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  • Seleno-methionine Incorporation (C3022)

    Introduction

    T7 Express Crystal is a metB1 derivative of T7 Express (NEB #C2566 ). 50 µg/ml L-seleno-methionine may be substituted with 100 µg/ml of D/L isomer mix. For this we recommend using Seleno-DL-methionine from Sigma (S-3875) as a 10 mg/ml stock in dH2O. For enhanced growth, add 0.0002% ferric ammonium citrate to minimal media. For this we recommend Ferric Ammonium Citrate from Fisher (#I72-500)/as this product also provides trace metals. Filter sterilize prior to use. Transformed cells may be plated on rich agar. To produce a starter culture, inoculate a single colony into 10 ml minimal media plus 50 µg/ml L-methionine – grow up to 24 hours to achieve saturation.

    Protocol

    1. Incorporation using a regulated expression system (e.g. T7-lac promoter):
      1. Add 10 ml starter culture to 1L minimal media supplemented with 50 µg/ml L-methionine. Grow to mid-log phase (O.D. 0.6–0.8).
      2. Pellet cells and resuspend in fresh, pre-warmed minimal expression media without methionine or seleno-methionine.
      3. Grow for 2.5 hours at 37°C to deplete cells of methionine (longer if using a lower temperature).
      4. Add 50 µg/ml L-seleno-methionine or 100 µg/ml D/L isomer mix.
      5. Incubate for 30 minutes at 37°C.
      6. Add inducer and incubate 3 hours to overnight.
    2. Incorporation with a constitutive expression system:
      To produce a starter culture, inoculate a single colony into 10 ml minimal media plus 50 µg/ml L-methionine – grow up to 24 hours to achieve saturation.
      1. Prepare minimal expression media with a 10:50 ratio of L-Met to L-SeMet to achieve maximal growth rate (e.g. 10 µg/ml L-methionine to 100 µg/ml D/L-selenomethionine). This ratio will result in approximately 83% SeMet labeling of expressed protein.
      2. Use the same expression conditions previously optimized for your protein of interest.
      3. If a higher level of SeMet labeling is desired, spike with 100 µg/ml D/L-selenomethionine at mid-log to late-log phase of growth.
    3. Minimal media (per liter):
      200 ml 5X M9 salts (Sambrook et al.)
      20 ml 20% glucose (0.4% final conc)
      0.1 ml 1 M CaCl2 (0.1 mM final conc)
      2 ml 1 M MgSO4 (2 mM final conc)
      0.2 ml 1% ferric ammonium citrate (0.0002% final conc)
      Met, SeMet or Met/SeMet mix
      Antibiotic for plasmid selection
      Sterilized dH2O to 1 L