Expression in non-melanogenic systems and purification of soluble variants of human tyrosinase.
Biol Chem
; 394(5): 685-93, 2013 May.
Article
em En
| MEDLINE
| ID: mdl-23241670
ABSTRACT
Mammalian tyrosinases are key enzymes of melanin formation. Their native forms undergo complex maturation and sorting processes before being integrated into the melanosomal membrane, which greatly complicates their heterologous expression in other cell types. In the present work, we constructed several differently truncated, soluble variants of human tyrosinase and studied their properties after expression in HEK 293 cells. In addition, we prepared two affinity-tagged forms of the enzyme for expression in the yeast Kluyveromyces lactis and HEK cells, respectively. A Strep-tagged variant was secreted by K. lactis in excellent yields but found to be inactive, whereas a His-tagged variant secreted by HEK 293 cells in an active state could be purified from cell supernatants to near homogeneity. The resulting preparation consisted of an inactive, probably unglycosylated species of about 57 kDa and several glycosylated forms with masses between 63 and 75 kDa, as confirmed by activity staining, Western blotting and mass spectrometry.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Kluyveromyces
/
Monofenol Mono-Oxigenase
Limite:
Humans
Idioma:
En
Revista:
Biol Chem
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2013
Tipo de documento:
Article
País de afiliação:
Alemanha