Expression, purification and characterization of soluble red rooster laforin as a fusion protein in Escherichia coli

BMC Biochem. 2014 Apr 2;15:8. doi: 10.1186/1471-2091-15-8.

Abstract

Background: The gene that encodes laforin, a dual-specificity phosphatase with a carbohydrate-binding module, is mutated in Lafora disease (LD). LD is an autosomal recessive, fatal progressive myoclonus epilepsy characterized by the intracellular buildup of insoluble, hyperphosphorylated glycogen-like particles, called Lafora bodies. Laforin dephosphorylates glycogen and other glucans in vitro, but the structural basis of its activity remains unknown. Recombinant human laforin when expressed in and purified from E. coli is largely insoluble and prone to aggregation and precipitation. Identification of a laforin ortholog that is more soluble and stable in vitro would circumvent this issue.

Results: In this study, we cloned multiple laforin orthologs, established a purification scheme for each, and tested their solubility and stability. Gallus gallus (Gg) laforin is more stable in vitro than human laforin, Gg-laforin is largely monomeric, and it possesses carbohydrate binding and phosphatase activity similar to human laforin.

Conclusions: Gg-laforin is more soluble and stable than human laforin in vitro, and possesses similar activity as a glucan phosphatase. Therefore, it can be used to model human laforin in structure-function studies. We have established a protocol for purifying recombinant Gg-laforin in sufficient quantity for crystallographic and other biophysical analyses, in order to better understand the function of laforin and define the molecular mechanisms of Lafora disease.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Carbohydrates / chemistry
  • Chickens / immunology*
  • Dual-Specificity Phosphatases / genetics
  • Dual-Specificity Phosphatases / isolation & purification
  • Dual-Specificity Phosphatases / metabolism
  • Escherichia coli / genetics*
  • Glycogen / metabolism
  • Humans
  • Inclusion Bodies / metabolism
  • Lafora Disease / genetics*
  • Male
  • Molecular Sequence Data
  • Mutation / genetics
  • Phosphorylation
  • Protein Binding
  • Protein Stability
  • Protein Tyrosine Phosphatases, Non-Receptor / genetics
  • Protein Tyrosine Phosphatases, Non-Receptor / isolation & purification
  • Protein Tyrosine Phosphatases, Non-Receptor / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism*
  • Sequence Alignment
  • Solubility

Substances

  • Carbohydrates
  • Recombinant Fusion Proteins
  • Glycogen
  • Dual-Specificity Phosphatases
  • Protein Tyrosine Phosphatases, Non-Receptor
  • EPM2A protein, human