Protein production by auto-induction in high-density shaking cultures

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Abstract

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).

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)

  • Y. Chao et al.

    Thermodynamic studies of the mechanism of metal binding to the Escherichia coli zinc transporter YiiP

    J. Biol. Chem.

    (2004)
  • C.H. Miller et al.

    S-ribosylhomocysteine cleavage enzyme from Escherichia coli

    J. Biol. Chem.

    (1968)
  • D.R. Wycuff et al.

    Generation of an AraC-araBAD promoter-regulated T7 expression system

    Anal. Biochem.

    (2000)
  • F.W. Studier et al.

    Use of T7 RNA polymerase to direct expression of cloned genes

    Methods Enzymol.

    (1990)
  • J.W. Dubendorff et al.

    Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor

    J. Mol. Biol.

    (1991)
  • S.K. Burley et al.

    Structural genomics: Beyond the human genome project

    Nat. Genet.

    (1999)
  • W.B. Wood

    Host specificity of DNA produced by Escherichia coli: Bacterial mutations affecting the restriction and modification of DNA

    J. Mol. Biol.

    (1966)
  • S. Eswaramoorthy et al.

    Structure of a yeast hypothetical protein selected by a structural genomics approach

    Acta Crystallogr. D Biol. Crystallogr.

    (2003)
  • D. Kumaran et al.

    Structure and mechanism of ADP-ribose-1″-monophosphatase (Appr-1″-pase), a ubiquitous cellular processing enzyme

    Protein Sci

    (2005)
  • J. Sambrook et al.

    Molecular Cloning: A Laboratory Manual

    (1989)
  • N.D. Meadow et al.

    The bacterial phosphoenolpyruvate: Glycose phosphotransferase system

    Annu. Rev. Biochem.

    (1990)
  • P.W. Postma et al.

    Phosphoenolpyruvate: Carbohydrate phosphotransferase systems

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