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Two-Hybrid Screening

Figure 1: Arrangement of adenylate cyclase toxin gene in Bordetella pertussi.

The bacterial two-hybrid system is a technology used to detect protein-protein interactions. It is based on the adenylate cyclase Adenylate CyclaseAdenylate cyclase is a transmembrane enzyme that will generate the potent second messenger cyclic adenosine monophosphate (cAMP). This signalling pathway is activated in a low-energy metabolic state. activity reconstitution in a ΔcyaA Eschericia coli strain. Reference: Karimova G, Pidoux J, Ullmann A, Ladant D. (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. 1998;95(10):5752-6. [PubMed]

Battesti A, Bouveret E. (2012) The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli. 2012;58(4):325-34.
DOI:10.1016/j.ymeth.2012.07.018 [ScienceDirect] In this system the two proteins are generally referred to as "Bait" and "Prey". Reference: Battesti A, Bouveret E. (2012) The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli. 2012;58(4):325-34.
DOI:10.1016/j.ymeth.2012.07.018 [ScienceDirect] If there is an interaction between the two, it will lead to cAMP synthesis. This will trigger transcription of a reporter system that leads to a detectable phenotypic change. Reference: Karimova G, Pidoux J, Ullmann A, Ladant D. (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. 1998;95(10):5752-6.
[PubMed]
We are using this technique to screen for functioning peptide aptamers that are able to bind our chosen target protein, thus functioning as an alternative to antibodies.

Figure 2: Interacting proteins can be detected using the bacterial two-hybrid system

Cenerally, The bacterial two-hybrid system exploits the catalytic activity of adenylate cyclase to generate cAMP. The system we use was first described 17 years ago by Kariomva et al.. Reference: Karimova G, Pidoux J, Ullmann A, Ladant D. (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. 1998;95(10):5752-6.
[PubMed] In the system the two domains of the adenylate cyclase toxin gene (cyaA) of Bordetella pertussis, called T18 and T25, are placed on two different plasmids. The domains are linked to the nucleotide sequence of the two proteins of interest, generating so-called hybrid genes. Reference: Karimova G, Pidoux J, Ullmann A, Ladant D. (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. 1998;95(10):5752-6.
[PubMed] If the "Prey" protein is able to interact with the "Bait" protein, the two catalytic domains will be brought into close proximity, enabling synthesis of cAMP from ATP. cAMP will bind to Catabolite Activating Protein (CAP). The complex can induce expression of a various set of reporter-genes controlled by a cAMP/CAP-dependent promoter. Reference: Karimova G, Pidoux J, Ullmann A, Ladant D. (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. 1998;95(10):5752-6. [PubMed]

Battesti A, Bouveret E. (2012) The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli. 2012;58(4):325-34.
DOI:10.1016/j.ymeth.2012.07.018 [ScienceDirect] If you are interested in a different type of application of 'the bacterial two-hybrid system' click here.

Figure 3: The RFP reporter system

The reporter system which we initially intended to use was a cAMP/CAP-dependent transcription of the gene encoding Red Fluorescent Protein (RFP). On the target plasmid transcription of RFP was controlled by the cAMP/CAP-sensitive promoter PcstA (BBa_K118011). In this reporter system protein-protein interactions would result in expression of red fluorescence.

Figure 4: The X-gal/lacZ reporter system

Due to difficulties using this promoter we changed to a different reporter system. We used the bacterial strain BHT101, which was adenylate cyclase deficient and contained a chromosomal LacZ-reporter system. In this reporter system cAMP will induce transcription of the LacZ gene, which encodes the enzyme β-galactosidase. If the bacteria is grown on plates containing X-gal X-galX-gal is the common name of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranosid, an analoge of the disaccharide lactose., β-galactosidase can hydrolyze X-gal to a blue colored substrate (consult Figure 4 for a schematic overview). Bacteria with this reporter system would become blue when the two proteins are able to interact with each other.

The system is suitable for screening of different libraries. The system has been used to determine interaction partners in genomic libraries. Reference: Battesti A, Bouveret E. (2012) The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli. 2012;58(4):325-34.
DOI:10.1016/j.ymeth.2012.07.018 [ScienceDirect] Our aim is, however, to generate a new interaction partner to a target protein; a peptide aptamer. This should be done through screening of a randomly generated nucleotide library against a chosen target protein.