Overexpression and purification of halophilic proteins in Haloferax volcanii

Abstract

Halophilic enzymes function optimally at high salt concentrations and are active at low water availability. Such conditions are encountered at elevated concentrations of solutes such as salts and sugars, and at high concentrations of organic solvents. However, expression in heterologous hosts such as Escherichia coli can cause problems, since halophilic proteins typically misfold and aggregate in conditions of low ionic strength. We have harnessed the sophisticated genetic tools available for the haloarchaeon Haloferax volcanii, to develop a system for the overexpression and purification of halophilic proteins under native conditions.

Keywords: Haloferax volcanii; His-tag; archaea; halophile; protein overexpression.

Figure 1

Conditional overexpression vector pTA963. pTA963 utilizes the tryptophan-inducible promoter p.tnaA. For N-terminally 6xHis tagged proteins, the 5′ end of the gene is ligated with the PciI site located downstream of a (CAC)₆ tract. PciI-compatible ends are generated by NcoI and BspHI, and are used where the second codon starts with G and A, respectively. For expression of native proteins, the 5′ end of the gene is ligated with the NdeI site downstream of the promoter.