title: Expression, purification and characterization of soluble red rooster laforin as a fusion protein in Escherichia coli.
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.
authors.forename: M Kathryn
authors.initials: MK
authors.lastname: Brewer
authors.forename: Satrio
authors.initials: S
authors.lastname: Husodo
authors.forename: Vikas V
authors.initials: VV
authors.lastname: Dukhande
authors.forename: Mary Beth
authors.initials: MB
authors.lastname: Johnson
authors.affiliation: Department of Molecular and Cellular Biochemistry and Center for Structural Biology, College of Medicine, University of Kentucky, 741 S, Limestone, Lexington, Kentucky 40536-0509, USA. matthew.gentry@uky.edu.
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Gentry
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002241:Carbohydrates / Q000737:chemistry; D002645:Chickens / Q000276:immunology*; D054637:Dual-Specificity Phosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D006003:Glycogen / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D020192:Lafora Disease / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D011485:Protein Binding; D055550:Protein Stability; D054558:Protein Tyrosine Phosphatases, Non-Receptor / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D012995:Solubility
chemical_list: D002241:Carbohydrates; D011993:Recombinant Fusion Proteins; D006003:Glycogen; D054637:Dual-Specificity Phosphatases; D054558:Protein Tyrosine Phosphatases, Non-Receptor; C114990:EPM2A protein, human