Effect of N-terminal solubility enhancing fusion proteins on yield of purified target protein

  • Martin Hammarström
  • Esmeralda A. Woestenenk
  • Niklas Hellgren
  • Torleif Härd
  • Helena Berglund
Article

Abstract

We have studied the effect of solubilising N-terminal fusion proteins on the yield of target protein after removal of the fusion partner and subsequent purification using immobilised metal ion affinity chromatography. We compared the yield of 45 human proteins produced from four different expression vectors: three having an N-terminal solubilising fusion protein (the GB1-domain, thioredoxin, or glutathione S-transferase) followed by a protease cleavage site and a His tag, and one vector having only an N-terminal His tag. We have previously observed a positive effect on solubility for proteins produced as fusion proteins compared to proteins produced with only a His tag in Escherichia coli. We find this effect to be less pronounced when we compare the yields of purified target protein after removal of the solubilising fusion although large target-dependent variations are seen. On average, the GB1+His fusion gives significantly higher final yields of protein than the thioredoxin+His fusion or the His tag, whereas GST+His gives lower yields. We also note a strong correlation between solubility and target protein size, and a correlation between solubility and the presence of peptide fragments that are predicted to be natively disordered.

Keywords

Escherichia coli fusion protein high throughput His tag solubility structural genomics 

Abbreviations

eGFP

enhanced green fluorescent protein

GST

glutathione S-transferase

IMAC

immobilised metal ion affinity chromatography

MBP

maltose binding protein

MES

2-(N-morpholino) ethanesulfonic acid

Ni-NTA

Ni2+-nitrilotriacetic acid

ORF

open reading frame

RBS

ribosome binding site

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Notes

Acknowledgements

We would like to acknowledge the contributions of John Löfblom and Kristina Bergström for initial cloning of genes 202 to 215, Harry Brumer and Hongbin Henriksson for the mass spectrometry analysis and Susanne van den Berg for construction of the vectors pTH8 and pTH18. This work was supported by the European Commission Integrated Project SPINE (QLG2-CT-2002-00988) as part of the Framework 5 Quality of Life and Management of Living Resources Program, by the Wallenberg Consortium North (WCN) and by the Swedish foundation for strategic research (SSF).

Supplementary material

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Supplementary material

References

  1. 1.
    Waugh D.S. (2005) Trends Biotechnol. 23: 316–320PubMedCrossRefGoogle Scholar
  2. 2.
    Hammarström M., Hellgren N., van den Berg S., Berglund H. and Härd T. (2002) Protein Sci. 11: 313–21PubMedCrossRefGoogle Scholar
  3. 3.
    Braun P., Hu Y., Shen B., Halleck A., Koundinya M., Harlow E. LaBaer J. (2002) Proc. Natl. Acad. Sci. U S A 99: 2654–9PubMedCrossRefGoogle Scholar
  4. 4.
    Shih Y.P., Kung W.M., Chen J.C., Yeh C.H., Wang A.H., Wang T.F. (2002) Protein Sci. 11: 1714–9PubMedCrossRefGoogle Scholar
  5. 5.
    Dyson M.R., Shadbolt S.P., Vincent K.J., Perera R.L., McCafferty J. (2004) BMC Biotechnol. 4: 32PubMedCrossRefGoogle Scholar
  6. 6.
    Woestenenk E.A., Hammarström M., van den Berg S., Härd T., Berglund H. (2004) J. Struct. Funct. Genomics 5: 217–229CrossRefGoogle Scholar
  7. 7.
    Smith D.B., Johnson K.S. (1988) Gene 67: 31–40PubMedCrossRefGoogle Scholar
  8. 8.
    LaVallie E.R., DiBlasio E.A., Kovacic S., Grant K.L., Schendel P.F. and McCoy J.M. (1993) Biotechnology (N Y) 11: 187–93CrossRefGoogle Scholar
  9. 9.
    Huth J.R., Bewley C.A., Jackson B.M., Hinnebusch A.G., Clore G.M. and Gronenborn A.M. (1997) Protein Sci. 6: 2359–64PubMedGoogle Scholar
  10. 10.
    Bedouelle H. and Duplay P. (1988) Eur. J. Biochem. 171: 541–9PubMedCrossRefGoogle Scholar
  11. 11.
    di Guan C., Li P., Riggs P.D. and Inouye H. (1988) Gene 67: 21–30PubMedCrossRefGoogle Scholar
  12. 12.
    Davis G.D., Elisee C., Newham D.M., Harrison R.G. (1999) Biotechnol. Bioeng. 65: 382–8PubMedCrossRefGoogle Scholar
  13. 13.
    Sachdev D. and Chirgwin J.M. (1999) J. Protein Chem. 18: 127–36PubMedCrossRefGoogle Scholar
  14. 14.
    Nominé Y., Ristriani T., Laurent C., Lefèvre J.F., Weiss E. and Travé G. (2001) Protein Expr. Purif. 23: 22–32CrossRefGoogle Scholar
  15. 15.
    Ashraf S.S., Benson R.E., Payne E.S., Halbleib C.M., Grøn H. (2004) Protein Expr. Purif. 33: 238–45CrossRefGoogle Scholar
  16. 16.
    Lechner M.S. and Laimins L.A. (1994) J. Virol. 68: 4262–73PubMedGoogle Scholar
  17. 17.
    Kapust R.B. and Waugh D.S. (2000) Protein Expr. Purif. 19: 312–8CrossRefGoogle Scholar
  18. 18.
    Woestenenk E.A., Hammarström M., Härd T., Berglund H. (2003) Anal. Biochem. 318: 71–9PubMedCrossRefGoogle Scholar
  19. 19.
    Braun P. and LaBaer J. (2003) Trends Biotechnol. 21: 383–8PubMedCrossRefGoogle Scholar
  20. 20.
    Busso D., Kim R. and Kim S.H. (2004) J. Struct. Funct. Genomics 5: 69–74CrossRefGoogle Scholar
  21. 21.
    Chambers S.P., Austen D.A., Fulghum J.R., Kim W.M. (2004) Protein Expr. Purif. 36: 40–7CrossRefGoogle Scholar
  22. 22.
    Savchenko A., Yee A., Khachatryan A., Skarina T., Evdokimova E., Pavlova M., Semesi A., Northey J., Beasley S., Lan N., Das R., Gerstein M., Arrowmith C.H., Edwards A.M. (2003) Proteins 50: 392–9PubMedCrossRefGoogle Scholar
  23. 23.
    Scheich C., Sievert V., Büssow K. (2003) BMC Biotechnol. 3: 12PubMedCrossRefGoogle Scholar
  24. 24.
    Trésaugues L., Collinet B., Minard P., Henckes G., Aufrère R., Blondeau K., Liger D., Zhou C.Z., Janin J., Van Tilbeurgh H., Quevillon-Cheruel S. (2004) J. Struct. Funct. Genomics 5: 195–204CrossRefGoogle Scholar
  25. 25.
    Goh C.S., Lan N., Douglas S.M., Wu B., Echols N., Smith A., Milburn D., Montelione G.T., Zhao H., Gerstein M. (2004) J. Mol. Biol. 336: 115–30PubMedCrossRefGoogle Scholar
  26. 26.
    Wang W., Malcolm B.A. (1999) Biotechniques 26: 680–2PubMedGoogle Scholar
  27. 27.
    Bateman A., Coin L., Durbin R., Finn R.D., Hollich V., Griffiths-Jones S., Khanna A., Marshall M., Moxon S., Sonnhammer E.L., Studholme D.J., Yeats C. and Eddy S.R. (2004) Nucleic Acids Res. 32 Database issue : D138–41CrossRefGoogle Scholar
  28. 28.
    Zhang G., Gurtu V., Kain S.R. (1996) Biochem. Biophys. Res. Commun. 227: 707–11PubMedCrossRefGoogle Scholar
  29. 29.
    Bradford M.M. (1976) Anal. Biochem. 72: 248–54PubMedCrossRefGoogle Scholar
  30. 30.
    Etchegaray J.P. and Inouye M. (1999) J. Bacteriol. 181: 5852–4PubMedGoogle Scholar
  31. 31.
    Stenström C.M., Jin H., Major L.L., Tate W.P., Isaksson L.A. (2001) Gene 263: 273–84PubMedCrossRefGoogle Scholar
  32. 32.
    Christendat D., Yee A., Dharamsi A., Kluger Y., Savchenko A., Cort J.R., Booth V., Mackereth C.D., Saridakis V., Ekiel I., Kozlov G., Maxwell K.L., Wu N., McIntosh L.P., Gehring K., Kennedy M.A., Davidson A.R., Pai E.F., Gerstein M., Edwards A.M. and Arrowsmith C.H. (2000) Nat. Struct. Biol. 7: 903–9PubMedCrossRefGoogle Scholar
  33. 33.
    Lesley S.A., Kuhn P., Godzik A., Deacon A.M., Mathews I., Kreusch A., Spraggon G., Klock H.E., McMullan D., Shin T., Vincent J., Robb A., Brinen L.S., Miller M.D., McPhillips T.M., Miller M.A., Scheibe D., Canaves J.M., Guda C., Jaroszewski L., Selby T.L., Elsliger M.A., Wooley J., Taylor S.S., Hodgson K.O., Wilson I.A., Schultz P.G. Stevens R.C. (2002) Proc. Natl. Acad. Sci. U S A 99: 11664–9PubMedCrossRefGoogle Scholar
  34. 34.
    Yokoyama S. (2003) Curr. Opin. Chem. Biol. 7: 39–43PubMedCrossRefGoogle Scholar
  35. 35.
    Scheich C., Leitner D., Sievert V., Leidert M., Schlegel B., Simon B., Letunic I., Büssow K., Diehl A. (2004) BMC Struct. Biol. 4: 4PubMedCrossRefGoogle Scholar
  36. 36.
    Linding R., Jensen L.J., Diella F., Bork P., Gibson T.J. and Russell R.B. (2003) Structure 11: 1453–9PubMedCrossRefGoogle Scholar
  37. 37.
    Uversky V.N., Gillespie J.R., Fink A.L. (2000) Proteins 41: 415–27PubMedCrossRefGoogle Scholar
  38. 38.
    Kapust R.B. and Waugh D.S. (1999) Protein Sci. 8: 1668–74PubMedCrossRefGoogle Scholar
  39. 39.
    Lu Z., DiBlasio-Smith E.A., Grant K.L., Warne N.W., LaVallie E.R., Collins-Racie L.A., Follettie M.T., Williamson M.J. and McCoy J.M. (1996) J Biol Chem 271: 5059–65PubMedCrossRefGoogle Scholar
  40. 40.
    Hirel P.H., Schmitter M.J., Dessen P., Fayat G., Blanquet S. (1989) Proc. Natl. Acad. Sci. U S A 86: 8247–51PubMedCrossRefGoogle Scholar
  41. 41.
    Read S.M. and Northcote D.H. (1981) Anal. Biochem. 116: 53–64PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  • Martin Hammarström
    • 1
    • 3
  • Esmeralda A. Woestenenk
    • 1
  • Niklas Hellgren
    • 1
  • Torleif Härd
    • 2
  • Helena Berglund
    • 1
    • 3
  1. 1.Department of BiotechnologyRoyal Institute of Technology (KTH)StockholmSweden
  2. 2.Department of Medical BiochemistryGöteborg UniversityGöteborgSweden
  3. 3.Department of Medical Biochemistry and BiophysicsKarolinska InstituteStockholmSweden

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