Expression of the functional carbohydrate-binding module (CBM) of human laforin.
Protein Expr Purif
; 74(2): 169-74, 2010 Dec.
Article
en En
| MEDLINE
| ID: mdl-20600946
ABSTRACT
Laforin is a human protein associated with the glycogen metabolism, composed of two structurally and functionally independent domains a phosphatase catalytic domain and a substrate-binding module with glycogen and starch affinity. The main goal of this work is the development of a methodology for the expression of the so far poorly characterized carbohydrate-binding module (CBM) of laforin, allowing its study and development of biomedical applications. The laforin's CBM sequence was originally cloned by PCR from a human muscle cDNA library. The recombinant protein, containing laforin's CBM fused to an Arg-Gly-Asp sequence (RGD), was cloned and expressed using vector pET29a and recovered as inclusion bodies (IBs). Refolding of the IBs allowed the purification of soluble, dimeric and functional protein, according to adsorption assays using starch and glycogen. Several other experimental approaches, using both bacteria and yeast, were unsuccessfully tested, pointing towards the difficulties in producing the heterologous protein. Indeed, this is the first work reporting the production of the functional CBM from human laforin.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Ingeniería de Proteínas
/
Proteínas Tirosina Fosfatasas no Receptoras
Límite:
Humans
Idioma:
En
Revista:
Protein Expr Purif
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2010
Tipo del documento:
Article
País de afiliación:
Portugal