The ParB gene (residues 1–139) was PCR-amplified from plasmid YB411 (21 (link)) with primers WTF1 (5′-ATATACCATGGCTGATCGCACGGTTGC-3′; the restriction site is underlined) and WTR139 (5′-CCGCAAGCTTGCTTCCCAGTGGGCGCCCG-3′) and cloned into pET28a using NcoI and HindIII sites. The full-length ParB protein contains a non-cleavable six-His-tag at the C-terminus encoded in the plasmid. ParB fragments spanning residues 60–139, 65–139 and 65–135 were generated with primers PF60 (5′-GCCCGAAGCTTCGGAGCCCCGAGGGGCGCG-3′) and PR139 (5′-CCGGAATTCTTAGCTTCCCAGTGGGCGCCCGC-3′), PF65 (5′-GCCCGAAGCTTCCGCGCGTTCTGAGGTCAAGAT-3′) and PR139 and PF65 and PR135 (5′-CCGGAATTCTTAGCGCCCGCGAGTAACGCCTCG-3′), respectively. The fragments were cloned into a modified pET-Duet1 plasmid (Novagen) using HindIII and EcoRI sites, in which ParB was fused to an upstream six-His-tagged DsbA with a PreScission-cleavable linker.
All proteins were expressed in Escherichia coli BL21-Gold (DE3) induced with 0.2 mM isopropyl β-d-1-thiogalactopyranoside at 16°C. Harvested cells were resuspended in buffer P300 (300 mM KCl and 50 mM phosphate, pH 7.6) and lysed by sonication. Cell lysate was clarified by centrifugation and loaded onto a HisTrap column (GE Healthcare). After a wash with 25 mM imidazole in P300, bound protein was eluted with 500 mM imidazole in P300 and pooled. The six-His-DsbA tag was cleaved from the ParB fragments by PreScission protease. ParB was loaded onto a heparin column (GE Healthcare) and eluted at ∼300 mM KCl in a gradient from 50 to 1000 mM KCl in buffer H (20 mM HEPES, pH 7.6). The protein was further purified with a Superdex 200 gel filtration column in a buffer containing 5 mM HEPES-K, pH 7.6, and 100 mM KCl. The protein monomer concentration was measured by its absorbance at 280 nm using a molar extinction coefficient of 2980 M−1 cm−1 for all ParB constructs. This value was calculated on the basis of amino acid composition. The protein was concentrated to 20 mg/ml in buffer containing 5 mM HEPES-K, pH 7.6, and 100 mM KCl and stored at −80°C as aliquots. The molar concentration of ParB protein is expressed for its dimeric form.