Design and Application of an Immobilized Protein Kinase

Abstract

Protein Kinase A (PKA) is a biologically important enzyme for cell regulation, often referred to as the “central kinase”. An immobilized PKA that retains native substrate specificity and activity would be a useful tool for laboratory scientists, as it would allow for targeted phosphorylation of various substrates while harnessing the benefits afforded by enzyme immobilization: namely ease of enzyme capture, repeated reuse, and increased enzyme stability in various temperature and pH conditions. In this research, we utilized a recombinant PKA fusion protein that incorporates the HaloTag covalent immobilization system to moderate enzyme immobilization. In addition, we investigated the importance of protein fusion order for optimal heterologous expression in E. coli. Furthermore, various applications of our immobilized PKA were demonstrated, including the phosphorylation of endogenous PKA substrates in a cell lysate. Overall, these results hold promise for a generalizable strategy for the production immobilized protein kinases and wide variety of applications that will be demonstrated through examinations of the reusability of the immobilized PKA under variable experimental conditions.