Abstract
A system for heterologous expression in Escherichia coli of dehaloalkane dehalogenase Dh1A from Xanthobacter autotrophicus strain GJ10 is presented. The strategy involved overexpression of E. coli chaperonins GroEL/ES which facilitated the production of soluble Dh1A. When active-site mutant forms were constructed they could not to any detectable degree be expressed in a soluble state in the absence of overproduced GroEL/ES. However, with the described expression system, wild-type Dh1A as well as variant forms randomly mutated in the active-site residues Phe172 and Trp175 were reliably produced. An introduced C-terminal (His)5-tag provided an immunological handle as well as a site for metal ion coordination utilized in affinity chromatography for the purification of recombinant Dh1A. The purified His-tagged enzyme, Dh1A-5His, was confirmed to be catalytically fully active when measuring the dehalogenase activity with dichloroethane as substrate.
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