Hyperthermophilic Protein Scaffolds for Engineering Molecular Recognition

Antibodies are the most commonly used binding molecules in biomolecular measurement. However, antibodies have several disadvantages such as low thermodynamic stability, a large multi-domain structure stabilized by disulfide bonds and high cost of production. As an alternative to antibodies, we are investigating the use of a set of small protein domains from extremophilic organisms as novel scaffolds for engineering molecular recognition, i.e. a “template” for generating protein variants that bind with high affinity and specificity to target species.

As part of this effort, we have identified and cloned multiple small protein domains from the hyperthermophilic organisms Thermotoga maritima, Sulfolobus solfataricus and Pyrococcus furiosus. The characteristic features of these domains are: (i) high thermal stability (ii) lack of cysteines; and (iii) low molecular weight (< ~10-12 kDa). Using a combination of yeast surface display and mRNA display, we are currently screening large libraries of these scaffold proteins to select variants that bind to model targets. Our goal is to create a versatile collection of validated hyperthermophilic protein scaffolds for molecular recognition in biomolecular measurement applications. Collectively, these protein scaffolds will provide a diverse set of binding interface topologies suited for various target species.