Yeast two hybrid system

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When two-hybrid screening ( English Yeast Two-Hybrid System , abbreviated Y2H ) is a biochemical technique for the investigation of protein-protein interactions .

principle

Scheme of the yeast two-hybrid system. The Bait - fusion protein binds to the GAL4 binding site in front of a reporter gene in the genome of a yeast cell. A Prey fusion protein must now be able to bind to this bait fusion protein. Only when such an interaction takes place (see below) does a functional reconstitution of the GAL4 transcription factor result , which results in the expression of the reporter gene. The interaction can be detected via this.

The yeast two-hybrid system is an in vitro method for the detection of protein-protein interactions in yeast , usually the baker's yeast Saccharomyces cerevisiae . The basis for this is a protein required for gene regulation , a so-called transcription factor . In Saccharomyces cerevisiae , the transcription factor GAL4 is normally used . This has two different domains , one for binding to the DNA (binding domain, GAL4-BD) and one that activates transcription (activation domain, GAL4-AD). Although the two domains are usually on the same polypeptide chain , they are effective even when they are brought together by two different proteins through non-covalent protein-protein interactions. To do this, use is made of two expression vectors that are compatible in yeast .

Each of the two plasmids carries a fusion gene constructed for the corresponding experiment. In the first case, this codes for a hybrid protein which consists of the GAL4-BD and which is followed by the amino acid sequence for which a potential binding partner is to be found ( bait protein, bait protein). The second plasmid encodes a hybrid protein that is composed of the GAL4-AD and then a possible binding partner for the bait protein ( Prey protein, prey protein). Both yeast two-hybrid plasmids replicate autonomously both in yeast and in E. coli ( shuttle vectors ).

A yeast strain that does not have a functioning GAL4 gene and carries one or more reporter genes is transformed with both plasmids. Genes , which are preceded by one or more binding sites for the GAL4 transcription factor and which code for proteins that can either produce certain amino acids or bases (e.g. histidine , uracil or adenine ) or enable optical recognition, act as reporters of this interaction (e.g. a color change catalyzed by the lacZ gene ). If there is an interaction between bait and prey , this usually results in a functional reconstitution of the GAL4 transcription factor, which results in the expression of the reporter genes. The latter can be detected by growth on appropriate selection media, e.g. B. on a medium lacking histidine, only yeasts in which bait and prey interact and thus express the enzyme for histidine synthesis grow as a result .

In a screening process, possible interaction partners can be identified in a more empirical approach with a cDNA bank as “Prey”, or the interaction for certain proteins can be specifically checked with this system in the so-called “single mating”. The DNA sequences can come from a DNA purification or a synthesis . There are variants of Y2H in E. coli and mammalian cells.

Advantages and disadvantages

The yeast two-hybrid system enables the investigation of protein-protein interactions in at least in vivo -like conditions, i. H. in the milieu of a cell and with post-translational modifications occurring in eukaryotes such as glycosylation (attachment of sugar chains), palmitoylation (attachment of fatty acids ) or folding by chaperones . Since yeast is also relatively cheap and robust to handle, many interaction partners, e.g. B. in screening approaches are checked.

The problem with the classic yeast two-hybrid system, however, is that the interaction of the proteins to be examined must take place in the yeast cell nucleus , since transcription can only take place there. However, it is possible that proteins fold differently in this milieu than in the area of ​​the cell in which they usually occur. The modifications of the yeast are also partly different from those of other eukaryotic organisms. This changed folding influences the structure and surface properties of the proteins, which can lead to incorrect test results:

  • Proteins that would normally not interact with each other do so due to the changed surface ( false positive )
  • Proteins that actually interact with each other cannot do this due to the changed folding in the cell nucleus ( false negative )

Furthermore, it is possible that two proteins interact in the Y2H experiment, but do not occur simultaneously in the cell cycle , in the cell organelle or in the cell type and therefore cannot be actual interaction partners.

For these reasons, the interpretation of Y2H results must be done with great care. Positive interactions always have to be verified with additional techniques from molecular biology, such as immunoprecipitation or FRET . The Y2H experiment still provides information about the possible interaction of two proteins, but no information about how this interaction takes place. For this, more detailed studies of the structure of the proteins involved are necessary.

The classic yeast two-hybrid system described here has other limitations. Certain proteins, such as membrane proteins, cannot be transported into the cell nucleus. Even protein complexes in which more than two proteins are involved cannot be investigated directly with classic Y2H experiments. In order to counter these problems, modified yeast two-hybrid systems have been developed in which only further steps enable the reconstitution of the transcription factor. There are also systems that are based on other principles, such as B. the split-ubiquitin system , which is particularly interesting for interaction studies of membrane proteins.

Individual evidence

  1. ↑ S. Fields, O. Song: A novel genetic system to detect protein-protein interactions . (abstract) In: Nature . 340, No. 6230, 1989, pp. 245-6. bibcode : 1989Natur.340..245F . doi : 10.1038 / 340245a0 . PMID 2547163 .
  2. ↑ a b c Hurt J, Thibodeau S, Hirsh A, Pabo C, Joung J: Highly specific zinc finger proteins obtained by directed domain shuffling and cell-based selection . In: Proc. Natl. Acad. Sci. USA . 100, No. 21, 2003, pp. 12271-6. bibcode : 2003PNAS..10012271H . doi : 10.1073 / pnas.2135381100 . PMID 14527993 . PMC 218748 (free full text).
  3. ^ V. Ratushny, E. Golemis: Resolving the network of cell signaling pathways using the evolving yeast two-hybrid system. In: BioTechniques. Volume 44, number 5, April 2008, ISSN  0736-6205 , pp. 655-662, doi : 10.2144 / 000112797 , PMID 18474041 , PMC 2526548 (free full text).
  4. ↑ B. Stynen, H. Tournu, J. Tavernier, P. Van Dijck: Diversity in genetic in vivo methods for protein-protein interaction studies: from the yeast two-hybrid system to the mammalian split-luciferase system. In: Microbiology and molecular biology reviews: MMBR. Volume 76, Number 2, June 2012, ISSN  1098-5557 , pp. 331-382, doi : 10.1128 / MMBR.05021-11 , PMID 22688816 , PMC 3372256 (free full text).