Hyperthermophlic Protein Scaffolds for Generating Affinity Reagents

The ability to create affinity reagents that specifically bind to (or recognize) a molecular target is critical for applied biotechnology as well as fundamental research in biology. For instance, the specific recognition of targets present in complex mixtures is a key step in the detection and/or quantification of molecular species. Examples include detection of disease biomarkers in blood or trace explosives in water, and measurement of protein concentrations in cell lysates for quantitative systems biology. Antibodies are the most commonly used affinity reagents. Antibodies, however, are expensive to produce and typically have low stability. Also, the generation of antibodies for new targets through the frequently used procedure of immunizing animals, is a lengthy and tedious process. As an alternative to antibodies, we are investigating the use of small protein domains from extremophilic organisms as scaffolds or “templates” for generating affinity reagents.

To this end, we have identified and cloned several small protein scaffolds from the hyperthermophilic organisms Thermotoga maritima, Sulfolobus solfataricus and Pyrococcus furiosus. These protein scaffolds are free from the limitations associated with antibodies because of their small size (~10-12 kDa), high thermal stability, and easy production in bacterial expression systems due to the lack of cysteines. We have successfully screened libraries of several protein scaffolds against multiple targets using a combination of yeast surface display and mRNA display. Taken together, our results show that these hyperthermophilic scaffolds can be used to generate affinity reagents for a wide range of targets.