Escherichia coli expression, refolding and characterization of human laforin.
Laforin is a unique human dual-specificity phosphatase as it contains an amino terminal carbohydrate binding module (CBM). Laforin gene mutations lead to Lafora disease, a progressive myoclonus epilepsy with an early fatal issue. Previous attempts to produce recombinant laforin faced various difficulties, namely the appearance of protein inclusion bodies, the contamination with bacterial proteins and a high tendency of the protein to aggregate, despite the use of fusion tags to improve solubility and ease the purification process. In this work, we have expressed human laforin in Escherichia coli in the form of inclusion bodies devoid of any fusion tags. After a rapid dilution refolding step, the protein was purified by two chromatographic steps, yielding 5-7mg of purified protein per liter of bacterial culture. The purified protein was shown to have the kinetic characteristics of a dual-specificity phosphatase, and a functional carbohydrate binding module. With this protocol, we were able for the first time, to produce and purify laforin without fusion tags in the amounts traditionally needed for the crystallographic structural studies paving the way to the understanding of the molecular mechanisms of laforin activity and to the development of novel therapies for Lafora disease.
Biocant, Molecular Biotechnology Unit, Parque Tecnológico de Cantanhede, Núcleo 4, Lote 3, Cantanhede, Portugal. pedro.castanheira@biocant.pt
Pedro
P
Castanheira
Susana
S
Moreira
Miguel
M
Gama
Carlos
C
Faro
D054637:Dual-Specificity Phosphatases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism*; D020192:Lafora Disease / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D011506:Proteins / Q000235:genetics / Q000378:metabolism*
D011506:Proteins; D054637:Dual-Specificity Phosphatases