Advertisement

Molecular Biotechnology

, Volume 15, Issue 1, pp 51–63 | Cite as

Expression and purification of recombinant proteins by fusion to maltose-binding protein

  • Paul Riggs
Protocol

Abstract

The pMAL vectors provide a method for purifying proteins from cloned genes by fusing them to maltose-binding protein (MBP, product of malE), which binds to amylose. The vectors use the tac promoter and the translation initiation signals of MBP to give high-level expression of the fusion, and an affinity purification for MBP to isolate the fusion protein. The pMAL polylinkers carry restriction sites to insert the gene of interest, and encode a site for a specific protease to separate MBP from the target protein after purification. Vectors with or without the malE signal sequence can be used, to express the protein cytoplasmically for the highest level of production or periplasmically to help in proper folding of disulfide-bonded proteins.

Index Entries

Primer restriction genomic DNA viral RNA PCR RT-PCR amplification 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Guan, C., Li, P., Riggs, P. D. and Inouye, H. (1987) Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein. Gene 67, 21–30.Google Scholar
  2. 2.
    Maina, C. V., Riggs, P. D., Grandea, A. G. III, Slatko, B. E., Moran, L. S., Tagliamonte, J. A., McReynolds, L. A. and Guan, C. (1988) A vector to express and purify foreign proteins in Escherichia coli by fusion to, and separation from, maltose binding protein. Gene 74, 365–373.PubMedCrossRefGoogle Scholar
  3. 3.
    Amann, E. and Brosius, J. (1985). ‘ATG vectors’ for regulated high-level expression of cloned genes in Escherichia coli. Gene 40, 183–190.PubMedCrossRefGoogle Scholar
  4. 4.
    Duplay, P., Bedouelle, H., Fowler, A., Zabin, I., Saurin, W. and Hofnung, M. (1984). Sequences of the malE gene and of its product, the maltose-binding protein of Escherichia coli K12. J. Biol. Chem. 259, 10,606–10,613.Google Scholar
  5. 5.
    Kellerman, O. K. and Ferenci, T. (1982) Maltose binding protein from E. coli. Methods in Enzymology 90, 459–463.CrossRefGoogle Scholar
  6. 6.
    Johnston, T. C., Thompson, R. B., and Baldwin, T. O. (1986). Nucleotide sequence of the luxB gene of Vibrio harveyi and the complete amino acid sequence of the beta subunit of bacterial luciferase. J. Biol. Chem. 261, 4805–4811.PubMedGoogle Scholar
  7. 7.
    Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103–119.PubMedCrossRefGoogle Scholar
  8. 8.
    Nagai, K. and Thøgersen, H. C. (1984) Generation of b-globin by sequence-specific proteolysis of a hybrid protein produced in Escherichia coli. Nature 309, 810–812.PubMedCrossRefGoogle Scholar
  9. 9.
    Nagai, K. and Thøgersen, H. C. (1987) Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherichia coli. Methods in Enzymol. 153, 461–481.Google Scholar
  10. 10.
    Collins-Racie, L. A., McColgan, J. M., Grant, K. L., DiBlasio-Smith, E. A., McCoy, J. M., and LaVallie, E. R. (1995) Production of recombinant bovine enterokinase catalytic subunit in Escherichia coli using the novel secretory fusion partner DsbA. Biotechnology 13, 982–987.PubMedCrossRefGoogle Scholar
  11. 11.
    Carter, P., Nilsson, B., Burnier, J. P., Burdick, D., and Wells, J. A. (1989) Engineering subtilisin BPN’ for site-specific proteolysis. Proteins, 6, 240–248.PubMedCrossRefGoogle Scholar
  12. 12.
    Matthews, D. J., and Wells, J. A. (1993) Substrate phage: selection of protase substrates by monovalent phage display. Science, 260, 1113–1117.PubMedCrossRefGoogle Scholar
  13. 13.
    Lauritzen, C., Tüchsen, E., Hansin, P.E., and Skovgaard, O. (1991) BPTI and N-terminal extended analogs generated by Factor Xa cleavage and cathepsin C trimming of a fusion protein expressed in Escherichia coli. Protein Expression and Purification 2, 372–378.PubMedCrossRefGoogle Scholar
  14. 14.
    Takagi, H., Morinaga, Y., Tsuchiya, M., Ikemura, G., and Inouye, M. (1988) Control of folding of proteins secreted by a high expression secretion vector, pIN-III-ompA: 16-fold increase in production of active subtilisin E in Escherichia coli. Bio/technology 6, 948–950.CrossRefGoogle Scholar
  15. 15.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Preparation and transformation of competent E. coli, in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 1.74–1.84.Google Scholar
  16. 16.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Small-scale preparations of plasmid DNA, in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 1.25–1.28.Google Scholar
  17. 17.
    Bloch, K. D. (1989) Digestion of DNA with restriction endonucleases, in Current Protocols in Molecular Biology (Ausebel, F.M. et al. eds). Greene Publishing & Wiley-Interscience, New York, NY, pp. 3.1.1–3.2.5.Google Scholar
  18. 18.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) SDS-polyacrylamide gel electrophoresis of proteins, in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 18.47–18.55.Google Scholar
  19. 19.
    Smith, J. A. (1989) One-dimensional gel electrophoresis of proteins, in Current Protocols in Molecular Biology (Ausebel, F.M. et al. eds.), Greene Publishing & Wiley-Interscience, New York, NY, pp. 10.2.1–10.2.7.Google Scholar
  20. 20.
    Sasse, J. (1989) Detection of proteins in gels, in Current Protocols in Molecular Biology (Ausebel, F.M. et al. eds). Greene Publishing & Wiley-Interscience, New York, NY, pp. 10.6.1–10.6.3.Google Scholar
  21. 21.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Transfer of proteins from SDS-polyacrylamide gels to solid supports: immunological detection of immobilized proteins (Western blotting), in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 18.60–18.75.Google Scholar
  22. 22.
    Winston, S. E., Fuller, S. A. and Hurrell, J. G. R. (1989) Western Blotting, in Current Protocols in Molecular Biology (Ausebel, F.M. et al. eds). Greene Publishing & Wiley-Interscience, New York, NY pp. 10.8.1–10.8.6.Google Scholar
  23. 23.
    Neu, H. C. and Heppel, L. A. (1965) The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J. Biol. Chem. 240, 3685–3692.PubMedGoogle Scholar
  24. 24.
    Smith, J. A. (1989) Quantitation of proteins, in Current Protocols in Molecular Biology (Ausebel, F.M. et al. eds.), Greene Publishing & Wiley-Interscience, New York, NY pp. 10.1.1–10.1.3.Google Scholar
  25. 25.
    Ellinger, S., Mach, M., Korn, K., and Jahn, G (1991) Cleavage and purification of prokaryotically expressed HIV gag and env fusion proteins for detection of HIV antibodies in the ELISA. Virology 180, 811–813.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2000

Authors and Affiliations

  1. 1.New England BiolabsBeverly

Personalised recommendations