High affinity protein-protein interactions have been successfully used in protein/enzyme docking onto scaffolds for in vitro biotransformation. The most widely used protein-protein docking pair is the cohesin-dockerin pair. However, cohesin and dockerin proteins comprise of ~140 and ~70 amino acids, respectively, limiting their use in some applications. Here, we engineered a protein-ligand pair with similar high affinity but much smaller size. Using the PDZ domain from Tax-interacting Protein-1 (Tip1, 102 amino acids) and its ligand (Tip1lig, 9 amino acids), we introduced disulfide bond interactions using molecular modeling. Four pairs of disulfide bonds were tested, and one pair showed dramatically increased affinity under non-reducing conditions. Protein hydrogel cross-linked with the mutant pair of Tip 1 and Tip1lig exhibited very high stability in aqueous solution. We demonstrate the use of this hydrogel in enzyme immobilization for biofuel cell applications. This pair of protein-ligand interaction should be useful for diverse scaffold docking applications.

Rational design of a redox-sensitive protein-ligand pair

245th National Meeting (2013)

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