- Oral Presentation
- Open Access
An optimized method to produce halophilic proteins in Escherichia coli
© Esclapez et al; licensee BioMed Central Ltd. 2006
- Published: 10 October 2006
- Recombinant Protein
- Ammonium Sulphate
- High Salt Concentration
The homologous and heterologous expression of genes is a prerequisite for most biochemical studies of protein function. Many systems have been carried out for protein production in members of the Bacteria and Eukarya, however members of the Archaea are less amenable to genetic manipulation. Only a few systems for high-level gene expression have been developed for halophilic microorganisms. Because of this, mesophilic hosts, in particular Escherichia coli, have been used to produce halophilic proteins for biochemical characterization and crystallographic studies. Expression in E. coli has the advantage to be faster and it will easily allow production on a commercial scale. In contrast, difficulties are encountered since enzymes from extreme halophiles require the presence of high salt concentration for activity and stability, and the overexpressed product will need either reactivation or refolding in a salt solution, and so the purification techniques should be compatible with the high salt concentration required.
For the last years, we have developed and refined a system to produce and purify large amounts of recombinants proteins from Haloferax mediterranei and Haloferax volcanii in the mesophilic host E. coli.
Purification of recombinant glucose dehydrogenase from H. mediterranei
Specific activity (U/mg)
Inclusion bodies refolding in 20 mM Tris-HCl pH 7.4, 2 M NaCl, 1 mM EDTA
(NH 4 ) 2 SO 4 Precipitation
The overexpression, refolding and purification method of halophilic proteins developed provides a fast, simple and efficient process that yields enzymes of high purity in large amounts.
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