Heterologous expression in Escherichia coli of soluble active-site random mutants of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 by coexpression of molecular chaperonins GroEL/ES.
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.
Widersten
M
M
Biomedical Center, Uppsala University, Uppsala, S-751 23, Sweden. micke@balder.bmc.uu.se
D001665:Binding Sites; D018835:Chaperonin 10 / Q000235:genetics*; D018834:Chaperonin 60 / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology; D006867:Hydrolases / Q000235:genetics*; D016296:Mutagenesis
D018835:Chaperonin 10; D018834:Chaperonin 60; D006867:Hydrolases; C046137:haloalkane dehalogenase