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.

FIG. 1.

Conditional overexpression vector pTA963. pTA963 features a tryptophan-inducible promoter from the tnaA gene (p.tnaA) (26) flanked by the L11e rRNA terminator (t.L11e) (38) and a synthetic terminator (t.Syn). For expression of native proteins, the coding sequence is inserted between the NdeI site downstream of the promoter and either the EcoRI or BamHI site. For N-terminal 6×His-tagged proteins, the 5′ end of the gene is ligated instead with the PciI site located downstream of a (CAC)₆ tract. PciI-compatible ends are generated by NcoI and BspHI and are used where the second codon starts with G and A, respectively. pTA963 uses a pHV2 replication origin and has hdrB and pyrE2 selectable markers; variants are available with pyrE2 only (pTA929) and without a (CAC)₆ tract (pTA962, or pTA927 for pyrE2 only).

FIG. 2.

Replacement of the pitA gene. (A) Protein sequence alignment of the central region of PitA (5), linking the N-terminal chlorite dismutase-like and C-terminal antibiotic biosynthesis monooxygenase-like domains, from selected species of haloarchaea. Histidine residues are indicated by a black background; conserved residues are indicated by gray shading. Hvo, H. volcanii; Hsa, Halobacterium salinarum; Hwa, Haloquadratum walsbyi; Hla, Halorubrum lacusprofundi; Nph, N. pharaonis. (B) Gene replacement construct pTA1106, containing pitA from N. pharaonis (pitA_Nph) flanked by upstream (US) and downstream (DS) regions of H. volcanii pitA (pitA_Hvo). (C) Colony hybridization of 5-fluoroorotic acid (5-FOA)-resistant H. volcanii clones, after pop-in/pop-out gene replacement with pTA1106. H. volcanii pitA sequences (amplified with pitAF/pitAR primers) were used as a probe, and clones failing to hybridize therefore carried the N. pharaonis pitA gene. (D) Verification of pitA replacement in H1154 and H1155 using PCR with primers specific for either H. volcanii (pitAF/pitAR) or N. pharaonis (NphPitAF/NphPitAR) genes. H26 genomic DNA was used as a negative control and pTA1106 plasmid DNA as a positive control. (E) Map of the H. volcanii pitA region, indicating MluI sites and restriction fragment sizes of the native pitA gene (pitA_Hvo) and the replacement with N. pharaonis pitA (pitA_Nph). (F) Verification of pitA replacement in H1154 and H1155 by MluI digestion and Southern blotting. The probe used is indicated in panel E.

FIG. 3.

Overexpression of RadA in a pitA_Nph replacement strain. (A) Induction regime for protein overexpression using the tryptophan-inducible p.tnaA promoter. H1173 was grown for the times indicated in either Hv-Ca or Hv-YPC broth supplemented with tryptophan at the concentrations shown. 6×His-tagged RadA was purified, and the relative protein yield was determined by quantification of Coomassie blue-stained bands displayed on polyacrylamide gels as in panel B. (B) 6×His-tagged RadA was overexpressed in strains with either the native pitA gene (PitA _Hvo; H1045) or replacement with N. pharaonis pitA (PitA _Nph; H1173). The induction regime used was 5 h in Hv-YPC plus 1 mM tryptophan and 1 h in Hv-YPC plus 3 mM tryptophan at 42°C. The control strains H989 and H1172 contained the empty vector (eV) pTA963. 6×His-tagged RadA was purified from the soluble fraction (Lysate) by affinity chromatography on an Ni²⁺ chelating column, and samples were taken from the flowthrough (Flow) and after washing with 20 mM imidazole (Wash). Bound protein was eluted using 50 mM and 500 mM imidazole. Two additional bands were identified by mass spectrometry, PitA and Cdc48d. (C) Protein sequence alignment of the C termini of Cdc48d from selected species of haloarchaea. Histidine residues are indicated by a black background; conserved residues are indicated by gray shading. (D) Contamination by Cdc48d is reduced by growth at <45°C. 6×His-tagged RadA was overexpressed in H1173 and purified by affinity chromatography on a Ni²⁺ chelating column as in panel B. The relative yields of RadA and Cdc48d (standardized to the RadA yield at 42°C) were determined by quantification of Coomassie blue-stained bands displayed on polyacrylamide gels. The averages and standard errors of two experiments are shown. (E) Overexpression of 6×His-tagged RadB results in less contamination by Cdc48d. 6×His-tagged RadB was overexpressed in strain H1174 and purified by affinity chromatography on a Ni²⁺ chelating column as in panel B.

FIG. 4.

Deletion of the mrr gene improves transformation with dam-methylated DNA. (A) Protein sequence alignment of the conserved cores of Mrr homologs from selected archaea and bacteria and Schizosaccharomyces pombe SPAC824.03c (9). Conserved residues are indicated by gray shading, and identical residues are indicated by a black background. Hvo, H. volcanii; Hma, Haloarcula marismortui; Hsa, H. salinarum; Hmu, Halomicrobium mukohataei; Hwa, H. walsbyi; Hla, H. lacusprofundi; Nph, N. pharaonis; Mac, Methanosarcina acetivorans; Mhu, Methanospirillum hungatei; Mth, Methanothermobacter thermautotrophicus; Dra, Deinococcus radiodurans; Efa, Enterococcus faecalis; Eco, E. coli; Lbr, Lactobacillus brevis; Mae, Microcystis aeruginosa; Mtu, Mycobacterium tuberculosis; Sth, Salmonella thyphimurium; Vch, Vibrio cholerae; Spo, S. pombe. (B) Phylogenetic tree representing evolutionary relationships between Mrr homologs, constructed using neighbor joining and rooted using S. pombe SPAC824.03c. Support for individual branches is indicated by bootstrap values (1,000-fold resampling); values of <50% are not recorded. Pairwise distances between sequences are uncorrected. (C) Colony hybridization of 5-FOA-resistant H. volcanii clones after pop-in/pop-out gene deletion with pTA1150. H. volcanii mrr sequences (amplified with mrrF/mrrR primers) were used as a probe, and clones failing to hybridize therefore had the mrr gene deleted. (D) Map of the H. volcanii mrr region indicating MluI sites and restriction fragment sizes. (E) Verification of mrr deletion in H1206 to H1209 by MluI digestion and Southern blotting; the probe used is indicated in panel D. (F) Transformation efficiencies of Δmrr strains H1206 and H1209 compared to the mrr⁺ strains H26 and H1155 using a methylated pTA354 plasmid purified from an E. coli dam⁺ strain or unmethylated pTA354 DNA from an E. coli dam mutant (31). The averages and standard errors of three experiments are shown. (G) 6×His-tagged RadA(A196V) was overexpressed in the Δmrr strain H1209 and purified by affinity chromatography on a Ni²⁺ chelating column as in Fig. 3B.