MexC2

MexC2 was made by PCR from S. cere, residues XXX (plus extra methionine) to XXX (C-terminus). Cloned into pMW172 NdeI/EcoRI.

Growth

  1. Transform BL21 DE3 [RIL] with pMW172/MexC2 DNA. Select on Amp plates.
  2. Pick a single colony into 1 l 2xTY/Amp. Grow overnight at 37°C with shaking.
  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

  1. Thaw pellets and lyse by sonication on ice (3 x 20s on 25% power with the medium tip should do it. Don't overcook).
  2. <20k, 2°C, 20 min>
  3. Dialyse at 4°C overnight versus 2l HiLo buffer.
  4. <20k, 2°C, 20 min>
  5. Load on to Q-Sepharose in HiLo buffer. Keep the flow-through. Elute MexC2 with a 200 ml gradient to HiHi. Pool MexC2-containing fractions (it elutes very early on, just after the flow-through but before (?) the gradient).
  6. Concentrate the MexC2-containing fractions in Centriprep-3 and apply to S-100 in HiLo buffer + 100 mM NaCl.
  7. Collect MexC2-containing fractions and concentrate (Centriprep/con 3).

Quantification

Coefficient at 280 nm = 8,250 M-1 cm-1.

Mr = 11,718.

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

Buffers

HiLo HiHi
20 mM Tris-Cl pH 8.5
1 mM EGTA
1 mM DTT
 		 20 mM Tris-Cl pH 8.5
1 mM EGTA
1 mM DTT
500 mM NaCl
RPG 2000/07/12

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