Mtr2

Growth

  1. Transform BL21 DE3 [RIL] with pET9-Mtr2 DNA (may be called Mex20).
  2. Pick a single colony into 1l 2xTY with 30 µg ml-1 kanamycin. Grow overnight at 37°C.

  3. Harvest by centrifugation at 5,000 rpm at 4°C for 10 minutes.

  4. Resuspend in 10 ml sucrose buffer per litre of media. Store at -20°C.

Purification

Add fresh PMSF throughout.
  1. Thaw pellets and lyse by French press (twice) without DNase or divalent cations. Shear DNA by sonication on ice (3 x 20s on 20% power with the half-inch tip should do it. Don't overcook).
  2. <20k, 2°C, 20 min>
  3. Adjust supernatant to pH 6 with Bis-tris and dialyse overnight against 1 l 'LoLo' buffer.
  4. <20k, 2°C, 20 min>. Keep the supernatant
    1. Load onto S-Sepharose in LoLo buffer. Collect the flow-through and pass it over the column.
    2. Wash in with 30 - 50 ml LoLo buffer.
    3. Wash with 50 ml HiLo buffer.
    4. Elute with a gradient to 500 mM NaCl (HiHi buffer) over 200 ml.
    5. Mtr2 elutes almost immediately the NaCl hits the column.
  5. Concentrate the Mtr2-containing fractions in Centriprep-10 and apply to S-100 in HiLo buffer + 100 mM NaCl.
  6. Collect Mtr2-containing fractions and concentrate (Centriprep/con 10).

Quantification

Coefficient at 280 nm = 16,500 M-1 cm-1.

Mr = 20,783.5.

Enter
A280 reading 		Enter
dilution factor 		  Conc (mM) Conc (mg ml-1)

Buffers

LoLo HiLo HiHi
20 mM Bis-Tris-Cl pH 6.0
1 mM EDTA
1 mM DTT
 		 20 mM Tris-Cl pH 8.5
1 mM EDTA
1 mM DTT
 		 20 mM Tris-Cl pH 8.5
500 mM NaCl
1 mM EDTA
1 mM DTT
RPG 2000/06/06

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