Improved strains and plasmid vectors for conditional overexpression of His-tagged proteins in Haloferax volcanii

Thorsten Allers*, Shahar Barak, Susan Liddell, Kayleigh Wardell, Moshe Mevarech

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)1759-1769
Number of pages11
JournalApplied and Environmental Microbiology
Volume76
Issue number6
DOIs
StatePublished - Mar 2010

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