RESUMO
[ OBJECTIVE] To establish a convenient halophilic protein expression and purification system based on the haloarchaeal-type PhaP and polyhydroxyalkanoate (PHA) granule. [METHODS] We cloned a strong haloarchaeal promoter and the phaP-tag into the haloarchaea- Escherichia coli shuttle vector pWL502, and then used the constructed vector to express the PhaP-tagged haloarchaeal proteins in the phaP-deleted strain Haloferax mediterranei AphaP. We purified the PhaP-fusion proteins, which were associated with PHA granules, by sucrose density gradient centrifugation. We also inserted a haloarchaeal intein-containing fragment between phaP and multiple cloning sites, and modulated the intein splicing activity by site-directed mutagenesis. [RESULTS] We successfully constructed two expression vectors, pPM and pIP, in which PhaP was used as N-terminal and C-terminal fusion tag, respectively. The haloarchaeal proteins were effectively expressed by both vectors. The PhaP-tagged proteins were easily purified through the strategy of PHA granulemediated protein purification. In addition, we found that the intein-containing fragment Hbt21 from Halobacterium sp. NRC-1 had maintained splicing activity in H. mediterranei, and its C-terminal cleavage could be blocked or attenuated by mutating the conserved asparagine ( N182) or serine (S183) , respectively. [ CONCLUSION] We have established a convenient and economical halophilic protein expression and purification system. We have also identified the splicing active sites of a haloarchaeal intein, which showed potential for removing the PhaP-tag from the purified proteins.
