title: Improved strains and plasmid vectors for conditional overexpression of His-tagged proteins in Haloferax volcanii.
abstract: Research into archaea will not achieve its full potential until systems are in place to carry out genetics and biochemistry in the same species. Haloferax volcanii is widely regarded as the best-equipped organism for archaeal genetics, but the development of tools for the expression and purification of H. volcanii proteins has been neglected. We have developed a series of plasmid vectors and host strains for conditional overexpression of halophilic proteins in H. volcanii. The plasmids feature the tryptophan-inducible p.tnaA promoter and a 6xHis tag for protein purification by metal affinity chromatography. Purification is facilitated by host strains, where pitA is replaced by the ortholog from Natronomonas pharaonis. The latter lacks the histidine-rich linker region found in H. volcanii PitA and does not copurify with His-tagged recombinant proteins. We also deleted the mrr restriction endonuclease gene, thereby allowing direct transformation without the need to passage DNA through an Escherichia coli dam mutant.
authors.affiliation: Institute of Genetics, School of Biology, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom. thorsten.allers@nottingham.ac.uk
authors.forename: Thorsten
authors.initials: T
authors.lastname: Allers
authors.forename: Shahar
authors.initials: S
authors.lastname: Barak
authors.forename: Susan
authors.initials: S
authors.lastname: Liddell
authors.forename: Kayleigh
authors.initials: K
authors.lastname: Wardell
authors.forename: Moshe
authors.initials: M
authors.lastname: Mevarech
mesh_terms: D015280:DNA Restriction-Modification Enzymes / Q000235:genetics; D019641:DNA, Archaeal / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods; D019622:Haloferax volcanii / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D015280:DNA Restriction-Modification Enzymes; D019641:DNA, Archaeal; D011993:Recombinant Fusion Proteins