Identification and characterization of interacting molecules

Details Identification and characterization of interacting molecules Identification and characterization of interacting molecules

2000-05-30

2003-12-16

Marschel, Ardin H. (Department: 1631)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S006120, C435S069100, C435S320100, C702S019000

Reexamination Certificate

active

06664048

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to an improved method for the identification and optionally the characterization of interacting molecules designed to detect positive clones from the rather large numbers of false positive clones isolated by two-hybrid systems. The method of the invention relies on a novel combination of selection steps used to detect clones that express interacting molecules from false positive clones. The present invention further relates to a kit useful for carrying out the method of the invention. The present invention provides for parallel, high-throughput or automated interaction screens for the reliable identification of interacting molecules.
Protein-protein interactions are essential for nearly all biological processes like replication, transcription, secretion, signal transduction and metabolism. Classical methods for identifying such interactions like co-immunoprecipitation or cross-linking are not available for all proteins or may not be sufficiently sensitive. Said methods further have the disadvantage that only by a great deal of energy, potentially interacting partners and corresponding nucleic acid fragments or sequences may be identified. Usually, this is effected by protein sequencing or production of antibodies, followed by the screening of an expression-library.
An important development for the convenient identification of protein-protein interactions was the yeast two-hybrid (2H) system presented by Fields and Song (1989). This genetic procedure not only allows the rapid demonstration of in vivo interactions, but also the simple isolation of corresponding nucleic acid sequences encoding for the interacting partners. The yeast two-hybrid system makes use of the features of a wide variety of eukaryotic transcription factors which carry two separable functional domains: one DNA binding domain as well as a second domain which activates the RNA-polymerase complex (activation domain). In the classical 2H system a so-called “bait” protein comprising of a DNA binding domain (GAL4bd or lex A) and a protein of interest “X” are expressed as a fusion protein in yeast. The same yeast cell also simultaneously expresses a so called “fish” protein comprising of an activation domain (GAL4ad or VP16) and a protein “Y”. Upon the interaction of a bait protein with a fish protein, the DNA binding and activation domains of the fusion proteins are brought into close proximity and the resulting protein complex triggers the expression of the reporter genes, for example, HIS3 or lacZ. Said expression can be easily monitored by cultivation of the yeast cells on selective medium without histidine as well as upon the activation of the lacZ gene. The genetic sequence encoding, for example, an unknown fish protein, may easily be identified by isolating the corresponding plasmid and subsequent sequence analysis. Meanwhile, a number of variants of the 2H system have been developed. The most important of those are the “one hybrid” system for the identification of promoter binding proteins and the “tri-hybrid” system for the identification of RNA-protein-interactions (Li and Herskowitz, 1993; SenGupta et al., 1996; Putz et al., 1996). It is understood in the art that to identify, detect or assay the variety of interactions found in biological systems, different 2H systems must be employed. Indeed, other 2H technologies have been developed to enable protein-protein interactions to be investigated in other organisms and/or different cell compartments. For example, in mammalin cells (Rossi et al, 1997; PNAS 94:8405-8410), in bacterial cells (Karimova et al., 1998; PNAS 95:5752-5756), in the cytoplasm of yeast cells (Johnsson & Varshavsky; 1996 U.S. Pat. No. 5,503,977) and in the periplasmic space of yeast cells (Fowlkes et al., 1998; U.S. Pat. No. 5,789,184).
These 2H systems for the identification of protein-protein-interaction, have, until today, only been carried out on a laboratory scale. The various steps of these systems need to be conducted serially. They are, therefore, quite time consuming. As a consequence, these 2H systems have so far proven unsuitable for the analysis of eukaryotic library vs library screens to investigate protein-protein networks. Although recent developments have taken into account these disadvantages (Bartel et al., 1996), a successful large scale search of interacting proteins, for example on the basis of a eukaryotic library vs. library screen, has not been reported. More importantly also, 2H systems suffer from the serious drawback that many false-positive clones not representing any interactions between binding partners are isolated. This is particularly inconvenient in cases where large numbers of clones are to be analyzed because in the case of a eukaryotic library vs library screen it is typical that several hundreds of thousands of clones have to be analyzed for the investigation of protein-protein networks.
The technical problem underlying the present invention was therefore to overcome these prior art difficulties and to furnish a system that reliably produces clones that express interacting molecules. This system should, moreover, be suitable for large-scale library vs library screens using a parallel, high-throughput or automated approach.
The solution to said technical problem is achieved by providing the embodiments characterized in the claims.
SUMMARY OF THE INVENTION
Accordingly, the present invention relates to a method for the identification of at least one member of a pair or complex of interacting molecules, comprising:
(a) providing host cells containing at least two genetic elements with different selectable and counterselectable markers, said genetic elements each comprising genetic information specifying one of said members, said host cells further carrying a readout system that is activated upon the interaction of said molecules;
(b) allowing at least one interaction, if any, to occur;
(c) selecting for said interaction by transferring progeny of said host cells to
(ca) at least two different selective media, wherein each of said selective media allows growth of said host cells only in the absence of at least one of said counterselectable markers and in the presence of a selectable marker; and
(cb) a further selective medium that allows identification of said host cells only on the activation of said readout system;
(d) identifying host cells containing interacting molecules that
(da) do not activate said readout system on any of said selective media specified in (ca); and
(db) activate the readout system on said selective medium specified in (cb); and
(e) identifying at least one member of said pair or complex of interacting molecules.
Preferably, said interaction is a specific interaction.


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