DNA-binding domain of the original bait protein, to detect false positives.
Another strategy is described by Le Douarin et al 1995 (7). When an apparent positive interaction is detected, the bait hybrid protein is removed by curing and a bait hybrid containing a different DNA-binding domain is introduced.
REFERENCES:
P. Bartel et al., "Elimination of false positives that arise in using the two-hybrid system", Biotechniques, vol. 14, No. 6, pp. 920-922, (1993).
B. Le Douarin et al., "A new version of the two-hybrid assay for detection of protein-protein interactions", Nucleic Acids Research, vol. 23, No. 5, pp. 876-878, (1995).
J. Harper et al., "The p21 Cdk-interacting protein Cip 1 is a potent inhibitor of G1 cyclin-dependent kinase", Cell, vol. 75, pp. 805-816, Nov. 19, 1993.
J. Luban et al. "The yeast two-hybrid system for studying protein-protein interactions", Current Opinion in Biotechnology, vol. 6, No. 1, pp. 59-64, (1995).
C. Bendixen et al., "A yeast mating-selection scheme for detection of protein-protein interactions", Nucleic Acids Research, vol. 22, No. 9, pp. 1778-1779, (1994).
T. Wilson et al., "A genetic method for defining DNA-binding domains: application to the nuclear receptor NGFI-B", Proc. Natl. Acad. Sci., vol. 90, pp. 9186-9190, Oct. 1993.
S. Dalton et al., "Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element", Cell, vol. 68, pp. 597-612, Feb. 7, 1992.
U. Yavuzer et al., "pWITCH: a versatile two-hybrid assay vector for the production of epitope/activation domain-tagged proteins both in vitro and in yeast", Gene, vol. 165, pp. 93-96, Nov. 1995.
Fallon Rachel Alison
Hurd Douglas
Jones Nicholas
White Michael
Amersham International plc
Schwartzman Robert A.
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