title: Purification, enzymatic activity and inhibitor discovery for recombinant human carbonic anhydrase XIV.
abstract: 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 CO2hydration 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.
authors: Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Saulėtekio 7, Vilnius LT-10221, Lithuania. Electronic address: vaida.jogaite@bti.vu.lt.
authors: Vaida
authors: V
authors: Juozapaitienė
authors: Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Saulėtekio 7, Vilnius LT-10221, Lithuania.
authors: Brigita
authors: B
authors: Bartkutė
authors: Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Saulėtekio 7, Vilnius LT-10221, Lithuania.
authors: Vilma
authors: V
authors: Michailovienė
authors: Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Saulėtekio 7, Vilnius LT-10221, Lithuania.
authors: Audrius
authors: A
authors: Zakšauskas
authors: Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Saulėtekio 7, Vilnius LT-10221, Lithuania.
authors: Lina
authors: L
authors: Baranauskienė
authors: Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Saulėtekio 7, Vilnius LT-10221, Lithuania.
authors: Sandra
authors: S
authors: Satkūnė
authors: Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Saulėtekio 7, Vilnius LT-10221, Lithuania.
authors: Daumantas
authors: D
authors: Matulis
mesh_terms: D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D002257:Carbonic Anhydrase Inhibitors; D008565:Membrane Proteins; D011994:Recombinant Proteins; D002256:Carbonic Anhydrases; C466621:carbonic anhydrase XIV
keywords: Carbonic anhydrase XIV; E. coli; Enzyme catalytic activity; Protein activity; Protein purification; Recombinant protein expression