Skip to main content

Advertisement

We’d like to understand how you use our websites in order to improve them. Register your interest.

Microarray-based analysis of recombinant protein production in E. coli

Background

The production of heterologous proteins in E. coli is a powerful tool in the generation of many important biotechnological and medical products. Despite its widespread use as an expression host, however, yields of correctly folded, functional protein are frequently low in E. coli. This is due largely to the formation of insoluble protein aggregates and to premature lysis of the bacterial cells. We, and others, have previously shown that the cell lysis phenomenon associated with recombinant protein production in E. coli is not a direct result of synthesis of heterologous proteins [1], [2]. Instead, protein production triggers a global stress response in the bacterium, but the mechanism by which cell lysis subsequently occurs remains unclear [3].

We have carried out a microarray-based study of the response of E. coli to production of two recombinant proteins. In this analysis, a murine scFv antibody fragment and a human renal enzyme were produced in the E. coli periplasm, followed by co-production in turn of the cation efflux protein CzrB from Thermus thermophilus and E. coli disulfide bond isomerase DsbC. These latter proteins had previously been demonstrated in our group to delay lysis of the host E. coli cells and increase yields of the two proteins [1], [4].

Results

Growth and functional yields of the two recombinant proteins were studied using standard techniques. Co-expression of czrB and dsbC led to delayed lysis of host E. coli cells and to improvements in functional yields of recombinant proteins (see Figure 1).

Figure 1
figure1

A Growth of E. coli cells producing the murine2H12 scFv fragment with (diamonds) and without (squares) co-production of DsbC. B Immunoblot detection of 2H12 scFv produced in the presence and absence of DsbC overproduction. Lane 1. Molecular weight marker; lanes 2–3. no DsbC; lanes 4–5. + DsbC; lanes 2,4. insoluble scFv; lanes 3,5. soluble scFv.

Subsequent to mRNA purification and microarray analysis, data mining identified a number of genes whose expression was significantly altered upon recombinant protein production. Phage shock proteins and numerous chaperones were significantly upregulated, while OmpF was the main downregulated protein. Genes whose expression reverted towards pre-induction levels upon co-production of CzrB and/or DsbC were also identified. We report results of manipulation of expression of a number of these genes in an attempt to increase functional yields of the two recombinant proteins in vivo.

Conclusion

A microarray-based analysis of recombinant protein production was utilised to identify changes in gene expression in E. coli upon induction. Manipulation of expression of a number of these genes has been used to increase functional protein yields in vivo.

References

  1. 1.

    Spada S, Pembroke JT, Wall JG: Isolation of a novel Thermus thermophilus metal efflux protein that improves E. coli growth under stress conditions. Extremophiles. 2002, 6: 301-8. 10.1007/s00792-001-0257-0.

  2. 2.

    Knappik A, Plückthun A: Engineered turns of a recombinant antibody improve its in vivo folding. Protein Eng. 1995, 8: 81-9.

  3. 3.

    Hoffmann F, Rinas U: Stress induced by recombinant protein production in Escherichia coli. Adv Biochem Eng Biotechnol. 2004, 89: 73-92.

  4. 4.

    Hu X, O'Dwyer R, Wall JG: Cloning, expression and characterisation of a single-chain Fv antibody fragment againstdomoic acid in Escherichia coli . J Biotechnol. 2005, 120 (1): 38-45. 10.1016/j.jbiotec.2005.05.018.

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to Ronan O'Dwyer.

Rights and permissions

Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution 2.0 International License (https://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and Permissions

About this article

Cite this article

O'Dwyer, R., Hu, X., Pelizzola, M. et al. Microarray-based analysis of recombinant protein production in E. coli. Microb Cell Fact 5, P4 (2006). https://doi.org/10.1186/1475-2859-5-S1-P4

Download citation

Keywords

  • Recombinant Protein
  • Heterologous Protein
  • Recombinant Protein Production
  • scFv Antibody
  • Functional Yield