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.

  • Research Article
  • Published:

Creating a bifunctional protein by insertion of β-lactamase into the maltodextrin-binding protein

Nature Biotechnology volume 15, pages 1276–1279 (1997)Cite this article

Abstract

Hybrid proteins were generated by inserting the penicillin-hydrolyzing enzyme, TEM β-lactamase (Bla), into the maltodextrin-binding protein (MalE). The inserted Bla was functionally accommodated by MalE when it was placed within permissive sites. The maltose binding and penicillinase activities of purified hybrids were indistinguishable from those of the wild-type MalE and Bla proteins. Moreover, these hybrids displayed an additional unexpected property: maltose stabilized the active site of inserted Bla.

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

Similar content being viewed by others

References

  1. Uhlèn, M. and Moks, T. 1990. Gene fusions for purpose of expression: an introduction. Methods Enzymol. 185: 129–143.

    Article  PubMed  Google Scholar 

  2. Nilsson, B., Forsberg, G.T., Moks, T.M., Hartmanis, M., and Uhlèn, M. 1992. Fusion proteins in biotechnology and structural biology. Curr. Opin. Struct. Biol. 2: 569–575.

    Article  CAS  Google Scholar 

  3. Wetlaufer, D.B. 1973. Nucreation, rapid folding, and globular intrachain regions in proteins. Proc. NatI. Acad. Sci. USA 70: 697–701.

    Article  CAS  Google Scholar 

  4. Russell, R.B. 1994. Domain insertion. Protein Eng. 7: 1407–1410.

    Article  CAS  PubMed  Google Scholar 

  5. Bork, P., Downing, K.A., Kieffer, B., and Campbell, I.D. 1996. Concept of modules and domains. Q. Rev. Biophys. 29: 119–167.

    Article  CAS  PubMed  Google Scholar 

  6. Nikaido, H. 1994. Maltose transport system of Escherichia coli: an ABC-type transporter. FEBS Lett. 346: 55–58.

    Article  CAS  PubMed  Google Scholar 

  7. Spurlino, J.C., Lu, G.Y. and Quiocho, F.A. 1991. The 2.3-Å resolution structure of the maltose-or maltodextrin-binding protein, a primary receptor of bacterial active transport and chemotaxis. J. Biol. Chem. 266: 5202–5219.

    CAS  PubMed  Google Scholar 

  8. Sharff, A.J., Rodseth, L.E., Spurlino, J.C., and Qiocho, F.A. 1992. Crystallographic evidence of a large ligand-induced hinge-twist motion between the two domains of the maltodextrin binding protein involved in active transport and chemotaxis. Biochemistry 31: 10657–10663.

    Article  CAS  PubMed  Google Scholar 

  9. Duplay, P., Szmelcman, S., Bedouelle, H., and Hofnung, M. 1987. Silent and functional changes in the periplasmic maltose-binding protein of Escherichia coli K12. J. Mol. Biol. 194: 663–673.

    Article  CAS  PubMed  Google Scholar 

  10. Betton, J.-M., Martineau, P., Saurin, W., and Hofnung, M. 1993. Location of tolerated insertions/deletions in the structure of the maltose binding protein. FEBS Lett. 325: 34–38.

    Article  CAS  PubMed  Google Scholar 

  11. Betton, J.-M. and Hofnung, M. 1994. In vivo assembly of active maltose binding protein from independently exported protein fragments. EMBO J. 13: 1226–1234.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Frère, J.M. 1995. Beta-lactamases and bacterial resistance to antibiotics. Mol. Microbiol. 16: 385–395.

    Article  PubMed  Google Scholar 

  13. Jelsch, C., Lenfant, F., Masson, J.M., and Samama, J.P. 1992. β-lactamaseTEM1 of E. coli: crystal stucture at 2.5 Å resolution. FEBS Lett. 299: 135–142.

    Article  CAS  PubMed  Google Scholar 

  14. Jelsch, C., Mourey, L., Masson, J.-M., and Samama, J.-P. 1993. Crystal structure of Escherichia coli TEM1 β-lactamase at 1.8 Å resolution. Proteins: Struct Funct. & Genet. 16: 364–383.

    Article  CAS  Google Scholar 

  15. Martineau, P., Guillet, J.-G., Leclerc, C., and Hofnung, M. 1992. Expression of heterologous peptides at two permissive sites of the MalE protein: antigenicity and immunogenicity of foreign B-cell and T-cell epitopes. Gene 113: 35–46.

    Article  CAS  PubMed  Google Scholar 

  16. Sharff, A.J., Rodseth, L.E., Szmelcman, S., Hofnung, M., and Quiocho, F.A. 1995. Refined structures of two insertion/deletion mutants probe function of the maltodextrin binding protein. J. Mol. Biol. 246: 8–13.

    Article  CAS  PubMed  Google Scholar 

  17. Martineau, P., Leclerc, C., and Hofnung, M. 1997. Modulating the immunological properties of a linear B-cell epitope by insertion into permissve sites of the MalE protein. Mol. Immun. 33: 1345–1358.

    Article  Google Scholar 

  18. Shuman, H.A. 1982. Active transport of maltose in Escherichia coli K12. J. Biol. Chem. 257: 5455–5461.

    CAS  PubMed  Google Scholar 

  19. Baneyx, F. and Georgiou, G., 1989. Expression, purification, and enzymatic characterization of a protein A β-lactamase hybrid protein. Enzyme Microb. Technol. 11: 559–567.

    Article  CAS  Google Scholar 

  20. Betton, J.-M. and Hofnung, M. 1996. Folding of a mutant maltose binding protein of E.coli which forms inclusion bodies. J. Biol. Chem. 271: 8046–8052.

    Article  CAS  PubMed  Google Scholar 

  21. Sigal, I.S., DeGrado, W.F., Thomas, B.J., and Petteway, S.R. 1984. Purification and properties of thiol β-lactamase. J. Biol. Chem. 259: 5327–5332.

    CAS  PubMed  Google Scholar 

  22. Laminet, A.A. and Pluckthun, A. 1989. The precursor of β-lactamase: purification, properties and folding kinetics. EMBO J. 8: 1469–1477.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bülow, L. and Mosbach, K. 1991. Multienzyme systems obtained by gene fusion. Trends Biotech 9: 226–231.

    Article  Google Scholar 

  24. Manoil, C. and Bailey, J. 1997. A simple screen for permissive sites in proteins: analysis of Escherichia coli lacpermease. J. Mol. Biol. 267: 250–263 .

    Article  CAS  PubMed  Google Scholar 

  25. Duplay, P., Bedouelle, H., Fowler, A., Zabin, I., Saurin, W., and Hofnung, M. 1984. Sequences of the malEgene and its product, the maltose-binding protein of Escherichia coliK12. J. Biol. Chem. 259: 10606–10613.

    CAS  PubMed  Google Scholar 

  26. Zhang, Y. and Broome-Smith, J.K. 1990. Correct insertion of a simple eukaryot-ic plasma-membrane protein into the cytoplasmic membrane of Escherichia coli. Gene 96: 51–57.

    Article  CAS  PubMed  Google Scholar 

  27. Ferenci, T. and Klotz, U. 1978. Affinity chromatographic isolation of the periplam-ic maltose-binding protein of Escherichia coli. FEBS Lett. 94: 213–217.

    Article  CAS  PubMed  Google Scholar 

  28. Melling, J. and Scott, G.K. 1972. Preparation of gram quantities of a purified R-factor-mediated penicillinase from Escherichia colistrain W3310. Biochem. J. 130: 55–62.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Pace, C.N., Vajdos, F., Fee, L., Grimsley, G. and Gray, T. 1995. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4: 2411–2423.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. O'Callaghan, C.H., Morris, A., Kirby, S.M. and Shingler, A.H. 1972 1972. Novel method for detection of β-lactamases by using a chromogenic cephalosporin substrate. Antimicrob. Agents Chemother. 1: 283–288.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Mitchinson, C. and Pain, R.H. 1985 1985. Effects of sulfate and urea on the stability and reversible unfolding of β-lactamase from Staphylococcus aureus. J. Mol. Biol. 184: 331–342.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Unité de Programmation Moléculaire et Toxicologie Génétique, Département des Biotechnologies, Institut Pasteur–CNRS URA1444, 25 rue du DocteurRoux, 75015, Paris, France

    Jean-Michel Betton & Maurice Hofnung

  2. Biochemistry Group, School of Biological Sciences, University of Sussex, Palmer, Brighton, BN1 9QG, UK

    Jansen P. Jacob & Jenny K. Broome-Smith

Authors
  1. Jean-Michel Betton
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jansen P. Jacob
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maurice Hofnung
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jenny K. Broome-Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Jean-Michel Betton or Maurice Hofnung.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Betton, JM., Jacob, J., Hofnung, M. et al. Creating a bifunctional protein by insertion of β-lactamase into the maltodextrin-binding protein. Nat Biotechnol 15, 1276–1279 (1997). https://doi.org/10.1038/nbt1197-1276

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1197-1276

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