Home Protocols Protein Expression Using Lemo21(DE3) (C2528)

Protein Expression Using Lemo21(DE3) (C2528)

Protocol

  1. Transform expression plasmid into Lemo21(DE3). Transformation plates must contain antibiotic to select the expression plasmid and 30µg/ml chloramphenicol to maintain pLemo, a pACYC184 derivative with a p15A origin of replication. Incubate overnight at 37°C.
  2. Resuspend a single colony in a 1 or 2 ml liquid culture with both antibiotics and grow overnight to produce a starter culture. Use media without glucose for optimal strain performance.
  3. To sample different expression levels, set up 10 ml expression cultures at the beginning of day 2 with various levels of L-rhamnose: for example 0, 100, 250, 500, 750, 1,000 and 2,000 µM for difficult targets. Inoculate each expression culture with 0.2 ml of starter culture.
  4. Incubate at 37°C until OD600 reaches 0.4–0.8.
  5. Induce with 40 µl of a 100 mM stock of IPTG (final concentration of 400 µM). IPTG should not be varied, only L-rhamnose concentration is varied. Induce for 5 hours to overnight at 30 or 37°C.
  6. Check for expression either by Coomassie stained protein gel, Western Blot or activity assay. Check expression in both the total cell extract (soluble + insoluble) and the soluble fraction only. In the case of over-expressed membrane protein, most of the target should be in the low-speed spin supernatant after cell breakage by French Press, cell disruption or sonication (in combination with EDTA-lysozyme treatment).
    *If a significant fraction of the target protein is insoluble (low speed pellet), repeat expression at a lower temperature (30°C). Membrane protein expression may be improved by early induction (OD600 = 0.4) at 20 to 25°C.
  7. For large scale, prepare 1 Liter of liquid medium with antibiotics and the optimal level of L-rhamnose determined in a small scale trial. Inoculate with a freshly grown colony or 10 ml of freshly grown culture. Incubate at 37°C until OD600 reaches 0.4 - 0.8. Add 400 µM IPTG and express protein using optimal time/temperature determined in a small scale trial.