Challenges and Limitations

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

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

Secondly there is a limitation to the size of the target protein. There doesn’t exist a defined size restriction, but theoretically speaking 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 there wouldn’t be generated cAMP and expression of the reporter would not be activated.

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 the 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 a bacterial protein, which E.coli has a copy of, it should be deleted before the screen is applied. This is to rule out the possibility that the T18-linked peptide aptamer should interact with the chromosomal gene-product of the target protein and fail 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 (1), meaning the system is not limited to bacterial protein. The target protein should, on the other hand, be non-toxic in great amounts. Since they are transformed into the bacterium on a high-copy plasmid, it’s essential that it does not elicit a toxic effect in the organism.

One of the advances of therapeutic use of peptide aptamers is that they are very small molecules. Because of its size peptide aptamers are less likely to be immunogenic (to elicit a host immune response) compared to a murine antibody. We hope to further reduce the chances of being perceived as ‘foreign’ since we have chosen human thioredoxin (hThx) as the scaffold protein of our peptide aptamers. But peptide aptamers will not be able to activate the rest of the immune system to help it exterminate the intruder like antibodies to. 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).