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Protein production by auto-induction in high-density shaking cultures》.pdf
Protein Expression and Purification 41 (2005) 207–234
/locate/yprep
Protein production by auto-induction in high-density shaking cultures
F. William Studier *
Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA
Received 7 January 2005
Available online 12 March 2005
Abstract
Inducible expression systems in which T7 RNA polymerase transcribes coding sequences cloned under control of a T7lac pro-
moter efficiently produce a wide variety of proteins in Escherichia coli. Investigation of factors that affect stability, growth, and
induction of T7 expression strains in shaking vessels led to the recognition that sporadic, unintended induction of expression in com-
plex media, previously reported by others, is almost certainly caused by small amounts of lactose. Glucose prevents induction by
lactose by well-studied mechanisms. Amino acids also inhibit induction by lactose during log-phase growth, and high rates of aer-
ation inhibit induction at low lactose concentrations. These observations, and metabolic balancing of pH, allowed development of
reliable non-inducing and auto-inducing media in which batch cultures grow to high densities. Expression strains grown to satura-
tion in non-inducing media retain plasmid and remain fully viable for weeks in the refrigerator, making it easy to prepare many
freezer stocks in parallel and use working stocks for an extended period. Auto-induction allows efficient screening of many clones
in parallel for expression and solubility, as cultures have only to be inoculated and grown to saturation, and yields of
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