Team:SDU-Denmark/Tour31
Two-Hybrid Screening
The bacterial two-hybrid system is a technology used to detect protein-protein interactions. It is based on adenylate cyclase activity reconstitution in a cyaA 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 cyclic adenosine-monophosphat (cAMP) synthesis. This will trigger transcription of a reporter system that leads to a detectable phenotypical 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 1: T18 and T25 each contain one of the coding sequences for the two catalytic subunits of adenylate cyclase.
The bacterial two-hybrid system commonly exploits the catalytic activity of adenylate cyclase to generate cAMP. In the system the two domains of the adenylate 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]
Before we could use the bacterial two-hybrid system to screen for functioning peptide aptamers, we needed to verify the system, that it could indeed detect protein-protein interactions.
To this purpose we chose two proteins that were known to interact and linked them to T18 and T25. For this positive control we used two leucine zippers (LeuZ) which are known to form homodimers.
The reporter system which we initially intended to use was a cAMP/CAP-dependent transcription of Red Fluorescent Protein (RFP). On the target plasmid transcription of RFP was controlled by the cAMP-sensitive promoter PcstA. Bacteria with the combination T18-LeuZ and T25-LeuZ (and potentially functioning peptide aptamers) should turn red.
Figure 2:
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 -galactosidase. If the bacteria is grown on plates containing the lactose analog 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (commonly referred to as just ‘X-gal’, thankfully), -galactosidase can convert X-gal to a blue colored substrate (consult figure # 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. We would do so by ordering a randomly generated nucleotide library. In the library every molecule should contain a different sequence of 60 base pairs. The sequence ordered was 3’-(…)-NNK-NNK-(…)-5’, meaning every third base should be either a guanine or cytosine. This should lower the risk of generating a stop-codon in the library. With this restriction it would mean that we would be able to generate different nucleotide sequences. Due to the degeneracy of the genetic code, however, this does not equal the possible peptide sequences that could be generated. If we temporary ignore the possibility of generating a stop codon in our 60 bp sequence, this would mean that we could be able to generate different amino acid sequences. It is very likely that somewhere in our library a nucleotide sequence would give rise to a functioning peptide aptamer that is able to bind our target protein.
References:
1. Karimova G, Pidoux J, Ullmann A, Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proceedings of the National Academy of Sciences of the United States of America. 1998;95(10):5752-6.
2. Battesti A, Bouveret E. The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli. Methods (San Diego, Calif). 2012;58(4):325-34.