Purification, enzymatic activity and inhibitor discovery for recombinant human carbonic anhydrase XIV

•Recombinant preparation of CA XIV, a difficult to produce protein, is described.•Large amount of CA XIV was affinity purified using sulfonamide-modified sepharose.•Inhibitor titration of activity demonstrated nearly 100% pure and active enzyme.•Nanomolar inhibitors of CA XIV, selective over CA I an...

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Bibliographic Details
Published in:	Journal of biotechnology Vol. 240; pp. 31 - 42
Main Authors:	Juozapaitienė, Vaida, Bartkutė, Brigita, Michailovienė, Vilma, Zakšauskas, Audrius, Baranauskienė, Lina, Satkūnė, Sandra, Matulis, Daumantas
Format:	Journal Article
Language:	English
Published:	Netherlands Elsevier B.V 20-12-2016
Subjects:	Carbonic anhydrase Carbonic Anhydrase Inhibitors - pharmacology Carbonic anhydrase XIV Carbonic Anhydrases - biosynthesis Carbonic Anhydrases - isolation & purification Carbonic Anhydrases - metabolism Crystallography, X-Ray Drugs E. coli Electrophoresis, Polyacrylamide Gel Enzyme Activation - drug effects Enzyme catalytic activity Escherichia coli Escherichia coli - genetics Humans Inhibitors Medical services Membrane Proteins - biosynthesis Membrane Proteins - isolation & purification Protein activity Protein purification Proteins Purification Recombinant Recombinant protein expression Recombinant Proteins - biosynthesis Recombinant Proteins - isolation & purification Recombinant Proteins - metabolism Titration Carbonic anhydrase XIV Protein activity Protein purification E. coli Enzyme catalytic activity Recombinant protein expression
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Summary:	•Recombinant preparation of CA XIV, a difficult to produce protein, is described.•Large amount of CA XIV was affinity purified using sulfonamide-modified sepharose.•Inhibitor titration of activity demonstrated nearly 100% pure and active enzyme.•Nanomolar inhibitors of CA XIV, selective over CA I and II, were designed. 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 CO2 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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0168-1656 1873-4863
DOI:	10.1016/j.jbiotec.2016.10.018