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The Targeted Expression of Nucleotide Sugar Transporters to the E. coli Inner Membrane

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Heterologous Gene Expression in E.coli

Part of the book series: Methods in Molecular Biology ((MIMB,volume 705))

Abstract

The heterologous expression of functional mammalian integral membrane proteins still represents a significant hurdle towards evaluating the relationship between their structure and function. We have therefore utilised the OmpA signal sequence to deliberately target the expression of a mammalian nucleotide sugar transporter, the murine CMP-sialic acid transporter, to the E. coli inner membrane. The functionality of the recombinant CMP-sialic acid transporter could then be evaluated either following the spheroplasting of E. coli cells or through the isolation of the E. coli inner membrane and the formation of mixed phosphatidylcholine-inner membrane proteoliposomes.

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References

  1. Grisshammer, R., Tate, C. G. (2003) Overexpression of integral membrane proteins. Biochim Biophys Acta 1610, 1.

    Article  Google Scholar 

  2. Link, A. J., Georgiou, G. (2007) Advances and challenges in membrane protein expression. AIChE J 53, 752–756.

    Article  CAS  Google Scholar 

  3. Drew, D., Froderberg, L., Baars, L., de Gier, J. W. (2003) Assembly and overexpression of membrane proteins in Escherichia coli. Biochim Biophys Acta 1610, 3–10.

    Article  PubMed  CAS  Google Scholar 

  4. Eckhardt, M., Muhlenhoff, M., Bethe, A., Gerardy-Schahn, R. (1996) Expression cloning of the Golgi CMP-sialic acid transporter. Proc Natl Acad Sci USA 93, 7572–7576.

    Article  PubMed  CAS  Google Scholar 

  5. Caffaro, C. E., Hirschberg, C. B. (2006) Nucleotide sugar transporters of the Golgi apparatus: from basic science to diseases. Acc Chem Res 39, 805–812.

    Article  PubMed  CAS  Google Scholar 

  6. Tiralongo, J., Abo, S., Danylec, B., Gerardy-Schahn, R., von Itzstein, M. (2000) A high-throughput assay for rat liver Golgi and Saccharomyces cerevisiae-expressed murine CMP-N-acetylneuraminic acid transport proteins. Anal Biochem 285, 21–32.

    Article  PubMed  CAS  Google Scholar 

  7. Tiralongo, J., Ashikov, A., Routier, F., Eckhardt, M., Bakker, H., Gerardy-Schahn, R., von Itzstein, M. (2006) Functional expression of the CMP-sialic acid transporter in Escherichia coli and its identification as a simple mobile carrier. Glycobiology 16, 73–81.

    Article  PubMed  CAS  Google Scholar 

  8. Maggioni, A., von Itzstein, M., Gerardy-Schahn, R., Tiralongo, J. (2007) Targeting the expression of functional murine CMP-sialic acid transporter to the E. coli inner membrane. Biochem Biophys Res Commun 362, 779–784.

    Article  PubMed  CAS  Google Scholar 

  9. Osborn, M. J., Munson, R. (1974) Separation of the inner (cytoplasmic) and outer membranes of Gram-negative bacteria. Methods Enzymol 31, 642–653.

    Article  PubMed  CAS  Google Scholar 

  10. White, S. H., von Heijne, G. (2004) The machinery of membrane protein assembly. Curr Opin Struct Biol 14, 397–404.

    Article  PubMed  CAS  Google Scholar 

  11. Witholt, B., Boekhout, M., Brock, M., Kingma, J., Heerikhuizen, H. V., Leij, L. D. (1976) An efficient and reproducible procedure for the formation of spheroplasts from variously grown Escherichia coli. Anal Biochem 74, 160–170.

    Article  PubMed  CAS  Google Scholar 

  12. Witholt, B., Heerikhuizen, H. V., De Leij, L. (1976) How does lysozyme penetrate through the bacterial outer membrane? Biochim Biophys Acta 443, 534–544.

    Article  PubMed  CAS  Google Scholar 

  13. Sambrook, J., Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, Vol. 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  14. Dumon-Seignovert, L., Cariot, G., Vuillard, L. (2004) The toxicity of recombinant proteins in Escherichia coli: a comparison of overexpression in BL21(DE3), C41(DE3), and C43(DE3). Protein Expr Purif 37, 203–206.

    Article  PubMed  CAS  Google Scholar 

  15. Miroux, B., Walker, J. E. (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260, 289–298.

    Article  PubMed  CAS  Google Scholar 

  16. Stark, G. R., Stein, W. H., Moore, S. (1960) Reactions of the cyanate present in aqueous urea with amino acids and proteins. J Biol Chem 235, 3177–3181.

    CAS  Google Scholar 

  17. Ragan, C. I. (1986) Analysis of membrane protein composition by gel electrophoresis. In (Ragan, C. I., Cherry, R. J. eds.), Techniques for the Analysis of Membrane Proteins. Chapman and Hall, London, pp. 1–25.

    Google Scholar 

  18. Mui, B., Chow, L., Hope, M. J. (2003) Extrusion technique to generate liposomes of defined size. Methods Enzymol 367, 3–14.

    Article  PubMed  CAS  Google Scholar 

  19. Vojta, A., Scheuring, J., Neumaier, N., Mirus, O., Weinkauf, S., Schleiff, E. (2005) Determination of liposome size: a tool for protein reconstitution. Anal Biochem 347, 24–33.

    Article  PubMed  CAS  Google Scholar 

  20. Olson, F., Hunt, C. A., Szoka, F. C., Vail, W. J., Papahadjopoulos, D. (1979) Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes. Biochim Biophys Acta 557, 9–23.

    Article  PubMed  CAS  Google Scholar 

  21. Kasahara, M., Hinkle, P. C. (1976) Reconstitution of D-glucose transport catalyzed by a protein fraction from human erythrocytes in sonicated liposomes. Proc Natl Acad Sci USA 73, 396–400.

    Article  PubMed  CAS  Google Scholar 

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Correspondence to Joe Tiralongo .

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Tiralongo, J., Maggioni, A. (2011). The Targeted Expression of Nucleotide Sugar Transporters to the E. coli Inner Membrane. In: Evans, Jr., T., Xu, MQ. (eds) Heterologous Gene Expression in E.coli. Methods in Molecular Biology, vol 705. Humana Press. https://doi.org/10.1007/978-1-61737-967-3_14

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  • DOI: https://doi.org/10.1007/978-1-61737-967-3_14

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61737-966-6

  • Online ISBN: 978-1-61737-967-3

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