Expression of the functional carbohydrate-binding module (CBM) of human laforin

• Author(s): Moreira, Susana ; Castanheira, Pedro ; Casal, Margarida ; Faro, Carlos ; Gama, F. M.
• Date: 2010
• Persistent ID: http://hdl.handle.net/1822/11476
• Origin: RepositóriUM - Universidade do Minho
• Subject(s): Laforin; Carbohydrate-binding module; RGD; Inclusion bodies
• Document Type: Article
• Language: English

Description

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.