Juozapaitienė Vaida, Bartkutė Brigita, Michailovienė Vilma, Zakšauskas Audrius, Baranauskienė Lina, Satkūnė Sandra, Matulis Daumantas
Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Saulėtekio 7, Vilnius LT-10221, Lithuania.
Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Saulėtekio 7, Vilnius LT-10221, Lithuania.
J Biotechnol. 2016 Dec 20;240:31-42. doi: 10.1016/j.jbiotec.2016.10.018. Epub 2016 Oct 20.
Human carbonic anhydrase XIV (CA XIV), a transmembrane protein, highly expressed in the central nervous system, is difficult to recombinantly express and purify in large scale for the measurements of inhibitor binding and drug design. CA XIV belongs to the family of twelve catalytically active CA isoforms in the human body. Disorders in the expression of CA XIV cause serious diseases and CA XIV has been described as a possible drug target for the treatment of epilepsy, some retinopathies, and skin tumors. In this study, the effect of different promoters, E. coli strains, and the length of recombinant CA XIV protein construct were analyzed for the production CA XIV in large scale by using affinity purification. Active site titration by inhibitors and the isothermal titration calorimery revealed over 96% purity of the protein. Enzymatic activity of the purified CA XIV was determined by following the CO hydration using the stopped-flow technique. Several inhibitors were discovered that exhibited selectivity towards CA XIV over other CA isoforms and could be developed as drugs.
