RU2529356C2 - Modified yeast-2-hybrid for effective study of interactions between proteins and their domains - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biological engineering, molecular biology, and biotechnology. What is presented is a yeast cell applicable for detecting an interaction between proteins and their domains co-transformed by two plasmids modified for expression of two proteins in the yeast cells, wherein a nucleotide sequence coding the first protein is cloned into one plasmid, while the nucleotide surface coding the second protein - into the other plasmid, wherein the nucleotide sequences coding the proteins are fused with the nucleotide sequences coding the activation and DNA-binding domains of the protein GAL4; the nucleotide sequences coding the studied proteins are separated from the nucleotide sequences coding the activation or DNA-binding domains of the protein GAL4 by means of a nucleotide sequence coding a peptide representing the sequence GluLeuGluAlaAlaAlaLysGluAlaAlaAlaLysGluAlaAlaAlaLysGluAlaAlaAla which is expressed in the form of a protein bridge between the studied protein and the protein GAL4 domains.

EFFECT: invention enables minimising the mutual interaction of the tested protein domains and DNA-binding/activation domains of a vector and can be used for detecting the mutual interaction between the protein molecules for medical and studying applications.

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Description

FIELD OF THE INVENTION

The present invention relates to the field of bioengineering, molecular biology and biotechnology, in particular to methods for testing interactions between protein molecules.

State of the art

One of the key problems of molecular and cellular biology is related to the study of interactions between proteins. Among all known methods for studying such interactions, the yeast two-hybrid system is the most effective and economical method, on the basis of which the first proteomic studies were carried out by many firms in an attempt to establish all possible interactions between yeast and Drosophilic proteins. The source material for the analysis are two plasmids into which the DNA sequences of two proteins are cloned into the desired reading frame (one of them is fused in a single translation frame with the DNA-binding domain of the transcription factor, and the second with its transcriptional activating domain), for interaction. In this case, physical binding of the studied proteins to each other leads to a rapprochement of the DNA-binding and activation domains and, as a result, expression of the reporter gene is activated or repressed. The most common variant of the two-hybrid system uses the DNA-binding and activation domains of the GAL4 protein and a yeast strain containing reporter genes with GAL4-binding sites in the promoter region (US 5283173, US 5468614). The main drawback of this system is associated with structural limitations imposed by closely spaced domains in the chimeric protein, which result in either the inactivation of the DNA-binding or activation domain of the GAL4 protein, or the corresponding domains of the studied proteins. As a result, more than half of all potential interactions are not detected, which significantly reduces the possibility of using the method.

The functioning of some modern yeast double-hybrid systems is provided by modifications that improve the detection of protein-protein interactions in the presence of possible false signals by using split systems (most often based on the ubiquitin molecule), which work regardless of the activation or repression of transcription of the reporter gene, which can have a strong the effect of test protein molecules (US 6562576, US 6911311, US 20090298089 A1).

There are also modifications of vectors used in the two-hybrid system, mainly aimed at the use of other domains (in contrast to the classical using the GAL4 activator) in the yeast two-hybrid system: for example, repression (US 5885779) or domains of protein participating in membrane signaling pathways cells (US 2010/0075326 A1). In addition, there are modifications of vectors based on the use of recombination and used in the two-hybrid system that simplify the cloning of cDNA into the corresponding plasmids (US 7323313). Finally, there is a known application of flexible linkers between artificial zinc finger domains, the use of which increases the affinity of chimeric proteins for their DNA targets (US 6,479,626).

However, there are no ways in which the vectors for the yeast two-hybrid system are modified so that the domains of the tested proteins are separated from the DNA-binding and / or activation domains by flexible protein bridges that isolate the domains from each other and thereby leveled the effect of steric effects.

SUMMARY OF THE INVENTION

The present invention provides a new method for reducing the interdependence of domains in a yeast two-hybrid system. The vectors used in the yeast two-hybrid system are modified so that the studied proteins are separated from the DNA-binding or activation domains of the GAL4 protein using a neutral flexible protein bridge. To confirm the effectiveness of the chosen approach, a number of known interactions that are not detected in the usual version of the method were tested using modified vectors.

The present invention can be used to detect interactions between protein molecules that can be used for medical and research purposes.

The essence of the described invention is illustrated by the drawing (figure 1), which shows the scheme of vectors designed for cloning in them cDNA sequences of the tested proteins and subsequent screening in a yeast two-hybrid system, where

A - vectors pG4AD_h1 and pG4AD_h2, in which sequences encoding the flexible protein bridge 1 or 2 are cloned below the GAL4 activation domain;

B - vector ph1_G4AD, in which a sequence encoding a flexible protein bridge 1 is cloned above the GAL4 activation domain;

B is the vector pG4BD_h1 and pG4BD_h2, in which sequences encoding the flexible protein bridge 1 or 2 are cloned below the GAL4 DNA binding domain;

G is a ph1_G4BD vector in which a sequence encoding a flexible protein bridge 1 is cloned above the DNA-binding domain of GAL4;

D is the vector plexABD_h1, in which a sequence encoding a flexible protein bridge 1 is cloned below the DNA-binding domain of lexA;

E is a ph1_lexABD vector in which a sequence encoding a flexible protein bridge 1 is cloned above the DNA-binding domain of lexA;

W - amino acid sequences of the used protein bridges 1 and 2.

Example 1. The creation of modifications of the vectors intended for the yeast two-hybrid system.

The yeast two-hybrid system is based on the use of transcription factors, which are characterized by a modular structure and consist of physically and functionally separable domains: a DNA-binding (BD - binding domain) and a domain that activates transcription (AD - activation domain). The physical separation of the BD and AD domains leads to inactivation of the transcription factor. BD and AD domains are fused to the studied proteins X (“bait”) and Y (“prey”), respectively. Reconstruction of the factor becomes possible in the case of the interaction of X with Y, then the transcription of genes that are dependent on the transcription factor used is activated (Bartel, Chien et al. 1993). To create a two-hybrid system, the transcription factor S. cerevisiae GAL4, which is divided into DNA-binding and activation domains, is most often used. Also, the bacterial repressor protein domain lexA is often used as a DNA binding motif.

The source material for the analysis are two plasmids into which, in the desired reading frame, the cDNA sequences of two proteins that are studied for interaction are cloned. The sequence of the first protein is cloned in a single frame with the BD domain in one plasmid, and the sequence of the second is in a single frame with the AD domain of Gal4 in another plasmid.

Yeast cells are co-transformed with two such plasmids, protein constructs are expressed in cells and sent via a special nuclear localization signal from the cytoplasm to the cell nucleus. The BD domain coupled to the second protein binds to promoters of reporter genes into which either GAL4 or lexA binding DNA sequences are inserted, and if the first and second proteins interact, then the AD domain is attracted to the regulatory part of the reporter gene and activates its expression.

It should be noted that in S. cerevisiae cells the fusion protein is expressed in a relatively small amount from the minimal constitutive ADH1 promoter; the sequence of the ADH1 termination signal is also present in the vector. A fusion protein is sent to the nucleus of S. cerevisiae cells using a nuclear localization signal that is part of BD or of heterologous origin (from the SV40 virus). Vectors are able to autonomously replicate in E. coli cells and in S. cerevisiae cells. The vectors contain the β-lactamase gene, which determines E. coli resistance to ampicillin. In the version of the vector with the GAL4 activation domain, there is the LEU2 gene, and with the DNA-binding domain, the TRP1 gene, which allows heterotrophic S. cerevisiae cells to grow under conditions limited to leucine or tryptophan, respectively.

An experiment involves several steps.

- Obtaining expression vectors - plasmids in which the cDNA sequences of the studied proteins are cloned in a single frame with a DNA-binding or activation domain.

- Cotransformation of yeast with two types of plasmids and plating of transformants on Petri dishes with a selective medium that does not contain selective amino acids (for example, tryptophan and leucine in the classical version). Mutations that violate the formation of functionally active proteins are artificially introduced into the genes of biosynthesis of these amino acids in the genome of the used yeast strain. These mutationless genes are expressed from transformed plasmids. As a result, selection occurs on the plates; if the cotransformation is successful, then separate colonies grow on the plate after 2-3 days.

- Several colonies are crossed by a draining stroke on Petri dishes with selective medium (for example, without tryptophan, leucine, histidine and adenine).

- Most often, the histidine and adenine biosynthesis genes ADE2 and HIS3, as well as the β-galactosidase gene LacZ are reporter. If the proteins interact, the ADE2 and HIS3 genes are activated and, therefore, colonies grow. If proteins do not interact, then colony growth is not observed. In addition, it is possible to simultaneously detect the level of activation of the LacZ gene.

The main limitation in the use of the yeast two-hybrid system is the frequent appearance of false negative and false positive results. There are several approaches to overcome these problems:

1) the use of several reporter genes,

2) the transfer in plasmids of the domains of the Gal4 / lexA protein to the C-terminus of the chimeric protein,

3) checking not a whole protein for interaction, but only its individual domains,

4) the use of inhibitors, which in certain concentrations exclude false-positive growth.

However, despite all these approaches, many interactions cannot be detected using the classical version of the yeast two-hybrid system due to the fact that, in the sequence of the chimeric protein, the domains of the tested protein appear to be tightly coupled to the sequences encoding the DNA-binding / activation domains. In this regard, it was suggested that the use of flexible protein bridges in the vectors for the yeast two-hybrid system separating the sequence of the tested protein from the sequences of the DNA-binding / activation domains will allow sterically isolating the protein domains and achieving a detectable signal for the interaction of protein molecules.

As modifiable vectors, pGAD424 and pGBT9 (Clontech) were selected for use in the yeast two-hybrid system and based on the GAL4 activator. Additionally, the corresponding lexA motif was used as the DNA binding domain, which was cloned into the pGBT9 vector instead of the GAL4 DNA binding domain.

DNA sequences encoding 21- and 22-amino acid linkers, which are quite flexible α-helices, were developed and synthesized. The developed amino acid sequences are shown in FIG.

Linker DNA sequences were cloned into vectors for the yeast two-hybrid system either at the N- (Fig. 1B, D, E) or at the C-terminus (Fig. 1A, C, D) relative to the DNA-binding (Fig. 1B, D , D, E) / activation (Fig.1A, B) domains. The HindIII restriction site was used to clone the DNA sequence encoding the protein bridge at the N-terminus of the DNA binding / activation domains. An EcoRI restriction site was used to clone the DNA sequence encoding the protein bridge at the C-terminus of the DNA binding / activation domain. The corresponding restriction sites were laid in the sequence of oligonucleotides used to synthesize the DNA sequence encoding the protein bridge. The oligonucleotide sequences were selected so that the open reading frame of the cloned fragments in the final constructs was not violated.

As a result, 8 vectors were obtained in which the sequences of the studied proteins can be cloned in a single reading frame with DNA-binding / activation domains or at the N- or C-terminus, and in all cases the protein domains of the tested proteins will be separated from the DNA-binding / activation domains with a 21- or 22-amino acid protein bridge.

Example 2. Testing of modified vectors for the detection of interactions between protein molecules.

Based on the modified vectors, plasmids were created to test the effectiveness of the developed approach. To do this, both the classical vectors for the yeast two-hybrid system (pGAD424 and pGBT9) and the modified ones (pG4AD_h1, pG4AD_h2, ph1_G4AD, pG4BD_h1, pG4BD_h2, ph1_G4BD, plexABD_h1, and DNA-tested were tested for DNA-labeled AB1 As a positive control of the correct functioning of all plasmids, the well-studied interaction of Drosophila proteins CTCF and CP190 was used. Initial plasmids were used as negative controls. The results of the verification of all variants of the vectors are presented in FIG. 2, from which it can be seen that only signals of the dCTCF-CP190 pairs are detected in a selective medium without histidine and adenine, and in the β-galactosidase test.

Then, proteins and individual protein domains were selected, for which it is known from in vitro experiments that they can form homodimers, however, in the classical version of the yeast two-hybrid system, a positive signal could not be detected. These are the full-sized CTCF proteins of Drosophila and humans and its N-terminal domains, Pita, Zw5 Drosophila proteins, and their N-terminal domains, which are C4-type zinc finger (the so-called ZAD domain). For all these proteins, in vitro experiments have shown that they can form homodimers, and N-terminal domains of these proteins are necessary for such dimerization. Figure 3 presents the results of a yeast two-hybrid system obtained using classical vectors into which the cDNA sequences of these proteins were cloned. It can be seen that in the case of individual domains in all experiments negative results were obtained, and only in some cases a positive signal is detected using full-sized proteins.

All studied sequences were cloned into modified vectors, and Fig. 4 presents the results of this variant of the yeast two-hybrid system. Thus, in many combinations in which the N-terminus of the tested proteins remained free and was separated from the DNA-binding / activation domains by a protein bridge, it was possible to detect positive signals that were previously undetectable. Based on the results obtained, it is indeed seen that the molecules of the studied proteins interact with each other due to the N-terminal domains.

Bibliography

1. Bartel, R., S.T. Chien, et al. (1993). ″ Elimination of false positives that arise in using the two-hybrid system. ″ Biotechniques 14 (6): 920-4.

Claims (1)

A yeast cell designed to detect the interaction between proteins and their domains, cotransformed with two plasmids modified to express two proteins in yeast cells, where the nucleotide sequence encoding the first protein is cloned into one plasmid and the nucleotide sequence encoding the second protein into another a plasmid where the nucleotide sequences encoding the proteins are fused to the nucleotide sequences encoding the activation and DNA-binding domains of the GAL4 protein, wherein eotidnye sequences encoding proteins under study, separated from nucleotide sequences encoding the activation or the DNA binding domain of GAL4 protein, using the nucleotide sequence encoding a peptide representing a sequence GluLeuGluAlaAlaAlaLysGluAlaAlaAlaLysGluAlaAlaAlaLysGluAlaAlaAla, which is expressed as a protein bridge between the test protein and the protein domains of GAL4.

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