title: A new procedure for the cloning, expression and purification of the β-carbonic anhydrase from the pathogenic yeast Malassezia globosa, an anti-dandruff drug target.
abstract: Malassezia yeasts are almost exclusively the single eukaryotic members of the fungal flora of the skin. Malassezia globosa and Malassezia restricta are found on the skin of practically all humans. Malassezia globosa is highly implicated in the pathogenesis of dandruff and its genome encodes for only one carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the β-class (MgCA). It has been indeed demonstrated that in many pathogenic microorganisms, CAs are essential for their life cycle and their inhibition can lead to growth impairment and defects. In the previous work, the recombinant MgCA was investigated for its inhibition profile with sulfonamides, which in models of dandruff infection were able to protect animals from the fungal infection, allowing us to propose this enzyme as a new antidandruff target. MgCA was cloned as GST-fusion protein, but the yield was rather low and the protein was often found in inclusion bodies. Here, we propose an alternative procedure consisting in cloning the recombinant MgCA as His-Tag fusion protein. This procedure resulted in a good method to express and purify the active recombinant MgCA, and the protein recovery was better with respect to that used for preparing MG-CA (β-CA cloned as GST-fusion protein).
authors.affiliation: a Institute of Biosciences and Bioresources, CNR , Naples , Italy .|b Polo Scientifico, Laboratorio Di Chimica Bioinorganica, Dipartimento Di Chimica, Università degliStudi Di Firenze , Florence , Italy .
authors.forename: Sonia
authors.initials: S
authors.lastname: Del Prete
authors.affiliation: a Institute of Biosciences and Bioresources, CNR , Naples , Italy .
authors.forename: Viviana
authors.initials: V
authors.lastname: De Luca
authors.affiliation: b Polo Scientifico, Laboratorio Di Chimica Bioinorganica, Dipartimento Di Chimica, Università degliStudi Di Firenze , Florence , Italy .
authors.forename: Daniela
authors.initials: D
authors.lastname: Vullo
authors.affiliation: c Department of Chemistry , College of Science, King Saud University , Riyadh , Saudi Arabia , and.
authors.forename: Sameh M
authors.initials: SM
authors.lastname: Osman
authors.affiliation: c Department of Chemistry , College of Science, King Saud University , Riyadh , Saudi Arabia , and.
authors.forename: Zeid
authors.initials: Z
authors.lastname: AlOthman
authors.affiliation: a Institute of Biosciences and Bioresources, CNR , Naples , Italy .
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Carginale
authors.affiliation: b Polo Scientifico, Laboratorio Di Chimica Bioinorganica, Dipartimento Di Chimica, Università degliStudi Di Firenze , Florence , Italy .|c Department of Chemistry , College of Science, King Saud University , Riyadh , Saudi Arabia , and.|d Polo Scientifico, Dipartimento Neurofarba, Sezione Di ScienzeFarmaceutiche, Università Degli Studi Di Firenze , Florence , Italy.
authors.forename: Claudiu T
authors.initials: CT
authors.lastname: Supuran
authors.affiliation: a Institute of Biosciences and Bioresources, CNR , Naples , Italy .
authors.forename: Clemente
authors.initials: C
authors.lastname: Capasso
mesh_terms: D002256:Carbonic Anhydrases / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D063807:Dandruff / Q000382:microbiology*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008290:Malassezia / Q000201:enzymology*
chemical_list: D002256:Carbonic Anhydrases
keywords: Carbonic anhydrases; Malassezia; enzyme kinetics; fusion protein; protonography