Cell-free expression of protein kinase a for rapid activity assays.
Functional protein analysis often calls for lengthy, laborious in vivo protein expression and purification, and can be complicated by the lack of stability of the purified protein. In this study, we demonstrate the feasibility of a simplified procedure for functional protein analysis on magnetic particles using cell-free protein synthesis of the catalytic subunit of human cAMP-dependent protein kinase as a HaloTag((R)) fusion protein. The cell-free protein synthesis systems provide quick access to the protein of interest, while the HaloTag technology provides efficient, covalent protein immobilization of the fusion protein, eliminating the need for further protein purification and minimizing storage-related stability issues. The immobilized cPKA fusion protein is assayed directly on magnetic beads and can be used in inhibitor analyses. The combination of rapid protein synthesis and capture technologies can greatly facilitate the process of protein expression and activity screening, and therefore, can become a valuable tool for functional proteomics studies.
Leippe
Donna M
DM
Research and Development, Promega Corporation, 2800 Woods Hollow Road, Madison, WI, 53711, USA. Email: donna.leippe@promega.com.
Zhao
Kate Qin
KQ
Hsiao
Kevin
K
Slater
Michael R
MR