Prbbbb:large scale expression v1

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Overview

This protocol describes how to express larger protein amounts from a 1l culture. The constructs are supposed to be under control of the T7 promoter. BL21 strains contain a genomic T7 polymerase that is under the control of the Lac repressor -- it is induced by IPTG and leaky expression is repressed by adding extra Glucose.

Culture Growth

If not mentioned otherwise, the medium must contain the appropriate antibiotic (or both if the plasmid contains two resistances).

day 1

  1. prepare LB (+appropriate antibiotic) Agar plates with Glucose added to 1% final concentration
    • (not Ampicillin alone, if possible -- Amp increases risk of plasmid loss)
    • (prepare stock of sterile-filtered 20% Glucose)
  2. re-streak BL21DE3 strains with your expression constructs to get fresh single colonies
    • grow over night @ 37°C but avoid overgrowing clones -- 13 or 14 h are best

day 2

  1. inoculate a single colony into 10-20 ml LB (or 2xTY) + 1% Glucose + (both) Antibiotic(s)
  2. grow starting culture over night @ 37 °C

day 3

  1. wash off beta-Lactamase and other secretions
    • pellet cells by centrifugation
    • re-suspend in same or lower amount of fresh medium
  2. inoculate 1l 2xTY + 1% Glucose + Antibiotic(s) from over night culture at 1:100 dilution
  3. incubate shaking @ 37°C until OD600 reaches 0.5 (usually 2-3 h)
  4. induce expression: add IPTG to final concentration of 0.1 - 1 mM (0.5mM as a start)
    • lower concentrations may be preferable for reduced stress-response
  5. let induced cultures grow 3h @ 37 C with shaking
  6. measure OD600 of cultures
  7. backup 50 µl culture for sequencing
  8. pellet by centrifugation
  9. store pellet @ -20°C

Modifications for lower temperature growth

  • grow cells at low temperatures before and after induction
  • shake over night after induction
  • reduce IPTG to ...
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