Hitrap q hp column

A cDNA encoding Schizosaccharomyces pombe Suc1 (provided by Eiichi Okumura [Tokyo Institute of Technology, Japan]) was cloned into the pET28-3C vector. Protein expression in the E. coli host and coarse purification using a Ni²⁺-charged column were performed as described above. The resultant suc1 fused with a His tag at their N-terminus was purified by using a Ni²⁺-charged Chelating Sepharose Fast Flow column. Peak fractions were pooled, further purified with a HiTrap Q HP column (Cytiva, 17115301), and dialyzed against buffer KHG150/10. Typically, 10 mg protein was obtained from a 1-liter culture and the concentration of stock aliquots ranged from 200 μM to 500 μM.

WTA were extracted from purified cell walls with TCA as described previously (Tomita et al., 2009 (link)) with some modifications. Briefly, lyophilized cell walls (50 mg; prepared as described above, keeping pH < 6.0 to avoid D-Ala hydrolysis from WTA) were incubated with 1 mL of 10% TCA at 4°C for 48 hr under rotation. The suspension was then centrifuged at 20.000 × g for 20 min at 4°C and the supernatant was recovered. WTAs were precipitated by addition of 5 V of ethanol and incubation overnight at –20°C. The pellet was purified by resuspension in TCA 10% and precipitation with ethanol. The final pellet was washed twice with cold ethanol, and the pellet was resuspended in MilliQ H₂O and lyophilized. WTAs were resuspended in 100 mM ammonium acetate buffer pH 4.8 (buffer A) and purified by anion exchange chromatography on a 1 mL HiTrap Q HP column (Cytiva) equilibrated with buffer A. WTAs were eluted with a gradient from 0 to 100% buffer B (buffer A containing 1 M NaCl) in 20 min. Fractions were collected and the presence of WTA was assessed in microplates with thymol-H₂SO₄ reagent (Engelhardt and Ohs, 1987 (link)), allowing detection of hexoses substituents of WTAs. The WTA-containing fractions were pooled and dialyzed against 10 mM ammonium acetate pH 4.8 with Float-a-Lyzer G2 dialysis devices (cut-off 500–1000 Da) (Spectra/Por) and lyophilized.

The Arabidopsis thaliana UBA1 open reading frame was cloned into the NdeI and XhoI sites of pET23a(+) in frame with the C-terminal His₆ tag by commercial gene synthesis (Genscript). AtUBA1 was expressed as a His₆-tagged protein from pET23a in E. coli BL21-AI^TM (Invitrogen^TM) at 18°C for 20 h with 800 µM IPTG and 0.2% (w/v) L-arabinose. AtUBA1 was purified by Immobilised Metal Affinity Chromatography (IMAC). The protein was purified using a HisTrap HP column (1 mL, Cytiva) and an equilibration buffer of 50 mM Tris-HCl pH 7.5, 400 mM NaCl, and 20 mM imidazole. The wash and elution buffers consisted of 50 mM Tris-HCl, 400 mM NaCl and 40 mM/200 mM imidazole. AtUBA1-containing fractions were dialysed into anion exchange start buffer (50 mM Tris-HCl, 50 mM NaCl, 1 mM DTT, 5% (v/v) glycerol) and purified using a HiTrap Q HP column (1 mL, Cytiva). The protein was eluted using a NaCl gradient (50-500 mM), and the resulting fractions were analysed by SDS-PAGE. AtUBA1 containing fractions were dialysed into size exclusion chromatography buffer (50 mM Tris-HCl, 200 mM NaCl, 1 mM DTT, 15% (v/v) glycerol), concentrated and then further purified by size exclusion chromatography on a 16/600 Superdex 200 pg column (GE Lifesciences) and an AKTA Pure chromatography system.

P14 (8 µM) was incubated with 4 mM NAD⁺ and either P14p6 or P14p8 (115 µM) for 2 h at room temperature in a 420 µL reaction volume consisting of 50 mM HEPES, pH 7.5, 400 mM NaCl, 2.5% glycerol, and 0.4 mM β-mercaptoethanol. At 30 min an additional 11.8 µL of concentrated P14 (285 µM) was added. The reaction was diluted to 1 mL in an SE buffer (10 mM potassium phosphate, pH 6.0 and 25 mM NaCl) and subjected to size exclusion chromatography at a rate of 0.75 mL/min using a Superdex Peptide 10/300 GL column (Cytiva, Marlborough, MA, USA) while collecting 250 µL fractions. MAR–peptide-containing fractions were pooled and brought up to 5 mL in an IE buffer (10 mM BIS-Tris, pH 5.0 and 25 mM NaCl) and further purified using anion exchange chromatography with a HiTrap Q HP column (Cytiva) and exchanged into an IE buffer + 1 M NaCl. ADPr–peptide fractions were desalted using a Sep-Pak C18 cartridge (Waters, Milford, CT, USA). Desalted peptides were diluted 1:1 with 100% acetonitrile, flash-frozen in liquid nitrogen, and lyophilized. Samples were stored at −80 °C immediately following drying to prevent ADPr hydrolysis.