Expression of the functional carbohydrate-binding module (CBM) of human laforin.
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.
Moreira
Susana
S
IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Braga, Portugal.
Castanheira
Pedro
P
Casal
Margarida
M
Faro
Carlos
C
Gama
Miguel
M
D050260:Carbohydrate Metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D054558:Protein Tyrosine Phosphatases, Non-Receptor / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D054558:Protein Tyrosine Phosphatases, Non-Receptor; C114990:EPM2A protein, human