Recombinant Production of 12 Catalytically Active Human CA Isoforms

Target-based drug design is based on the search of chemical compounds that would strongly and specifically bind to the target protein. The target protein is chosen based on biochemical and cellular investigation of a disease and a demonstration that chemical inhibitor/activator could be of help for the patient. Biochemical and biophysical interaction experiments require relatively large amount of the target protein. In this chapter, we overview recombinant production of the 12 catalytically active human carbonic anhydrase (CA) isoforms, beginning with the cloning of the CA gene sequences, construction of the catalytic domain encoding sequences, search for the best expression conditions in bacterial and mammalian cell cultures, and finally—the purification of the enzymes through Ni2+-chelation chromatography or ligand-affinity chromatography using an immobilized benzenesulfonamide headgroup. The purity of every CA isoform has been confirmed by SDS-PAGE, HR-MS, and the specific activity determined by the stopped-flow inhibition of Open image in new window hydration assay. Tens to hundreds of milligrams of highly purified CAs have been produced and subsequently used to crystallize the proteins and determine their interaction with synthetic compounds, and the inhibition of enzymatic activity as described in the following chapters.