(570e) Engineered Ca2+-Binding Protein for Chemoenzymatic Tagging and Bioorthogonal Surface Conjugation

Calcium (Ca²⁺)-binding proteins are involved in many biological processes, including vesicle release, cell proliferation, and apoptosis. One of the most widely studied Ca²⁺-binding proteins, calmodulin (CaM), is a ubiquitous Ca²⁺ sensor found in all eukaryotes that binds and activates more than 100 other proteins. In addition to playing a central role in intracellular signal transduction, CaM is used in affinity chromatography to purify both natural binding partners and proteins that have been engineered to carry a CaM-binding peptide. Previous studies of CaM biochemical function, which have focused on the binding of Ca²⁺ to CaM and subsequent binding of CaM to downstream proteins, have generally employed established protein labeling strategies, often leading to a decrease in CaM activity. Here we report the engineering of CaM to display a recognition sequence for N-myristoyl transferase (NMT) that enables the covalent attachment of an azide fatty acid to the engineered CaM protein N-terminus. This chemoenzymatic tagging approach proceeds co-translationally in E. coli with greater than 95% of the expressed CaM labeled with the tag.  We show that neither the NMT recognition sequence nor the presence of the azide tag decreases CaM activity. The presence of the azide group allows CaM to be conjugated to surfaces directly from complex cell lysate mixtures with the bioorthogonal azide-alkyne cycloaddition reaction (also known as click chemistry). The conjugation of azide-tagged CaM to cyclooctyne-functionalized resins provides a streamlined process for making CaM-affinity chromatography resins for rapid protein purification. Additionally, we show that azide-tagged CaM maintains activity when conjugated to the resin, and that azide-CaM resin outperforms traditional CaM resins for purification of a CaM-binding protein in a one-step procedure.

We expect our results to be translatable to the conjugation of other azide-tagged proteins to other platforms, such as protein microarrays and microtiter plates. Future work also includes optimization of the amount of azide-tagged CaM conjugated to the resin and purification of other CaM-binding proteins from multiple sources. Together, our methods and results have the potential to dramatically streamline the affinity resin manufacturing process.