RU2762410C1 - Modification of the yeast-2-hybrid system making it possible to test interactions between proteins within multicomponent protein complexes - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a yeast cell designed to detect interactions between proteins and their domains in multicomponent protein complexes. The yeast cell is co-transformed by three plasmids modified to express several proteins in yeast cells. In this case, the nucleotide sequence encoding the DNA-binding domain of the GAL4 protein is fused with the nucleotide sequence encoding the first protein and cloned into one plasmid. The nucleotide sequence encoding the activation domain of the GAL4 protein is fused with a nucleotide sequence encoding the second and third proteins and having a single reading frame with one promoter and terminator, and cloned into a second plasmid, while a nucleotide sequence encoding a self-cleaving peptide is embedded between the second and third proteins, which makes it possible for several proteins or their domains to be expressed simultaneously in a single reading frame with one promoter and terminator. The nucleotide sequence encoding the non-hybrid protein has been cloned into a third plasmid. In this case, the self-cleaving peptide is a 2A-peptide of the horse B rhinitis virus, having the amino acid sequence GlySerGlyGlyAlaThrAsnPheSerLeuLeuLysLeuAlaGlyAspValGluLeuAsnProGlyPro, presented in SEQ ID NO: 1.

EFFECT: invention can be used to detect interactions between protein molecules as part of multicomponent protein complexes that can be used for medical and research purposes.

1 cl, 2 dwg, 2 ex

Description

The technical field to which the invention relates

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

The invention was created with the financial support of the Ministry of Science and Higher Education of the Russian Federation under the Agreement No. 075-15-2019-1661 of October 31, 2019.

State of the art

One of the key problems in molecular and cellular biology is related to the study of interactions between proteins. Among all the known methods for studying such interactions, the yeast two-hybrid system is the fastest and most economical method, on the basis of which the first proteomic studies were carried out with an attempt to establish all possible interactions between yeast proteins and Drosophila proteins.

The yeast two-hybrid system is based on the use of transcription factors, which are characterized by modular structure and consist of physically and functionally separable domains: a DNA-binding domain (BD - binding domain) and a transcription activating domain (AD - activation domain). The physical separation of the BD and AD domains leads to inactivation of the transcription factor: BD alone is unable to activate transcription, and AD alone is unable to bind to its specific sites. Domains BD and AD are fused with the studied proteins X (bait) and Y (prey), respectively. Factor reconstruction becomes possible in the case of interaction between X and Y, then transcription of genes dependent on the used transcription factor is activated (Bartel, Chien et al. 1993). To create a two-hybrid system, the transcription factor Saccharomyces cerevisiae GAL4 is most often used, divided into DNA-binding and activation domains.

The starting material for the analysis is two plasmids, into which the cDNA sequences of the two proteins to be studied for interaction are cloned in the desired reading frame. 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 - in a single frame with the AD-domain of GAL4 in another plasmid.

Yeast cells are cotransformed with such plasmids, protein constructs are expressed in cells and directed by a special nuclear localization signal from the cytoplasm to the cell nucleus. The BD domain connected to the second protein binds to the promoters of the reporter genes, into which the GAL4-binding DNA sequences are inserted, and if the first and second proteins interact, then as a result the AD domain is attracted to the regulatory part of the reporter gene and activates its expression.

In S. cerevisiae cells, the tested proteins are expressed in relatively small amounts from the minimal constitutive ADH1 promoter; the vector also contains an ADH1 termination signal sequence. The expressed protein is directed to the nucleus of S. cerevisiae cells by a nuclear localization signal that is part of the BD or is of heterologous origin (from the SV40 virus). The vectors are capable of autonomous replication in E. coli and S. cerevisiae cells. The vectors contain the β-lactamase gene, which makes E. coli ampicillin resistant. The vector with the activation domain GAL4 contains the LEU2 gene, with the DNA-binding domain, the TRP1 gene, and an additional non-fusion protein, the URA3 gene, which allow heterotrophic S. cerevisiae cells to grow under conditions limited by leucine, tryptophan, or uracil, respectively.

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 (US5283173A, US5468614A).

There are also vector modifications used in the yeast two-hybrid system, mainly aimed at using other domains (in contrast to the classical one using the GAL4 activator): for example, repression (US5885779A) or domains of proteins participating in signaling pathways associated with cell membranes (US20100075326A1) ... In addition, there are vector modifications based on the use of recombination and simplifying the cloning of cDNA into the corresponding plasmids used in the two-hybrid system (US7323313B2). In RU2529356C, the nucleotide sequences encoding the proteins under study are separated from the nucleotide sequences encoding the activation or DNA-binding domain of the GAL4 protein using the nucleotide sequence encoding the peptide, which is expressed as a protein bridge between the protein under investigation and the GAL4 protein domains.

To study the interactions of proteins in ternary complexes, in addition to two fusion proteins fused with the DNA-binding and activation domains of GAL4, an additional non-fusion protein is used. The non-fusion protein is expressed either from one of the vectors encoding the fusion protein or from a third additional vector. In the case of four- or more component protein systems, each additional non-fusion protein must be cloned with its own promoter and terminator into one of the three vectors used, which significantly complicates the cloning scheme (Stynen et al. 2012).

In a multicomponent yeast two-hybrid system, an additional non-fusion protein or proteins is used to connect two fusion proteins that cannot directly interact with each other, or to stabilize the weak interaction of two fusion proteins.

The essence of the invention

The classical two-hybrid yeast system allows testing double or triple protein interactions, which significantly reduces the applicability of the method, since many proteins in a living organism function only as part of multicomponent protein complexes. The existing modifications of the method for multicomponent systems (four or more proteins) imply the cloning of additional proteins in a separate reading frame with a separate promoter and terminator, which complicates the use of the method.

The present invention provides a new method for the simultaneous expression of several proteins or their domains in a single reading frame with a single promoter and terminator in a yeast two-hybrid system. The vectors used in the yeast two-hybrid system are modified so that the coding sequences of the studied proteins are in a single reading frame and are connected to each other by short self-cleaving 2A peptides. 2A peptides are short amino acid sequences that terminate the translation of a polypeptide without dissociation of the ribosome from mRNA, resulting in continued translation of the next polypeptide. The mechanism of their self-cleavage consists in stopping the ribosomes at the glycine residue, releasing the upstream protein bound to the 2A-peptide, and translating the downstream protein with proline as the first residue. Using 2A peptide sequences, simultaneous expression of several proteins can be achieved. 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 technical result of the invention is modified plasmids containing a nucleotide sequence encoding a self-cleaving peptide, which allows, during cotransformation into yeast cells, to simultaneously express several proteins and to detect interactions between proteins and their domains in multicomponent protein complexes.

The proposed invention can be used to detect the interaction between protein molecules in the composition of multicomponent protein complexes that can be used for medical and research purposes.

The essence of the described invention is illustrated by a drawing (figure 1).

Brief Description of Drawings

Figure 1. Schematic of a vector for expressing test proteins, separated by a sequence encoding a self-cleaving 2A-peptide, and subsequent screening in a yeast two-hybrid system, where

A - a vector in which proteins are cloned in a single reading frame with the GAL4 activation domain, separated by a sequence encoding a self-cleaving 2A-peptide;

B - amino acid sequence of the self-cleaving 2A-peptide ERBV-1.

Figure 2. Results of yeast two-hybrid analysis with classical and modified vectors

Implementation of the invention

Example 1. Creation of vector modifications intended for a yeast two-hybrid system.

The term "self-cleaving peptide" should be understood as short amino acid sequences that terminate the translation of the polypeptide without dissociation of the ribosome from the mRNA, as a result of which the translation of the next polypeptide continues.

The vector to be modified was pGAD424 (Clontech), intended for use in a yeast two-hybrid system and based on the GAL4 activation domain.

Optimized 2A peptide of horse rhinitis B virus (ERBV-1) was chosen as a self-cleaving peptide, for which the rate of self-cleavage efficiency in Saccharomyces cerevisiae cells is one of the highest among the known sequences (more than 90%) (Souza-Moreira et al. 2018 ). The amino acid sequence of the optimized ERBV-1 β2A peptide is shown in Figure 1B. (SEQ ID NO: 1).

The implementation of the invention implies a multi-stage experiment.

1. Obtaining expression vectors - two plasmids in which the cDNA sequences of the studied proteins are cloned in a single frame with a DNA-binding and activation domain, and a third additional plasmid with a non-fusion protein.

Cotransformation of yeast with three types of plasmids and inoculation of transformants on Petri dishes with a selective medium that does not contain selective amino acids (tryptophan, leucine and uracil). In the genes for the biosynthesis of these amino acids in the genome of the yeast strain used, mutations were artificially introduced that disrupt the formation of functionally active proteins. These genes without mutations are expressed from the transformed plasmids. As a result, selection takes place on the plates, if the cotransformation was successful, then individual colonies grow on the plate after 2-3 days.

S. cerevisiae yeast strain Pj69-4A is subcultured into a 15 ml tube into a liquid YPD medium (2% bactopeptone, 1% yeast extract, 2% glucose) using a sterile loop. Are built up in a thermostated shaker at 250-300 rpm and a temperature of 30 ° C to an optical density OD600 ~ 1. Then the culture is diluted 10 times with YPDA medium and grown for 3 hours under the same conditions. 1.5 ml of the resulting cell suspension is cotransformed with three plasmids. The cells are precipitated by centrifugation at 4000 g for 10 seconds, the supernatant is removed. 1 ml of 0.1 M lithium acetate is added to the sediment, then incubated for 10 minutes on a shaker at a temperature of 30 ° C, centrifuged at 4000 g, 10 seconds, the supernatant is removed. To the cell pellet, 270 μl of 50% PEG 3380 dissolved in 0.1 M lithium acetate, 25 μg of denatured salmon DNA and 50 μl of a mixture of three plasmids in 0.1 M lithium acetate, 0.4-0.8 μg each are added successively ... The mixture is resuspended and incubated in a shaker at 30 ° C for 30 minutes. Then incubated at 42 ° C for 2 minutes. The tubes are cooled on ice, centrifuged at 4000 g for 10 seconds, the supernatant is removed. 150 μL of deionized water (mQ) is added to the sediment, resuspended and plated on a plate with selective medium (0.67% yeast medium without amino acids, 1.5% agar, 2% glucose, 0.030% L-isoleucine, 0.015% L-valine , 0.002% L-arginine, 0.002% L-histidine monohydrate chloride, 0.003% L-lysine chloride, 0.002% L-methionine, 0.005% L-phenylalanine, 0.02% L-threonine, 0.003% L-tyrosine, 0.002% adenine sulfate). The dishes are placed in a thermostat at 30 ° C for three days, until the appearance of individual colonies.

2. Several colonies are subcultured with a depleting streak on Petri dishes with selective medium (0.67% yeast medium without amino acids, 1.5% agar, 2% glucose, 0.030% L-isoleucine, 0.015% L-valine, 0.002% L-arginine , 0.003% L-lysine chloride, 0.002% L-methionine, 0.005% L-phenylalanine, 0.02% L-threonine, 0.003% L-tyrosine, 0.002% adenine sulfate). The cups are placed in a thermostat at 30 ° C for several days.

3. Most often, the genes for the biosynthesis of histidine HIS3 and adenine ADE2, as well as the gene for β-galactosidase LacZ, are reporter. If the proteins interact, then the ADE2 and HIS3 genes are activated, and therefore the colonies grow. If the proteins do not interact, then the growth of colonies is not observed. In addition, the activation level of the LacZ gene can be detected in parallel.

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

To test the efficiency of the self-cleaving peptide 2A ERBV-1 in the yeast dihybrid system, the DUB module of the Drosophila melanogaster SAGA complex, consisting of three proteins Eny2, Sgf11, and Nonstop, was chosen.

On the basis of the modified vectors, plasmids were created to test the effectiveness of the developed approach. For this, cDNA sequences of the tested proteins were cloned into vectors for the yeast two-hybrid system. The interaction of Drosophila proteins Eny2, Sgf11, and Nonstop, which form the DUB module of the SAGA complex, was used as a positive control for the correct functioning of all plasmids and the efficiency of self-cleavage of the ERBV-1 2A peptide. The activation domain of the GAL4 protein, the Eny2 cDNA, the cDNA of the ERBV-1 self-cleaving peptide 2A, and the Sgf11 cDNA are in the same translation frame. The DNA-binding domain of the GAL4 protein is fused in a single translation frame with the cDNA of the UBP Nonstop domain. The parental plasmids were used as negative controls. The results of testing all variants of vectors are presented in Fig. 2, from which it can be seen that positive signals are detected only in the pair UBPNonstop - Eny2_2A_Sgf11 on a selective medium without histidine. The assembly of the protein complex requires three separate proteins, which is only possible when the 2A peptide is cleaved.

Then the Ataxin7 protein was tested, which supposedly can bind the proteins of the DUB-module to form a quaternary complex. The DNA-binding domain of the GAL4 protein is fused in a single translation frame with the Ataxin7 cDNA. The Nonstop protein is cloned in the third non-fusion vector, Y3H. From the results of yeast two-hybrid analysis, it follows that Ataxin7 does not interact with any of the Nonstop, Eny2, or Sgf11 proteins separately, and Ataxin7 does not bind to any of the possible pairs of proteins of the DUB module. And only with the simultaneous presence of all three proteins Nonstop, Eny2 and Sgf11, which is possible only when the 2A-peptide is cleaved, a quadruple complex with Ataxin7 is formed (Fig. 2).

List of cited literature

Stynen et al. (2012). "Diversity in Genetic In Vivo Methods for Protein-Protein Interaction Studies: from the Yeast Two-Hybrid System to the Mammalian Split-Luciferase System." Microbiol Mol Biol Rev. 76 (2): 331-382.

Bartel et al. (1993). "Elimination of false positives that arise in using the two-hybrid system." Biotechniques 14 (6): 920-4.

Souza-Moreira et al. (2018). "Screening of 2A peptides for polycistronic gene expression in yeast." FEMS Yeast Research 18 (5).

<110> Federal State Budgetary Institution of Science Institute of Gene Biology Russian Academy of Sciences

<120> Modification of the yeast two-hybrid system that allows testing of interactions between proteins within multicomponent protein complexes

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<213> ERBV-1

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<223> Horse rhinitis B virus 2A peptide (ERBV-1)

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Gly Ser Gly Gly Ala Thr Asn Phe Ser Leu Leu Lys Leu Ala Gly Asp 16

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Val Glu Leu Asn Pro Gly Pro 23

twenty

Claims (6)

Yeast cell designed to detect interactions between proteins and their domains in multicomponent protein complexes, cotransformed with three plasmids modified to express several proteins in yeast cells, where the nucleotide sequence encoding the DNA-binding domain of the GAL4 protein is fused with the nucleotide sequence encoding the first protein and cloned into one plasmid; where the nucleotide sequence encoding the activation domain of the GAL4 protein is fused with the nucleotide sequence encoding the second and third proteins and having a single reading frame with one promoter and terminator, and cloned into the second plasmid, with a nucleotide sequence encoding a self-cleaving a peptide that allows the simultaneous expression of several proteins or their domains in a single reading frame with a single promoter and terminator; and where the nucleotide sequence encoding the non-fusion protein is cloned into a third plasmid; the self-cleaving peptide is the horse rhinitis B virus 2A peptide having the amino acid sequence GlySerGlyGlyAlaThrAsnPheSerLeuLeuLysLeuAlaGlyAspValGluLeuAsnProGlyPro as set forth in SEQ ID NO: 1.

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