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Engineering Proteins to Enhance their Partition Coefficients in Aqueous Two-Phase Systems

Bio/Technology volume 9, pages 642–646 (1991)Cite this article

Abstract

We describe a novel method to partition recombinant proteins into the polymerrich top phase in poly(ethylene glycol) (PEG)4000/potassium phosphate aqueous two-phase systems. The concept is based on fusion of a gene fragment encoding a short peptide sequence to the product gene of interest thereby changing the partitioning properties of the expressed product protein as a fusion to the peptide. The model protein in this study, ZZ, is a two domain molecule based on staphylococcal protein A (SPA) which distributes evenly in PEG/salt systems. A tetrapeptide sequence, AlaTrpTrpPro (designated the partitioning peptide), was designed by molecular modeling techniques to include exposed tryptophan residues and to have a coding DNA sequence which is possible to polymerize in an obligate head-to-tail fashion at the DNA level. Gene fragments encoding one and three partitioning peptides, respectively, were fused to the 3′ end of the ZZ gene and the fusion proteins were produced intracellularly in Escherichia coli. The partition coefficients of ZZ proteins containing zero, one and three fused partitioning peptides were determined in three PEG 4000/potassium phosphate aqueous two-phase systems of different compositions. In all three phase systems, there were dramatic effects on the partition coefficient by the fused partitioning peptides. In the phase system with the largest effects, the partition coefficient was enhanced from 1.6 to 11.6 by fusing one tetrapeptide sequence to the 147 amino acid model ZZ protein. By the fusion of three partitioning peptides, the coefficient was increased to 96. These results show that fused partitioning peptides have a dramatic effect on the partitioning of a protein in aqueous two-phase systems which may open up new possibilities to utilize aqueous two-phase extraction techniques as a primary recovery step of recombinant proteins.

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References

  1. Hustedt, H., Kroner, K.H. and Kula, M.R. 1985. Applications of phase partitioning in biotechnology, p. 529–587. In: Partitioning in Aqueous Two-Phase Systems. Theory, Methods, Uses and Applications to Biotechnology. H. Walter, D. E. Brooks and D. Fischer (Eds.). Academic Press, Orlando, FL.

    Google Scholar 

  2. Kula, M.R. 1990. Trends and future prospects of aqueous two-phase extraction. Bioseparation 1: 181–189.

    CAS  PubMed  Google Scholar 

  3. Hustedt, H. 1986. Extractive enzyme recovery with simple recycling of phase forming chemicals. Biotechnol. Lett. 8: 791–796.

    Article  CAS  Google Scholar 

  4. Kroner, K.H., Hustedt, H. and Kula, M.R. 1984. Extractive enzyme recovery: Economic considerations. Process Biochem. 19: 170–179.

    Google Scholar 

  5. Huddleston, J.G. and Lyddiatt, A. 1991. Aqueous two phase systems in biochemical recovery: Systematic analysis, design and implementation of practical processes for recovery of proteins. Appl. Biochem. Biotechnol. In press.

    Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Johansson, G. and Tjerneld, F. 1989. Affinity partitioning between aqueous phases - a tool for large scale purification of enzymes. J. Biotechnol. 11: 135–142.

    Article  CAS  Google Scholar 

  8. Uhlén, M. and Moks, T. 1990. Gene fusions for the purpose of expression - an introduction. Methods Enzymol. 185: 129–143.

    Article  Google Scholar 

  9. Köhler, K., Veide, A. and Enfors, S.-O. 1991. Partitioning of β-galactosidase fusion proteins in PEG/potassium phosphate aqueous two-phase systems. Enz. Microb. Technol. 13: 204–209.

    Article  Google Scholar 

  10. Diamond, A.D., Lei, X. and Hsu, J.T. 1989. Reversing the amino acid sequence of a dipeptide changes its partition in an aqueous two-phase system. Biotechnol. Technol. 3: 271–274.

    Article  CAS  Google Scholar 

  11. Eiteman, M. and Gainer, J. 1989. A correlation for prediction of partition coefficients in poly (ethylene glocol)-salt aqueous two-phase systems, p. 54. In: 6th Int. Conf. on Partitioning in Aqueous Two-Phase Systems. M. R. Kula and W. Müller (Eds). Assmanshausen, Germany.

    Google Scholar 

  12. Ljungquist, C., Breitholtz, A., Brink-Nilsson, H., Moks, T., Uhlén, M. and Nilsson, B. 1989. Immobilization and affinity purification of recombinant proteins using histidine peptide fusions. Eur. J. Biochem. 186: 563–569.

    Article  CAS  Google Scholar 

  13. Hultman, T., Ståhl, S., Moks, T. and Uhlén, M. 1988. Approaches to solid phase DNA sequencing. Nucl. Acids Res. 7: 629–638.

    CAS  Google Scholar 

  14. Nilsson, B. and Abrahmsén, L. 1990. Fusions to staphylococcal protein A. Methods Enzymol. 185: 144–161.

    Article  CAS  Google Scholar 

  15. See, Y.P. and Jackowski, G. 1989. Estimated molecular weights of polypeptides by SDS gel electrophoresis, p. 1–22. In: Protein Structure, a Practical Approach. T. E. Creighton (Ed.). IRL Press. Oxford, England.

    Google Scholar 

  16. Gill, S.C. and von Hippel, P.H. 1989. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182: 319–326.

    Article  CAS  Google Scholar 

  17. Nilsson, B., Moks, T., Jansson, B., Abrahmsén, L., Elmblad, A., Holmgren, E., Henrichson, C., Jones, T. and Uhlén, M. 1987. A synthetic IgG-binding domain based on the staphylococcal protein A. Protein Engineering. 1: 107–113.

    Article  CAS  Google Scholar 

  18. Creighton, T.E. 1984. Proteins, Structure and Molecular Properties. W. H. Freeman and Company, New York.

    Google Scholar 

  19. Laskowski, M., Jr. 1966. Measurement of accessibility of protein chromophores by solvent perturbation of their ultraviolet spectra. Biochem. Soc. Sym. Fed. Proc. 25: 20–27.

    CAS  Google Scholar 

  20. Stone, F.W. and Strata, J.J. 1966. 1,2-Epoxide polymers, p. 103–145. In: Encyclopedia of Polymer Science and Technology. H. F. Mark, G. Gaylord, and N. M. Biales (Eds.). Wiley Interscience, New York.

    Google Scholar 

  21. Molyneux, P. 1975. Synthetic polymers, p. 569–757. In: Water, a Comprehensive Treatise Volume 4. F. Franks (Ed.). Plenum Press, New York.

    Chapter  Google Scholar 

  22. van Oss, C.J., Chaudhury, M.K. and Good, R.J. 1987. The mechanism of partition in aqueous media. Sep. Science and Technol. 22: 1515–1526.

    Article  CAS  Google Scholar 

  23. Carter, P. 1990. Site-specific proteolysis of fusion proteins, p. 181–193. In: Protein Purification: From Molecular Mechanism to Large-Scale Processes. M. R. Ladisch, R. C. Willson, C. C. Painton, and S. E. Builder (Eds.). Symposium Series Vol. 427, American Chemical Society, Washington, D.C.

    Chapter  Google Scholar 

  24. Maniatis, T., Fritsch, E. and Sambrook, J. 1982. Molecular Cloning: a Laboratory Handbook, Cold Spring Harbor Laboratory Press, New York.

    Google Scholar 

  25. Altman, J.D., Henner, D., Nilsson, B., Anderson, S. and Kuntz, I.D. 1991. Intracellular expression of BPTI-fusion proteins and single-step cleavage/affinity purification by chymotrypsin. Submitted.

    Google Scholar 

  26. Carter, P. 1987. Improved oligonucleotide-directed mutagenesis using M13 vectors. Methods Enzymol. 154: 382–403.

    Article  CAS  Google Scholar 

  27. Nygren, P.-Å., Eliasson, M., Palmcrantz, E., Abrahmsén, L. and Uhlén, M. 1988. Analysis and use of the serum albumin binding region of streptococcal protein G. J. Mol. Recognition. 1: 69–74.

    Article  CAS  Google Scholar 

  28. Boyer, H. and Roulland-Dussoix, D. 1969. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J. Mol. Biol. 41: 459–472.

    Article  CAS  Google Scholar 

  29. Maurer, R., Meyer, B.J. and Ptashne, M. 1980. Gene regulation at the right (OR) of bacteriophage λ I. OR3 autogenous negative control by represser. J. Mol. Biol. 139: 147–161.

    Article  CAS  Google Scholar 

  30. Hellebust, H., Uhlén, M. and Enfors, S. 1990. The interaction between the heat shock protein Dna K and recombinant staphylococcal protein A. J. Bacteriol. 172: 5030–5034.

    Article  CAS  Google Scholar 

  31. Skoog, B. 1979. Determination of polyethylene glycols 4000 and 6000 in plasma protein preparations. Vox Sang. 37: 345–349.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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Author notes

  1. Andres Veide and Björn Nilsson: Corresponding authors.

Authors and Affiliations

  1. Department of Biochemistry and Biotechnology, Royal Institute of Technology, S-100 44, Stockholm, Sweden

    Kristina Köhler, Charlotta Ljungquist, Akihiko Kondo, Andres Veide & Björn Nilsson

  2. Department of Applied Chemistry, Kyushu Institute of Technology, Sensuicho, Tobata, Kitakyushu, 804, Japan

    Akihiko Kondo

  3. Department of Biochemistry, Kabi Pharmacia KabiGen, S-112 87, Stockholm, Sweden

    Björn Nilsson

Authors
  1. Kristina Köhler
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  2. Charlotta Ljungquist
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  3. Akihiko Kondo
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  4. Andres Veide
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  5. Björn Nilsson
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Köhler, K., Ljungquist, C., Kondo, A. et al. Engineering Proteins to Enhance their Partition Coefficients in Aqueous Two-Phase Systems. Nat Biotechnol 9, 642–646 (1991). https://doi.org/10.1038/nbt0791-642

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