Challenges and Limitations

Limitations of the bacterial two-hybrid system

Using the bacterial two hybrid system as a screening method for peptide aptamers has 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 cellular 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 cellular 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 RFP-reporter system. Even though a functioning peptide aptamer is generated, the large target protein forhindre interaction between T18 and T25, simple due to relative space between the two. Thus there wouldn’t be generated cAMP and expression of RFP 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.

Therapeutic use

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).