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  • 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.