Protein Expression Using BL21(DE3), Large Scale (C2527)

Abstract

This is the protocol for a large-scale Protein Expression Using BL21(DE3) Competent E. coli cells (C2527).

Before start

Determine the optimal time/temperature for the protein expression in a small scale trial.

Steps

Transform expression plasmid into BL21(DE3).

Plate on antibiotic selection plates.

Incubate at 37°C.

Inoculate 1L with a freshly grown colony or with 10mL.

Incubate at 37°C until OD₆₀₀ reaches 0.4–0.8.

Induce with 4µL or 40µL of 100millimolar (mM) (final concentration of 40micromolar (µM) or 400micromolar (µM)).

Express protein using optimal time/temperature determined in a small scale trial (3 to 5h 0m 0s at 37°C).

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.

Note

If a fraction of the target protein is insoluble, repeat expression at a lower temperature (15 to 30°C) or test expression in Lemo21(DE3) (If a fraction of the target protein is insoluble, repeat expression at a lower temperature (15 to 30°C) or test expression in Lemo21(DE3) (NEB #C2528).).

Abstract

Before start

Steps

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