Rational design of multifunctional hetero-hexameric proteins for hydrogel formation and controlled delivery of bioactive molecules.

A hetero-hexameric protein system is developed in this study, which not only functions as cross-linkers for hydrogel formation but also offers docking sites for controlled delivery of bioactive molecules. First, a hexameric protein with two, four, and six tax-interacting protein-1 (TIP-1), respectively (named as 2T, 4T, and 6T), is designed and obtained. As the hexapeptide ligand (WRESAI) can specifically bind to TIP-1 with high affinity, the hexameric proteins of 2T, 4T, and 6T can be used to crosslink the self-assembling nanofibers of Nap-GFFYGGGWRESAI, leading to formation of injectable biohybrid hydrogels with tunable mechanical properties. Furthermore, a hetero-hexameric protein containing four TIP-1 and two C-terminal moiety of the pneumococcal cell-wall amidase LytA (C-LytA) proteins is designed and engineered (named as 4T2C). The 4T2C proteins can not only serve as cross-linkers for hydrogel formation but also provide docking sites for loading and controlled release of model drug Rhoda-GGK'. This study opens up new opportunities for further development of multifunctional hetero- recombinant protein-based hydrogels for biological applications.