Protein Expression Using BL21(DE3), Large Scale (C2527)
New England Biolabs
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.
