Development of PhaP-tagged protein expression and purification systems for extremely halophilic archaea

Abstract:

Abstract:［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 ΔphaP．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．