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Single protein production (SPP) system in Escherichia coli

Nature Protocols volume 2, pages 18021810 (2007) | Download Citation

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Abstract

Here, we provide a detailed protocol for the single protein production (SPP) system, which is designed to produce only a single protein of interest in living Escherichia coli cells. Induction of MazF, an mRNA interferase that cleaves RNA at ACA nucleotide sequences, results in complete cell growth arrest. However, if mRNA encoding a protein of interest is engineered to be devoid of ACA base triplets and is induced at 15 °C using pCold vectors in MazF-expressing cells, only the protein from this mRNA is produced at a yield of 20–30% of total cellular protein; other cellular protein synthesis is almost completely absent. In theory, any protein can be produced by the SPP system. Protein yields are typically unaffected even if the culture is condensed up to 40-fold, reducing the cost of protein production by up to 97.5%. The SPP system has a number of key features important for protein production, including high-yield and prolonged production of isotope-labeled protein at a very high signal-to-noise ratio. The procedure can be completed in 7 d after cloning of an ACA-less target gene into the expression system.

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Acknowledgements

We thank Dr. G. Montelione for his valuable discussions and Dr. S. Phadtare for critical reading of the manuscript.

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Affiliations

  1. Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.

    • Motoo Suzuki
    • , Lili Mao
    •  & Masayori Inouye

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Competing interests

Dr. Inouye is a member of the board of directors of Takara Bio Inc.

Corresponding author

Correspondence to Masayori Inouye.

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https://doi.org/10.1038/nprot.2007.252

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