Implications of intracrystalline OC17 on the protection of lattice incorporated proteins.

Biogenic CaCO 3 formation is regulated by crystallization proteins during crystal growth. Interactions of proteins with nascent mineral surfaces trigger proteins to be incorporated into the crystal lattice. As a result of incorporation, these intracrystalline proteins are protected in the lattice, an example of which is ancient eggshell proteins that have persisted in CaCO 3 for thousands of years even under harsh environmental conditions. OC17 is an eggshell protein known to interact with CaCO 3 during eggshell formation during which OC17 becomes incorporated into the lattice. Understanding protein incorporation into CaCO 3 could offer insights into protein stability inside crystals. Here, we study the protection of OC17 in the CaCO 3 lattice. Using thermogravimetric analysis we show that the effect of temperature on intracrystalline proteins of eggshells is negligible below 250 °C. Next, we show that lattice incorporation protects the OC17 structure despite a heat-treatment step that is shown to denature the protein. Because incorporated proteins need to be released from crystals, we verify metal chelation as a safe crystal dissolution method to avoid protein denaturation during reconstitution. Finally, we optimize the recombinant expression of OC17 which could allow engineering OC17 for engineered intracrystalline entrapment studies.