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Cold-shock induced high-yield protein production in Escherichia coli

Nature Biotechnology volume 22, pages 877–882 (2004)Cite this article

Abstract

Overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability1,2. Here, we apply the unique features of the cspA gene to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock. Several proteins were produced with very high yields, including E. coli EnvZ ATP-binding domain (EnvZ-B) and Xenopus laevis calmodulin (CaM). The pCold vector system can also be used to selectively enrich target proteins with isotopes to study their properties in cell lysates using NMR spectroscopy. We have cloned 38 genes from a range of prokaryotic and eukaryotic organisms into both pCold and pET14 (ref. 3) systems, and found that pCold vectors are highly complementary to the widely used pET vectors.

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Figure 1: Structures of pCold vectors I, II, III and IV.
Figure 2: SDS-PAGE patterns of E. coli EnvZ ATP-binding domain (EnvZ-B), X. laevis calmodulin (CaM) and E. coli trigger factor expressed using pCold vectors.
Figure 3: 600 MHz 15N-HN HSQC spectra of E. coli EnvZ-B.
Figure 4: 600 MHz NMR spectra of X. laevis CaM.
Figure 5: Summary of triple-resonance NMR data obtained on whole-cell lysates establishing sequence-specific resonance assignments for some 80% of 15N, HN, Hα, 13Cα, 13Cβ and 13C′ nuclei.

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Acknowledgements

We thank Y. Chiang for helpful discussions. This work was supported by NIH grants R21-GM067061 (to M.I.) and P50-GM62413 (to G.T.M.), a grant from Takara-Bio Inc., Japan (to M.I.), and a Canadian Institutes of Health Research (CIHR) grant (to M.I.). M.I. is a CIHR senior investigator.

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Authors and Affiliations

  1. Department of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, 08854, New Jersey, USA

    Guoliang Qing, Bing Xia, Sangita Phadtare, Haiping Ke, Gaetano T Montelione & Masayori Inouye

  2. Northeast Structural Genomics Consortium, Rutgers University, 679 Hoes Lane, Piscataway, 08854, New Jersey, USA

    Li-Chung Ma, G V T Swapna, Thomas Acton, Gaetano T Montelione & Masayori Inouye

  3. Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine (CABM), Rutgers University, 679 Hoes Lane, Piscataway, 08854, New Jersey, USA

    Li-Chung Ma, G V T Swapna, Thomas Acton & Gaetano T Montelione

  4. Division of Molecular and Structural Biology, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, M5G 2M9, Ontario, Canada

    Ahmad Khorchid, Tapas K Mal & Mitsuhiko Ikura

  5. Takara Bio Inc., Otsu, 520-2193, Shiga, Japan

    Masanori Mitta Takayama

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  1. Guoliang Qing
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Correspondence to Masayori Inouye.

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Supplementary information

Supplementary Fig. 1

SDS-PAGE patterns of FR genes expressed in pET14 and pColdI vectors (see Table 1) (PDF 140 kb)

Supplementary Table 1

Summary of triple-resonance NMR experiments and key parameters used for determining backbone resonance assignments for Xenopus calmodulin in whole-cell lysate. (PDF 7 kb)

Supplementary Table 2

Sequence specific backbone 1H, 15N, and 13C, and 13Cβ resonance assignments a for Xenopus calmodulin at pH 6.8 and temperature of 10 °C, determined by triple resonance NMR experiments on whole-cell lysates. (PDF 11 kb)

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Qing, G., Ma, LC., Khorchid, A. et al. Cold-shock induced high-yield protein production in Escherichia coli. Nat Biotechnol 22, 877–882 (2004). https://doi.org/10.1038/nbt984

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