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Open Access Research article

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

M Kathryn Brewer, Satrio Husodo, Vikas V Dukhande, Mary Beth Johnson and Matthew S Gentry*

Author Affiliations

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

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BMC Biochemistry 2014, 15:8  doi:10.1186/1471-2091-15-8

Published: 2 April 2014

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.

Keywords:
Laforin; Lafora disease; Phosphatase; Carbohydrate-binding module; Glycogen