Protein production by auto-induction in high density shaking cultures.
Inducible expression systems in which T7 RNA polymerase transcribes coding sequences cloned under control of a T7lac promoter 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 complex 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 aeration 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 saturation 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 target protein are typically several-fold higher than obtained by conventional IPTG induction. Auto-inducing media have been developed for labeling proteins with selenomethionine, 15N or 13C, and for production of target proteins by arabinose induction of T7 RNA polymerase from the pBAD promoter in BL21-AI. Selenomethionine labeling was equally efficient in the commonly used methionine auxotroph B834(DE3) (found to be metE) or the prototroph BL21(DE3).
Studier
F William
FW
Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA. studier@bnl.gov
D000596:Amino Acids / Q000494:pharmacology; D001431:Bacteriological Techniques; D015169:Colony Count, Microbial; D003470:Culture Media; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D004734:Energy Metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007544:Isopropyl Thiogalactoside; D007785:Lactose / Q000494:pharmacology; D010710:Phosphates; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D003470:Culture Media; D010710:Phosphates; D011994:Recombinant Proteins; D014764:Viral Proteins; D007544:Isopropyl Thiogalactoside; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D007785:Lactose