Table 1 Summarised data for expression constructs and purification protocols, oligomerisation states, and Zn stoichiometry of Zur proteins from different organisms
OrganismExpression and purificationOligomerisationStoichiometry (Zn per monomer)Ref.
Mycobacterium tuberculosisGST-fusion; glutathione sepharose 4B resinn.d.; assumed dimeric1.8 ± 0.2 (Bradford assay and FAAS)[39]
Mycobacterium tuberculosisHis-tagged; Ni-IMAC, TEV-cleavage, SECDimer: X-ray crystallography3 (X-ray crystallography)
1 (post EDTA treatment; μPIXE)
[15]
Streptomyces coelicolorHis-tagged; Ni-charged Chelex-100, elution with imidazole, dialysis against 5 mM EDTADimer (analytical ultracentrifugation). No indication of self-association.n.d.[16]
Streptomyces coelicolorNo tag; Ni-charged NTA column; elution with imidazole gradient; dialysis; no EDTA during purificationDimer (X-ray crystallography and SEC) for WT, D, and M site mutants. C90S mutant monomeric3 (X-ray crystallography)
2.4 (WT; ICP-OES)
1.2 (EDTA treated)
1.5 (H84A mutant; D site)
1.0 (C79S mutant; M site)
0.25 (C90S mutant; C site)
[38]
Bacillus subtilisCell lysis in presence of 2 mM EDTA and 2 mM DTT;
heparin affinity, SEC, AEX, dialysis
Dimer (SEC)0.5–0.8 initially bound;
additional 1.4 Zn by titration, giving approximately 2 Zn per monomer ([P] by A277, [M] by PAR assay and titration with FluoZin3)
[40,41]
Streptococcus suisHis- or GST-tag.
Ni-IMAC or glutathione Sepharose 4B (PBS). GST-tag removed
Dimer (chemical cross-linking assays)n.d.[42]
Escherichia coliTag-less expression.
Lysis in the presence of 6 M urea, and 100 mM DTT, then refolded in presence of 100 μM Zn2+. AEX, HIC and SEC
n.d., assumed dimeric1.4–1.8 (standardised Bradford assay and ICP-AES)[43]
Escherichia coliSimilar to [43]Dimer (X-ray crystallography)
C103S mutant monomeric
2 (X-ray crystallography)
2.8 (WT; ICP-MS)
0.7 (EDTA-treated WT)
2.1 (C103S mutant)
0.0 (EDTA-treated C103S mutant)
2.5 (C88S mutant)
0.6 (EDTA-treated C88S mutant)
[17]
Salmonella entericaHis-tagged.
Lysis: 1 mM EDTA
IMAC, SEC, heparin affinity
n.d., assumed dimericCa. 1 (ICP-MS)
Capacity to bind up to two more per monomer
[44,45]
Paracoccus denitrificansMBP fusion tag. Lysis: 1 mM EDTA. Amylose resin column. Tag cleaved by Factor Xa protease, AEX. Apo-Zur by dialysisDimer (SEC)Ca. 1 (ICP-OES), capacity to bind one more Zn per monomer[46]
Synechocystis sp. PCC6803Tag-less expression.
Lysis: 100 mM NaCl, 5 mM DTT, 1 mM EDTA, heparin affinity and SEC
n.d., assumed dimeric1.02 ± 0.15 (ICP-MS)
Capacity to bind at least one more Zn per monomer
[47]
Anabaena sp. PCC 7120His-tagged. Cell lysis in presence of 2M GdnHCl (pH 8).
Zn-IMAC, dialysis pH 5.5
Mostly monomer, some dimer (SDS–PAGE, denaturing conditions)1 (ICP-MS)
Up to two more binding sites per monomer
[48]

Abbreviations: AEX: anion-exchange chromatography; DTT: dithiothreitol; EDTA: ethylenediaminetetraacetic acid; FAAS: flame atomic absorption spectroscopy; GdnHCl: guanidinium hydrochloride; GST: Glutathione S-transferase; HIC: hydrophobic interaction chromatography; His-tag: polyhistidine-tag; ICP-AES/ICP-OES: inductively coupled plasma atomic/optical emission spectroscopy; ICP-MS: inductively coupled plasma mass spectrometry; IMAC: immobilised metal ion affinity chromatography; MBP: maltose-binding protein; n.d: not determined; μPIXE: micro-proton-induced X-ray emission; PAR: pyridyl-azo-resorcinol; SDS–PAGE: sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SEC: size-exclusion chromatography; TEV: tobacco etch virus; WT: wild type.