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Heterologous Expression in Escherichia coli of SolubleActive-Site Random Mutants of Haloalkane Dehalogenase from Xanthobacter autotrophicus GJ10 by Coexpression of Molecular Chaperonins GroEL/ES
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. (Widersten)
1998 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 13, no 3, 389-395 p.Article in journal (Refereed) Published
Abstract [en]

A system for heterologous expression in Escherichia coli of dehaloalkane dehalogenase DhlA from Xanthobacter autotrophicus strain GJ10 is presented. Thestrategy involved overexpression of E. coli chaperonins GroEL/ES which facilitated the production of soluble DhlA. 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 DhlA 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 DhlA. The purified His-tagged enzyme, DhlA-5His, was confirmed to be catalytically fully active when measuring the dehalogenase activity with dichloroethane as substrate.

Place, publisher, year, edition, pages
1998. Vol. 13, no 3, 389-395 p.
National Category
Natural Sciences
URN: urn:nbn:se:uu:diva-158468DOI: 10.1006/prep.1998.0913OAI: oai:DiVA.org:uu-158468DiVA: diva2:439609
Available from: 2011-09-08 Created: 2011-09-08 Last updated: 2011-09-13Bibliographically approved

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Publisher's full texthttp://www.sciencedirect.com/science/article/pii/S1046592898909135

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