Design and Application of an Immobilized Protein Kinase

Protein Kinase A (PKA) is a biologically important enzyme for cell regulation, often referred to as the “central kinase”. An immobilized PKA that retains substrate specificity and activity would be a useful tool for laboratory scientists, enabling targeted phosphorylation without interference from downstream kinase contamination or kinase autophosphorylation in sensitive assays. Moreover, it might also offer the benefits of robustness and reusability that are often associated with immobilized enzyme preparations. In this work, we describe the creation of a recombinant PKA fusion protein that incorporates the HaloTag covalent immobilization system. We demonstrate that protein fusion design, including affinity tag placement, is critical for optimal heterologous expression in E. coli. Furthermore, we demonstrate various applications of our immobilized PKA, including the phosphorylation of recombinant PKA substrates such as VASP and endogenous PKA substrates in a cell lysate. These results hold promise as a generalizable strategy for the production and application of immobilized protein kinases involving specific sequestration of a fusion protein by a solid support material.