Cloning and Overexpression of Pyrococcus Furiosus Endoglucanase A Gene (egIA) in Escherichia Coli

Abstract

This study describes the cloning and high-level expression of an Endoglucanase A Gene (egIA) from a hyperthermophilic archeon Pyrococcus Furiosus. An expression plasmid pET-EgIA was constructed for the production of recombinant EgIA in E.Coli B12 (DE3) under the control of T7lac promoter. Following induction, ~35kDa protein expressed at levels greater than 20% of the total E.Coli cellular proteins. The expressed protein, however, was in the form of inclusion bodies with little enzymatic activity, which was solubilized using higher concentration of denaturing agent (8M urea) followed by its refolding to an active state. A 7-8 fold increase in enzyme activity corresponding to 285U/mg specific activity could be achieved after refolding. The refolded egIA, partially purified by heat treatment upto ~92%, is being investigated for applications like hydrolysis of cellulose, a major component of plant biomass. Local, upscale and cheap production of these cellulolytic enzymes can help in reducing the costs of many processes in various industries like poultry and textile.