title: [Development of PhaP-tagged protein expression and purification systems for extremely halophilic archaea ].
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 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.
authors.forename: Jinhua
authors.initials: J
authors.lastname: Wul
authors.forename: Shuangfeng
authors.initials: S
authors.lastname: Cai
authors.forename: Hailong
authors.initials: H
authors.lastname: Liu
authors.forename: Jing
authors.initials: J
authors.lastname: Hou
authors.forename: Jing
authors.initials: J
authors.lastname: Han
authors.forename: Jian
authors.initials: J
authors.lastname: Zhou
authors.forename: Xiang
authors.initials: X
authors.lastname: Hua
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D019623:Haloferax mediterranei / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D054813:Polyhydroxyalkanoates / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D012965:Sodium Chloride / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C118828:PHAP protein, Bacteria; D054813:Polyhydroxyalkanoates; D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride