Skip to main content

Thank you for visiting 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.

Different Approaches to Stabilize a Recombinant Fusion Protein


We have used a fusion protein between staphylococcal protein A and E. coli β–galactosidase as a model system to investigate different approaches to stabilize recombinant gene products. First, growth conditions were adapted to preferentially produce insoluble inclusion bodies. This material was resistant to proteolysis, but no active fusion protein could be recovered. Secondly, biochemical characterization revealed that the soluble fusion protein was cleaved by a membrane–bound protease at a Lys–Arg peptide bound in the linker region. A new linker region was therefore engineered lacking the protease sensitive sequence. The new fusion protein was found to be resistant to degradation and high levels of soluble and active fusion protein could be produced. Finally, a mutant E. coli strain lacking the outer membrane protease OmpT was tested as a production strain, and a stable fusion protein could be recovered. These results demonstrate the use of several independent strategies to avoid degradation of recombinant proteins in heterologous hosts.

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

Access options

Rent or buy this article

Get just this article for as long as you need it


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


  1. Itakura, K., Hirose, T., Crea, R., Riggs, A.D., Heyneker, H.L., Bolivar, F. and Boyer, H.W. 1977. Expression in Escherichia coli of a chemically synthesized gene for the hormone somastotin. Science 198:1056–1063.

    Article  CAS  Google Scholar 

  2. Talmagde, K. and Gilbert, W. 1982. Cellular location affects protein stability in Escherichia coli. Proc. Natl. Acad. Sci USA 79:1830–1833.

    Article  Google Scholar 

  3. Goff, S.A. and Goldberg, A.L. 1985. Production of abnormal proteins in E. coli stimulates the transcription of Ion and other heat shock genes. Cell 41:587–595.

    Article  CAS  Google Scholar 

  4. Kitano, K., Fujimoto, S., Nakao, M., Witanable, T., and Nakao, Y. 1987. Intracellular degradation of recombinant proteins in relation to their location in Escherichia coli cells. J. Biotechnol. 5:77–86.

    Article  CAS  Google Scholar 

  5. Grodberg, J. and Dunn, J.J. 1988. ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J. Bacteriol. 170:1245–1253.

    Article  CAS  Google Scholar 

  6. Harris, T.J.R. 1983. Expression of eukaryotic genes in E. coli, p. 127–185. In: Williamson, R. (Ed.), Genetic Engineering Vol. 4: Academic Press, London.

    Google Scholar 

  7. Moks, T., Abrahmsén, L., Österlöf, B., Josephson, S. ., Östling, M., Enfors, S.-O., Persson, I., Nilsson, B., and Uhlén, M. 1987. Large scale affinity purification of human insulin-like growth factor I from culture medium of Escherichia coli. Bio/Technology 5:379–382.

    CAS  Google Scholar 

  8. Veide, A., Lindbäck, T. and Enfors, S.-O. 1984. Continuous extraction of β-D-galactosidase from Escherichia coli in an aqueous two-phase system: effects of biomass concentration on partitioning and mass transfer. Enzyme Microb. Technol. 6:325–330.

    Article  CAS  Google Scholar 

  9. Hellebust, H., Veide, A., and Enfors, S.-O. 1988. Proteolytic degradation of fused protein A-β-galactosidase in Escherichia coli. J. Biotechnol. 7:185–198.

    Article  CAS  Google Scholar 

  10. Nilsson, B., Abrahmsén, L. and Uhlén, M. 1985. Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors. EMBO J. 4:1075–1080.

    Article  CAS  Google Scholar 

  11. Strandberg, L., Veide, A., and Enfors, S.-O. 1987. Production of the hybrid protein staphylococcal protein A/ Escherichia coli β-galacto-sidase with E. coli. J. Biotechnol. 6:225–238.

    Google Scholar 

  12. Veide, A., Strandberg, L. and Enfors, S.-O. 1987. Extraction of β-galactosidase fused protein A in aqueous two-phase systems. Enzyme Microb. Technol. 9:730–738.

    Article  CAS  Google Scholar 

  13. Löfdahl, S., Guss, B., Uhlén, M., Philipson, L., and Lindberg, M. 1983. Gene for staphylococcal protein A. Proc. Natl. Acad. Sci. USA 80:697–701.

    Article  Google Scholar 

  14. Strandberg, L., Hellebust, H., and Enfors, S.-O. 1988. Factors influencing the production of a β-galactosidase fused protein in E. coli. Biotechnological aspects of protein production by cultured cells. Prague, July 6–8. In press.

  15. Zabeau, M. and Stanley, K.K. 1982. Enhanced expression of cro-β-galactosidase fusion proteins under the control of the PR promoter of bacteriophage lambda. EMBO J. 1:1217–1224.

    Article  CAS  Google Scholar 

  16. Uhlén, M., Guss, B., Nilsson, B., Gatenbeck, S., Philipson, L., and Lindberg, M. 1984. Complete sequence of the Staphylococcal gene encoding protein A, a gene evolved through multiple duplications. J. Biol. Chem. 259:1695–1702.

    Google Scholar 

  17. Guarente, L., Lauer, G., Roberts, T.M., and Ptashne, M. 1980. Improved methods for maximizing expression of a cloned gene: a bacterium that synthesizes rabbit β-globulin. Cell 20:543–553.

    Article  CAS  Google Scholar 

  18. Brake, A.J., Fowler, A.V., Zabin, I., Kania, J., and Müller-Hill, B. 1978. β-Galactosidase chimeras: Primary structure of a lac repressor-β-galactosidase protein. Proc. Natl. Acad. Sci. USA 75:4824–4827.

    Article  CAS  Google Scholar 

  19. Farabaugh, P.J. 1978. Sequence of the lacI gene. Nature 274:765–769.

    Article  CAS  Google Scholar 

  20. Kalnins, A., Otto, K., Rüther, U., and Müller-Hill, B. 1983. Sequence of the lacZ gene of Escherichia coli. EMBO J. 2:593–597.

    Article  CAS  Google Scholar 

  21. Shuman, H.A., Silhavy, T.J., and Beckwith, J.R. 1980. Labeling of proteins with β-galactosidase by gene fusion. J. Biol. Chem. 255:169–174.

    Google Scholar 

  22. Langley, K.E., Villarejo, M.R., Fowler, A.V., Zamenhof, P.J., and Zabin, I. 1975. Molecular basis of β-galactosidase alpha complementation. Proc. Natl. Acad. Sci. USA 72:1254–1257.

    Article  CAS  Google Scholar 

  23. Rüther, U. 1982. pUR 250 allows rapid chemical sequencing of both DNA strands of its insert. Nucleic Acid Res. 10:5765–5772.

    Article  Google Scholar 

  24. Earhart, C.F., Lundrigan, M., Pickett, C.L., and Pierce, J.R. 1979. Escherichia coli K-12 mutants that lack major outer membrane protein a. FEMS Microbiol. Lett. 6:277–280.

    Article  CAS  Google Scholar 

  25. Maniatis, T., Fritsch, E.F., and Sambrook, J. 1982. Molecular cloning, A laboratory manual. Cold Spring Harbour, NY.

    Google Scholar 

  26. Casadaban, M.J., Martinez-Arias, A., Shapioa, S.K., and Chou, J. 1983. β-Galactosidase gene fusion for analyzing gene expression in Escherichia coli and yeast. Meth. in Enzymol. 100:293–308.

    Article  CAS  Google Scholar 

  27. Holme, T., Arvidson, S., Lindholm, B., and Pavlu, B. 1970. Enzymes-laboratory-scale production. Process Biochem. Sept.:62–66.

  28. Laemmli, U.K. 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.

    Article  CAS  Google Scholar 

  29. Veide, A., Smeds, A.-L., and Enfors, S.-O. 1983. A process for large-scale isolation of β-galactosidase from E. coli in an aqueous two-phase system. Biotechnol. Bioeng. 25:1789–1800.

    Article  CAS  Google Scholar 

  30. Matsudaira, P. 1987. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262:10035–10038.

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations


Rights and permissions

Reprints and Permissions

About this article

Cite this article

Hellebust, H., Murby, M., Abrahmsén, L. et al. Different Approaches to Stabilize a Recombinant Fusion Protein. Nat Biotechnol 7, 165–168 (1989).

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI:

This article is cited by


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