Protein production by auto-induction in high-density shaking cultures
Section snippets
Background and introduction
DNA sequencing projects have provided coding sequences for hundreds of thousands of proteins from organisms across the evolutionary spectrum. Recombinant DNA technology makes it possible to clone these coding sequences into expression vectors that can direct the production of the corresponding proteins in suitable host cells. An inducible T7 expression system is highly effective and widely used to produce RNAs and proteins from cloned coding sequences in the bacterium Escherichia coli [1, 2].
Bacterial strains and plasmids
Escherichia coli strains used for testing growth and expression were primarily BL21(DE3) and B834(DE3). B834 is a restriction-modification defective, galactosenegative, methionine auxotroph of E. coli B [10]. BL21is a Met+ derivative of B834 obtained by P1 transduction [1]. DE3 lysogens contain a derivative of phage lambda that supplies T7 RNA polymerase by transcription from the lacUV5 promoter in the chromosome [1]. BL21-AI (Invitrogen) is a derivative of BL21 that supplies T7 RNA polymerase
Growth of shaking cultures to high density
Shaking cultures are convenient for growing many cultures in parallel, and rapid growth to high densities is desirable for maximizing the yield and efficiency of producing target proteins. Complex media containing enzymatic digests of casein and yeast extract are extensively used because they support growth of a wide range of E. coli strains with different nutritional requirements, and cultures typically grow 2–3 times faster than in simple mineral salts media with glucose as the sole carbon
Non-inducing media
Besides our new barrel of N-Z-amine, a sample of Bacto tryptone (Difco) also had inducing activity, suggesting that inducing activity may be fairly common in enzymatic digests of casein. Addition of excess glucose to complex media that have inducing activity prevents induction of target protein [6], but cultures eventually become acid enough to stop growth and can lose viability. At intermediate glucose concentrations, cultures became induced if the pH rose at saturation, indicating that
Unintended induction is almost certainly due to lactose in the medium
Media made with N-Z-amine from the old barrel did not have inducing activity. Apparently, something in the new N-Z-amine was causing induction (rather than something in the old N-Z-amine preventing induction) because increasing the concentration of new N-Z-amine in the medium also increased the inducing activity, as judged by 4107 plaque size and time of appearance (Table 2). Grossman et al. [6] had concluded that unintended induction was not due to the presence of lactose in the medium.
High-density cultures for preparation of plasmids
The high-density culture conditions developed for auto-induction also are convenient for preparation of plasmid DNAs. Rich media such as ZYM-505 support growth of the plasmid-containing strains we work with to culture densities of A600 ∼10 or higher when 1.5–2.5 ml culture is grown in an 18 × 150 mm tube shaken at 300–350 rpm. Lactose is omitted unless auto-induction is desired. The presence of 0.05% glucose ensures rapid initial growth with little lag. Typically, yields of plasmid DNA have
Discussion
The phenomenon of unintended induction was sporadic, being found in some lots of complex media but not others [6]. Furthermore, different portions of the same culture might produce widely different levels of target protein, depending on the rate of aeration (Table 8). The realization that lactose is responsible for unintended induction made it possible to develop non-inducing media in which T7 expression strains remain stable and viable all the way to saturation, and reliable auto-inducing
Acknowledgments
I am grateful for the enthusiasm and expert technical support of my co-workers and colleagues. Clones for expressing yeast proteins for structural genomics were constructed and tested by IPTG induction for expression and solubility by Sue-Ellen Gerchman, with help in the later stages from Eileen Matz, who constructed the clones of T7 and human proteins. Auto-induction and purification of normal and SeMet-labeled yeast proteins was by Helen Kycia. Nancy Manning performed innumerable gel
References (44)
- et al.
Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes
J. Mol. Biol.
(1986) Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system
J. Mol. Biol.
(1991)- et al.
Regulation of sCD4-183 gene expression from phage-T7-based vectors in Escherichia coli
Gene
(1995) - et al.
Spontaneous cAMP-dependent derepression of gene expression in stationary phase plays a role in recombinant expression instability
Gene
(1998) - et al.
Regulation of coliphage T3 and T7 RNA polymerases by the lac repressor-operator system
Gene
(1989) - et al.
On the mechanism of inhibition of phage T7 RNA polymerase by lac repressor
J. Mol. Biol.
(1998) - et al.
Vectors for selective expression of cloned DNAs by T7 RNA polymerase
Gene
(1987) - et al.
Expression of chicken linker histones in E. coli: Sources of problems and methods for overcoming some of the difficulties
Protein Expr. Purif.
(1994) Flux analysis and control of the central metabolic pathways in Escherichia coli
FEMS Microbiol. Rev.
(1996)- et al.
Lactose fed-batch overexpression of recombinant metalloproteins in Escherichia coli BL21 (DE3): Process control yielding high levels of metal-incorporated, soluble protein
Protein Expr. Purif.
(1995)
Thermodynamic studies of the mechanism of metal binding to the Escherichia coli zinc transporter YiiP
J. Biol. Chem.
S-ribosylhomocysteine cleavage enzyme from Escherichia coli
J. Biol. Chem.
Generation of an AraC-araBAD promoter-regulated T7 expression system
Anal. Biochem.
Use of T7 RNA polymerase to direct expression of cloned genes
Methods Enzymol.
Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor
J. Mol. Biol.
Structural genomics: Beyond the human genome project
Nat. Genet.
Host specificity of DNA produced by Escherichia coli: Bacterial mutations affecting the restriction and modification of DNA
J. Mol. Biol.
Structure of a yeast hypothetical protein selected by a structural genomics approach
Acta Crystallogr. D Biol. Crystallogr.
Structure and mechanism of ADP-ribose-1″-monophosphatase (Appr-1″-pase), a ubiquitous cellular processing enzyme
Protein Sci
Molecular Cloning: A Laboratory Manual
The bacterial phosphoenolpyruvate: Glycose phosphotransferase system
Annu. Rev. Biochem.
Phosphoenolpyruvate: Carbohydrate phosphotransferase systems
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