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An optimized method to produce halophilic proteins in Escherichia coli


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.


Halophilic proteins have been overexpressed using the pET3a vector in E. coli BL21(DE3), for instance glucose dehydrogenase, glutamate dehydrogenase, nitrite reductase, extracellular α-amylase and isocitrate lyase from H. mediterranei and isocitrate dehydrogenase from H. volcanii. The recombinant proteins were always obtained as inclusion bodies (see Figure 1), which were solubilised in the presence of urea. In most cases, the proteins were refolded by rapid dilution in a high salt concentration buffers. The purification procedure of the recombinant proteins was based on the halophilic properties of this kind of enzymes. On the whole, the method consists of a precipitation using ammonium sulphate and a chromatography on DEAE-cellulose in the presence of that salt. The elution with sodium/potassium chloride yielded proteins in a pure and highly concentrated form (see Figure 2 and Table 1). Halophilic recombinant proteins have been characterized and shown the same biochemical characteristics as the enzymes isolated from H. mediterranei and H. volcanii [13]. The high protein concentrations obtained has allowed us to carry on crystallization assays. In particular, the glucose dehydrogenase from H. mediterranei has been crystallized by the hanging-drop method using sodium citrate as the precipitant [4, 5].

Figure 1
figure 1

Expression of recombinant isocitrate dehydrogenase from H. volcanii under different temperatures. Lane 1. Molecular weight standards. Lane 2. Wild-type isocitrate dehydrogenase. Lanes 3. Uninduced insoluble fraction. Lanes 4 and 5. Induced insoluble fraction at 37°C. Lanes 6 and 7. Induced insoluble fraction at 25°C.

Figure 2
figure 2

Purification of recombinant glucose dehydrogenase from H. mediterranei. Lane 1: Molecular weight standards. Lane 2: Wild type glucose dehydrogenase. Lane 3: Inclusion body fraction. Lane 4: (NH4)2SO4 precipitation supernatant. Lane 5: (NH4)2SO4 precipitation pellet. Lane 6: Active fractions from DEAE-cellulose.

Table 1 Purification of recombinant glucose dehydrogenase from H. mediterranei


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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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution 2.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Esclapez, J., Bonete, M.J., Camacho, M. et al. An optimized method to produce halophilic proteins in Escherichia coli. Microb Cell Fact 5 (Suppl 1), S22 (2006).

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