TY - JOUR
T1 - Improved strains and plasmid vectors for conditional overexpression of His-tagged proteins in Haloferax volcanii
AU - Allers, Thorsten
AU - Barak, Shahar
AU - Liddell, Susan
AU - Wardell, Kayleigh
AU - Mevarech, Moshe
PY - 2010/3
Y1 - 2010/3
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=77749304005&partnerID=8YFLogxK
U2 - 10.1128/AEM.02670-09
DO - 10.1128/AEM.02670-09
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C2 - 20097827
AN - SCOPUS:77749304005
SN - 0099-2240
VL - 76
SP - 1759
EP - 1769
JO - Applied and Environmental Microbiology
JF - Applied and Environmental Microbiology
IS - 6
ER -