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

  • Published:
Journal of Structural and Functional Genomics

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.

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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

References

  1. Waugh D.S. (2005) Trends Biotechnol. 23: 316–320

    Article  PubMed  CAS  Google Scholar 

  2. Hammarström M., Hellgren N., van den Berg S., Berglund H. and Härd T. (2002) Protein Sci. 11: 313–21

    Article  PubMed  CAS  Google Scholar 

  3. Braun P., Hu Y., Shen B., Halleck A., Koundinya M., Harlow E. LaBaer J. (2002) Proc. Natl. Acad. Sci. U S A 99: 2654–9

    Article  PubMed  CAS  Google Scholar 

  4. Shih Y.P., Kung W.M., Chen J.C., Yeh C.H., Wang A.H., Wang T.F. (2002) Protein Sci. 11: 1714–9

    Article  PubMed  CAS  Google Scholar 

  5. Dyson M.R., Shadbolt S.P., Vincent K.J., Perera R.L., McCafferty J. (2004) BMC Biotechnol. 4: 32

    Article  PubMed  CAS  Google Scholar 

  6. Woestenenk E.A., Hammarström M., van den Berg S., Härd T., Berglund H. (2004) J. Struct. Funct. Genomics 5: 217–229

    Article  CAS  Google Scholar 

  7. Smith D.B., Johnson K.S. (1988) Gene 67: 31–40

    Article  PubMed  CAS  Google Scholar 

  8. LaVallie E.R., DiBlasio E.A., Kovacic S., Grant K.L., Schendel P.F. and McCoy J.M. (1993) Biotechnology (N Y) 11: 187–93

    Article  CAS  Google Scholar 

  9. Huth J.R., Bewley C.A., Jackson B.M., Hinnebusch A.G., Clore G.M. and Gronenborn A.M. (1997) Protein Sci. 6: 2359–64

    PubMed  CAS  Google Scholar 

  10. Bedouelle H. and Duplay P. (1988) Eur. J. Biochem. 171: 541–9

    Article  PubMed  CAS  Google Scholar 

  11. di Guan C., Li P., Riggs P.D. and Inouye H. (1988) Gene 67: 21–30

    Article  PubMed  Google Scholar 

  12. Davis G.D., Elisee C., Newham D.M., Harrison R.G. (1999) Biotechnol. Bioeng. 65: 382–8

    Article  PubMed  CAS  Google Scholar 

  13. Sachdev D. and Chirgwin J.M. (1999) J. Protein Chem. 18: 127–36

    Article  PubMed  CAS  Google Scholar 

  14. Nominé Y., Ristriani T., Laurent C., Lefèvre J.F., Weiss E. and Travé G. (2001) Protein Expr. Purif. 23: 22–32

    Article  CAS  Google Scholar 

  15. Ashraf S.S., Benson R.E., Payne E.S., Halbleib C.M., Grøn H. (2004) Protein Expr. Purif. 33: 238–45

    Article  CAS  Google Scholar 

  16. Lechner M.S. and Laimins L.A. (1994) J. Virol. 68: 4262–73

    PubMed  CAS  Google Scholar 

  17. Kapust R.B. and Waugh D.S. (2000) Protein Expr. Purif. 19: 312–8

    Article  CAS  Google Scholar 

  18. Woestenenk E.A., Hammarström M., Härd T., Berglund H. (2003) Anal. Biochem. 318: 71–9

    Article  PubMed  CAS  Google Scholar 

  19. Braun P. and LaBaer J. (2003) Trends Biotechnol. 21: 383–8

    Article  PubMed  CAS  Google Scholar 

  20. Busso D., Kim R. and Kim S.H. (2004) J. Struct. Funct. Genomics 5: 69–74

    Article  CAS  Google Scholar 

  21. Chambers S.P., Austen D.A., Fulghum J.R., Kim W.M. (2004) Protein Expr. Purif. 36: 40–7

    Article  CAS  Google Scholar 

  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: 392–9

    Article  PubMed  CAS  Google Scholar 

  23. Scheich C., Sievert V., Büssow K. (2003) BMC Biotechnol. 3: 12

    Article  PubMed  Google Scholar 

  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: 195–204

    Article  Google Scholar 

  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: 115–30

    Article  PubMed  CAS  Google Scholar 

  26. Wang W., Malcolm B.A. (1999) Biotechniques 26: 680–2

    PubMed  CAS  Google Scholar 

  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 : D138–41

    Article  CAS  Google Scholar 

  28. Zhang G., Gurtu V., Kain S.R. (1996) Biochem. Biophys. Res. Commun. 227: 707–11

    Article  PubMed  CAS  Google Scholar 

  29. Bradford M.M. (1976) Anal. Biochem. 72: 248–54

    Article  PubMed  CAS  Google Scholar 

  30. Etchegaray J.P. and Inouye M. (1999) J. Bacteriol. 181: 5852–4

    PubMed  CAS  Google Scholar 

  31. Stenström C.M., Jin H., Major L.L., Tate W.P., Isaksson L.A. (2001) Gene 263: 273–84

    Article  PubMed  Google Scholar 

  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: 903–9

    Article  PubMed  CAS  Google Scholar 

  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: 11664–9

    Article  PubMed  CAS  Google Scholar 

  34. Yokoyama S. (2003) Curr. Opin. Chem. Biol. 7: 39–43

    Article  PubMed  CAS  Google Scholar 

  35. Scheich C., Leitner D., Sievert V., Leidert M., Schlegel B., Simon B., Letunic I., Büssow K., Diehl A. (2004) BMC Struct. Biol. 4: 4

    Article  PubMed  Google Scholar 

  36. Linding R., Jensen L.J., Diella F., Bork P., Gibson T.J. and Russell R.B. (2003) Structure 11: 1453–9

    Article  PubMed  CAS  Google Scholar 

  37. Uversky V.N., Gillespie J.R., Fink A.L. (2000) Proteins 41: 415–27

    Article  PubMed  CAS  Google Scholar 

  38. Kapust R.B. and Waugh D.S. (1999) Protein Sci. 8: 1668–74

    Article  PubMed  CAS  Google Scholar 

  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: 5059–65

    Article  PubMed  CAS  Google Scholar 

  40. Hirel P.H., Schmitter M.J., Dessen P., Fayat G., Blanquet S. (1989) Proc. Natl. Acad. Sci. U S A 86: 8247–51

    Article  PubMed  CAS  Google Scholar 

  41. Read S.M. and Northcote D.H. (1981) Anal. Biochem. 116: 53–64

    Article  PubMed  CAS  Google Scholar 

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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).

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Author notes

  1. Martin Hammarström & Helena Berglund

    Present address: Department of Medical Biochemistry and Biophysics, Karolinska Institute, SE-171 77, Stockholm, Sweden

Authors and Affiliations

  1. Department of Biotechnology, Royal Institute of Technology (KTH), SE-106 91, Stockholm, Sweden

    Martin Hammarström, Esmeralda A. Woestenenk, Niklas Hellgren & Helena Berglund

  2. Department of Medical Biochemistry, Göteborg University, 440, SE-405 30, Göteborg, Sweden

    Torleif Härd

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  1. Martin Hammarström
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  2. Esmeralda A. Woestenenk
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  3. Niklas Hellgren
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Correspondence to Helena Berglund.

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Hammarström, M., Woestenenk, E.A., Hellgren, N. et al. Effect of N-terminal solubility enhancing fusion proteins on yield of purified target protein. J Struct Funct Genomics 7, 1–14 (2006). https://doi.org/10.1007/s10969-005-9003-7

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  • DOI: https://doi.org/10.1007/s10969-005-9003-7

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