High-Throughput Purification of Protein Kinases from Escherichia coli and Insect Cells.

Abstract

Protein kinases are major targets for the development of new medicines and play key roles in cellular signaling. The flexible nature of these proteins, posttranslational modifications, and thelarge size of some protein kinases pose a particular challenge obtaining homogeneous, active recombinant protein kinases suitable for functional or structural studies. Here we describe our expertise expressing protein kinases in two frequently used host systems: E. coli and insect cells using the baculovirus expression vector system. In particular, we will discuss and provide detailed methods on construct design, high-throughput cloning, parallel expression testing and scale up as well as purification and co-expression strategies leading to stable and homogeneous recombinant protein samples.