Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Strategies for protein coexpression in Escherichia coli

Nature Methods volume 3, pages 55–64 (2006)Cite this article

  • Calf intestinal phosphatase (NEB)

  • Complete protease inhibitor cocktail (Roche)

  • Deoxynucleoside triphosphate (dNTP) solution (each at 10 mM)

  • E. coli competent cells, DH5α and BL21 (DE3)

  • Isopropylthiogalactoside (IPTG; 1 M)

  • Luria broth (LB) agar and liquid medium

  • Lysozyme (1 mg/ml)

  • Pfu DNA polymerase and 10× Pfu buffer (Stratagene)

  • PCR primers (see Step 1 for details)

  • QIAprep Spin Miniprep Kit (Qiagen)

  • QIAprep Gel Extraction Kit (Qiagen)

  • QIAquick Nucleotide Removal Kit (Qiagen)

  • Rapid Ligation Kit (Roche) or T4 DNA ligase with ligase buffer (NEB)

  • Restriction endonucleases, appropriate for the vectors (NEB)

  • Selective antibiotics (ampicillin, chloramphenicol, streptomycin and/or kanamycin)

  • TBS (100 mM Tris-HCl (pH 8.0), 250 mM NaCl), ice-cold

  • Template DNA (plasmid or genomic cDNA)

  • Vector DNA: pETDuet-1, pACYCDuet-1, pCDFDuet-1, pRSFDuet-1 and/or pCOLADuet-1 (Novagen)

  • Additional vectors: pGEX (any, Amersham Biosciences), pMAL (any, New England BioLabs)

Calf intestinal phosphatase (NEB)

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2: Example of application.

References

  1. Li, C., Schwabe, J.W., Banayo, E. & Evans, R.M. Coexpression of nuclear receptor partners increases their solubility and biological activities. Proc. Natl. Acad. Sci. USA 94, 2278–2283 (1997).

    Article  CAS  Google Scholar 

  2. Rivas, F.V. et al. Purified Argonaute2 and an siRNA form recombinant human RISC. Nat. Struct. Mol. Biol. 12, 340–349 (2005).

    Article  CAS  Google Scholar 

  3. Bross, P. et al. Co-overexpression of bacterial GroESL chaperonins partly overcomes non-productive folding and tetramer assembly of E. coli–expressed human medium-chain acyl-CoA dehydrogenase (MCAD) carrying the prevalent disease-causing K304E mutation. Biochim. Biophys. Acta 1182, 264–274 (1993).

    Article  CAS  Google Scholar 

  4. Woestenenk, E.A., Hammarstrom, M., van den Berg, S., Hard, T. & Berglund, H. His tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors. J. Struct. Funct. Genomics 5, 217–229 (2004).

    Article  CAS  Google Scholar 

  5. Kapust, R.B. & Waugh, D.S. Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci. 8, 1668–1674 (1999).

    Article  CAS  Google Scholar 

  6. Selzer, G., Som, T., Itoh, T. & Tomizawa, J. The origin of replication of plasmid p15A and comparative studies on the nucleotide sequences around the origin of related plasmids. Cell 32, 119–129 (1983).

    Article  CAS  Google Scholar 

  7. Davison, J. Mechanism of control of DNA replication and incompatibility in ColE1-type plasmids—a review. Gene 28, 1–15 (1984).

    Article  CAS  Google Scholar 

  8. Ho, T.Q., Zhong, Z., Aung, S. & Pogliano, J. Compatible bacterial plasmids are targeted to independent cellular locations in Escherichia coli. EMBO J. 21, 1864–1872 (2002).

    Article  CAS  Google Scholar 

  9. Song, J.J., Smith, S.K., Hannon, G.J. & Joshua-Tor, L. Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305, 1434–1437 (2004).

    Article  CAS  Google Scholar 

  10. Liu, J. et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441 (2004).

    Article  CAS  Google Scholar 

  11. Meister, G. et al. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol. Cell 15, 185–197 (2004).

    Article  CAS  Google Scholar 

  12. Held, D., Yaeger, K. & Novy, R. New coexpression vectors for expanded compatibilities in E. coli. in Novations 18, 4–6 (2003).

    Google Scholar 

Download references

Acknowledgements

We thank R.-M. Xu for constructive conversations on coexpression, E. Enemark and D. Chitwood for comments on the manuscript, and members of the Joshua-Tor laboratory for helpful discussions. This work was supported by National Institutes of Health grant GM072689 (to L.J.).

Author information

Authors and Affiliations

  1. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, 11724, New York, USA

    Niraj H Tolia & Leemor Joshua-Tor

Authors
  1. Niraj H Tolia
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Leemor Joshua-Tor
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Niraj H Tolia.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tolia, N., Joshua-Tor, L. Strategies for protein coexpression in Escherichia coli. Nat Methods 3, 55–64 (2006). https://doi.org/10.1038/nmeth0106-55

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth0106-55

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing