Hisprep ff 16 10

exo+ and exo− versions of P. abyssi PolB (Gene ID: GI:1495739) were produced and purified as already described [33 (link)].
Briefly, PolB exo+ and exo− were overproduced by addition of 1 mM IPTG in E. coli strain Rosetta 2(DE3)pLysS grown overnight in Lysogeny broth (LB) at 30 °C. The cell extracts were treated by heating for 20 min at 75 °C, and His-tagged PolB was purified by using an affinity column (HisPrep FF 16/10, GE Healthcare, Chicago, MI, USA) and by hydrophobic chromatography (HiPrep Phenyl (low sub) FF 16/10, GE Healthcare). After dialysis and concentration, the final protein sample was stored in 30 mM Tris-HCl, pH 7.5; 0.6 mM DTT; 60 mM NaCl and 40% glycerol. Protein concentration was calculated by measuring absorbance at 280 nm. The recombinant PaPolB has a predicted extinction coefficient of 122,050 M⁻¹·cm⁻¹ and a molecular weight of 89,400.31 Da (ProtParam, ExPASy, [53 ]).

Two different purification strategies on the Äkta system (GE Healthcare) were performed for three of the produced enzymes: immobilised metal ion affinity chromatography (IMAC) for AaGE1 and WcGE1, and ion exchange chromatography (IEX) for PcGE1. Purification via IMAC was performed on HisPrepFF 16/10 or HisTrap Excel columns (GE Healthcare) according to the manufacturer’s recommendations. Elution was performed in one step with a buffer containing 500 mM imidazole, and flow-through and elution fractions showing UV absorbance at 280 nm were buffer-exchanged and examined for GE activity. For anion exchange chromatography, a 1 mL Q-Sepharose HiTrapFF column (GE Healthcare) was used. For PcGE1 (theoretical pI of 5.7) 0.02 M Tris-HCl, pH 8.5, was chosen as loading buffer and elution was performed with loading buffer containing an increasing gradient of NaCl up to a final concentration of 1 M. Fractions of all steps showing UV absorbance at 280 nm were collected and analysed for GE activity. Purity of target enzymes was assessed using SDS-PAGE (Bio-Rad) followed by Coomassie Brilliant Blue R-250 staining or imaging on a Chemidoc Touch Stain-free Imager (Bio-Rad). Presence of N-glycosylation on purified proteins was determined by digestion with PNGase F (New England Biolabs) according to the manufacturer’s protocol.

exo+ and exo− versions of P. abyssi PolD, DP1 (small subunit, Gene ID: 1495007) and DP2 (large subunit, Gene ID: 1495008),were produced and purified as already described [44 (link),55 (link)]. Briefly, PolD exo+ and exo− were overproduced by addition of 1 mM IPTG in E. coli strain grown overnight in Lysogeny broth (LB) at 20 °C. The cell extracts were treated by for 30 min at 70 °C, and his-tagged PolD was purified by using a strong anion exchange column (HiLoad 26/10 Q sepharose, GE Healthcare, Chicago, MI, USA), two affinity columns (HisPrep FF 16/10 and HiTrap Heparin, GE Healthcare, Chicago, MI, USA) and a size exclusion column (Superdex 200, GE Healthcare, Chicago, MI, USA). After concentration, the final protein sample was stored in 35 mM Tris-HCl, pH 7.5; 0.7 mM β-mercaptoethanol; 35 mM NaCl and 30% glycerol. Protein concentration was calculated by measuring absorbance at 280 nm. The recombinant PaPolD exo+ has a predicted extinction coefficient of 63,720 M⁻¹·cm⁻¹ and a molecular weight of 69,395.36 Da for DP1; 152,990 M⁻¹·cm⁻¹ and 146,368.57 Da for DP2 (ProtParam, ExPASy, [53 ]). The recombinant PaPolD exo− has a predicted extinction coefficient of 65,210 M⁻¹·cm⁻¹ and a molecular weight of 74,834.07 Da for DP1; 152,990 M⁻¹·cm⁻¹ and 144,205.25 Da for DP2 (ProtParam, ExPASy, [53 ]).

The IMAC purification of the histidine/asparagine‐tagged human NID1 was performed with a HisPrep FF 16/10 affinity chromatography column (GE Healthcare), controlled by the FPLC system Äkta Explorer 10 (GE Healthcare). NID1 elution was indicated by the increased absorption at the wavelengths 280 and 256 nm. Protein‐containing elution fractions were pooled and desalted using a HiPrep 26/10 desalting column (GE Healthcare), controlled by the Äkta Purifier 100 (GE Healthcare). Subsequently, the protein solution was washed and concentrated with ultrafiltration units (Vivaspin 20, Sartorius), sterile filtered (SCGP00525, Millipore), and stored at −80 °C until further use.

P. abyssi PCNA (Gene ID: 1496768) was produced and purified as described previously [54 (link)]. Briefly, PCNA was overproduced by addition of 1 mM IPTG in E. coli strain BL21-CodonPlus-RIL grown for 4 h in Lysogeny broth (LB) at 37 °C. The cell extracts were treated by heating at 80 °C for 10 min, and His-tagged PCNA was purified by using a strong anion exchange column (HiPrep Q FF 16/10, GE Healthcare, Chicago, MI, USA) and an affinity column (HisPrep FF 16/10, GE Healthcare, Chicago, MI, USA). After dialysis and concentration, the final protein sample was stored in 40 mM Tris-HCl, pH 8.0; 0.8 mM DTT; 240 mM NaCl; 0.16 mM EDTA and 20% glycerol. Protein concentration was measured by colorimetric assay based on the Bradford dye-binding method (BioRad protein assay dye reagent, BioRad, Hercules, CA, USA). The recombinant PaPCNA has a predicted extinction coefficient of 7450 M⁻¹·cm⁻¹ and a molecular weight of 29,433.77 Da (ProtParam, ExPASy, [53 ]).