Expression and Purification of M. smegmatis DnaN (β subunit) 

This protocol was used in the work “Investigating the composition and recruitment of the mycobacterial ImuA′–ImuB–DnaE2 mutasome” (DOI: 10.7554/eLife.75628) to purify the Mycobacterium smegmatis β clamp (DnaN) for biochemical experiments.

1. Expression

• Express the dnaN gene from Mycobacterium smegmatis (DNA polymerase III β subunit) in E. coli BL21(DE3) cells for 16 hours at 16 °C. In this paper, the expression vector pETNKI-his-3C-LIC-kan from the NKI-LIC vector suite (Luna-Vargas et al. 2011, DOI: 10.1016/j.jsb.2011.03.017) was used. 

2. Cell Lysis

• Resuspend bacterial cell pellets in Ni-25-2 buffer:
  • 25 mM imidazole
  • 25 mM HEPES pH 7.5
  • 500 mM NaCl
  • 5 mM β-mercaptoethanol
  • 5% glycerol
• Use a buffer volume equivalent to 3× the cell pellet weight.
• Lyse cells by sonication on ice using a VibraCell VC 750 (Sonics Inc.):
  • 100% tip output
  • 6-second pulses with 4-second pauses
  • Continue until complete lysis
• Centrifuge lysate:
  • 35,000 rpm
  • 20 minutes
  • 4 °C
  • Rotor: 75 Ti (Beckman Optima XL)
• Collect supernatant (soluble fraction).
• Sonicate supernatant again for 1 minute.
• Filter through a 0.45 µm membrane (Millipore).

3. Ni²⁺ Affinity Chromatography

• Load clarified lysate onto a HisTrap column.
• Wash with:
  • 4 column volumes (CV) of Ni-25-2 buffer
• Elute protein using a linear gradient 25–100% Ni-500-2 buffer:
  • 500 mM imidazole
  • 25 mM HEPES pH 7.5
  • 500 mM NaCl
  • 5 mM β-mercaptoethanol
  • 5% glycerol
  • Over 6 CV

4. Anion Exchange Chromatography

• Pool β-containing fractions.
• Dilute sample to 50 mM NaCl using Q-A2 buffer:
  • 50 mM HEPES pH 7.5
  • 0.5 mM EDTA
  • 5 mM β-mercaptoethanol
• Load onto HiTrap Q column pre-equilibrated in 5% Q-B2 buffer
  • 50 mM HEPES pH 7.5
  • 1 M NaCl
  • 0.5 mM EDTA
  • 5 mM β-mercaptoethanol
• Elute using a gradient:
  • 5–50% Q-B2
  • Over 6 CV

5. Size-Exclusion Chromatography

• Load fractions of interest onto Superdex 200 26/60 column (GE Healthcare).
• Use gel filtration buffer:
  • 50 mM HEPES pH 7.5
  • 150 mM NaCl
  • 5 mM β-mercaptoethanol

6. Concentration and Storage

• Concentrate protein using Amicon Ultra centrifugal filters (Millipore):
  • 50 kDa molecular weight cut-off
• Aliquot purified protein.
• Flash-freeze in liquid nitrogen.
• Store at −80 °C.