"I like the challenge of trying different things and wondering whether it's going to work or whether I'm going to fall flat on my face." - Johnny Depp

Challenges and Limitations

Using the bacterial two hybrid system as a screening method for peptide aptamers proposes several limitations.

Firstly E.coli has to be able to produce the target protein and the protein must have the same conformation as it would in its native species and environment. Preferably the target protein would have to be a cytosolic protein. Receptor proteins and other surface-bound proteins most likely do not have the same conformation in an intracellular environment as they do when exposed on the cell-surface. Since this screening method is functions intracellularly, the target proteins are limited to intracellular proteins.

Secondly there is a limit to the size of the target protein. There is no defined size restriction, but theoretically speaking a peptide aptamer screening for a large target protein could fail to activate the reporter system. Even though a functioning peptide aptamer is generated, the large target protein could prevent interaction between T18 and T25, simple due to relative space between the two. Thus no cAMP would be generated and therby expression of the reporter remains inactive.

The biggest limitation of the bacterial two hybrid system is that it can only screen for peptide aptamers against proteins. The targets have to be encoded in a nucleotide sequence. But many other molecules besides proteins are immunogenic antigens, such as lipopolysaccharides or sialic acids on the surface of bacteria. In principle a peptide aptamer could be generated to target these types of molecules, but not by using the bacterial two-hybrid system.

Furthermore we cannot eliminate the possibility that it might affect our results (or future results) whether the peptide aptamer and target protein is linked to the N-terminal or C-terminal of T18 and T25. To exclude this possibility parallel experiments should be conducted with the different combinations. Ideally the bacterial strain, in which the screening is conducted, should lack the chromosomal gene copy of the target protein. If the target protein is highly produced in E.coli, the gene encoding it should be deleted before the screen is applied. This is to rule out the possibility of a T18-linked peptide aptamer interacting with the chromosomal gene-product of the target protein and thusfail to activate the reporter system.

However, the bacterial two hybrid system is not limited to bacterial target proteins. The system has been shown to detect interactions between eukaryotic proteins as well 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] . The target protein should, on the other hand, be non-toxic to E.coli in great amounts. Since it is transformed into the bacterium encoded in a high-copy plasmid.

One of the advances of peptide aptamers in a potential therapeutic use is that they are very small molecules. Because of their size peptide aptamers are less likely to be immunogenic compared to a murine antibody. We hope to further reduce the chances of our peptide aptamer being perceived as ‘foreign molecule’ since we have chosen human thioredoxin (hThx) as the scaffold protein of our peptide aptamers. But a peptide aptamer bound to its specific target will not be able to activate the rest of the immunesystem to help it exterminate an intruder. To heighten the therapeutic use of peptide aptamers one should experiment with conjugating the effector region of human antibodies to the peptide aptamers (please visit our page “Medical Aspects” for more on the subject).