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Specific aggregation of partially folded polypeptide chains: The molecular basis of inclusion body composition

Nature Biotechnology volume 14, pages 1283–1287 (1996)Cite this article

Abstract

During expression of many recombinant proteins, off-pathway association of partially folded intermediates into inclusion bodies competes with productive folding. A common assumption is that such aggregation reactions are nonspecific processes. The multimeric intermediates along the aggregation pathway have been identified for both the P22 tailspike and P22 coat protein. We show that for a mixture of proteins refolding in vitro, folding intermediates do not coaggregate with each other but only with themselves. This indicates that aggregation occurs by specific interaction of certain conformations of folding intermediates rather than by nonspecific coaggregation, providing a rationale for recovering relatively pure protein from the inclusion body state.

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References

  1. Marston, F.A.O. 1986. The purification of eukaryotic polypeptides synthesized in Escherichia coli . Biochem. J. 240: 1–12.

    Article  CAS  Google Scholar 

  2. DeBernardez-Clark, E. and Georgiou, G. 1991. Inclusion bodies and recovery of proteins from the aggregated state, pp. 1–20 in Protein Refolding. Georgiou, G. and DeBernardez-Clark, E. (eds.). American Chemical Society Symposium Series No. 470, Washington, D.C.

    Google Scholar 

  3. Thomas, P.J., Qu, B.-H., and Pedersen, P.L. 1995. Defective protein folding as a basis of human disease. TIBS 20: 456–459.

    CAS  PubMed  Google Scholar 

  4. Wetzel, R. 1994. Mutations and off-pathway aggregation of proteins. Trends in Biotech. 12: 193–198.

    Article  CAS  Google Scholar 

  5. Mitraki, A. and King, J. 1989. Protein folding intermediates and inclusion body formation. Bio/Technology 7: 690–697.

    CAS  Google Scholar 

  6. Zettlmeissl, G., Rudolph, R. and Jaenicke, R. 1979. Reconstitution of lactic dehydrogenase. Noncovalent aggregation vs. reactivation. 1. Physical properties and kinetics of aggregation. Biochem. 18: 5567–5571.

    Article  CAS  Google Scholar 

  7. Cleland, J.L. 1993. Impact of protein folding on biotechnology, pp. 1–21 in Protein folding: In vivo and in vitro. Cleland, J.L. (ed.). American Chemical Society Symposium Series No. 526. Washington, DC.

    Chapter  Google Scholar 

  8. Brems, D.N., Plaisted, S.M., Havel, H.A., and Tomich, C.S.C. 1988. Stabilization of an associated folding intermediate of bovine growth hormone by site-directed mutagenesis. PNAS USA 85: 3367–3371.

    Article  CAS  Google Scholar 

  9. Lehrman, S.R., Tuls, J.L., Havel, H.A., Haskell, R.J., Putnam, S.D., and Tomich, C.S.C. 1991. Site-directed mutagenesis to probe protein folding: Evidence that the formation of a bovine growth hormone folding intermediate are dissociable processes. Biochem. 30: 5777–5784.

    Article  CAS  Google Scholar 

  10. Mitraki, A., Betton, J.-M., Desmadril, M., and Yon, J.M. 1987. Quasi-irreversibility in the unfolding-refolding transition of phosphoglycerate kinase induced by guanidine hydrochloride. Eur. J. Biochem. 163: 29–34.

    Article  CAS  Google Scholar 

  11. Mitraki, A., Fane, B., Haase-Pettingell, C., Sturtevant, J., and King, J. 1991. Global suppression of protein folding defects and inclusion body formation. Science 253: 54–58.

    Article  CAS  Google Scholar 

  12. Mitraki, A., Danner, M., King, J. and Seckler, R. 1993. Temperature-sensitive mutations and second-site suppressor substitutions affect folding of the P22 tailspike protein in vitro. J. Biol. Chem. 268: 20071–20075.

    CAS  PubMed  Google Scholar 

  13. Danner, M. and Seckler, R. 1993. Mechanism of phage P22 tailspike protein folding mutations. Protein Science 2: 1869–1881.

    Article  CAS  Google Scholar 

  14. Wetzel, R. and Chrunyk, B.A. 1994. Inclusion body formation by interteukin-1 β depends on the thermal sensitivity of a folding intermediate. FEES Letters 350: 245–248.

    Article  CAS  Google Scholar 

  15. Cleland, J.L., Hedgepeth, C., and Wang, D.I.C. 1992. Polyethylene glycol enhanced refolding of bovine carbonic anhydrase B: reaction stoichiometry and refolding model. J. Biol. Chem. 19: 13327–13334.

    Google Scholar 

  16. Tian, G., Vainberg, I.E., Tap, W.D., Lewis, S.A., and Cowan, N.J. 1995. Specificify in chaperonin-mediated protein folding. Nature 375: 250–253.

    Article  CAS  Google Scholar 

  17. Goldberg, M.E., Rudolph, R., and Jaenicke, R. 1991. A kinetic study of the competition between renaturation and aggregation during the refolding of denatured-reduced egg white lysozyme. Biochem. 30: 2790–2797.

    Article  CAS  Google Scholar 

  18. London, J., Skrzynia, C., and Goldberg, M. 1974. Renaturation of Eschericia coli tryptophanase after exposure to 8M urea. Eur. J. Biochem. 47: 409–415.

    Article  CAS  Google Scholar 

  19. De Young, L.R., Dill, K.A., and Fink, A.L. 1993. Aggregation and denaturation of apomyoglobin in aqueous urea solutions. Biochem. 32: 3877–3886.

    Article  CAS  Google Scholar 

  20. Jarrett, J.T. and Lansbury, P.T., Jr. 1992. Amyloid fibril formation requires a chemically discriminating nucleation event: studies of an amyloidogenic sequence from the bacterial protein osm B. Biochem. 31: 12345–12352.

    Article  CAS  Google Scholar 

  21. Kocisko, D.A., Priola, S.A., Raymond, G.J., Chesbro, B., Lansbury, P.T., and Caughey, B. 1995. Species specificity in the cell-free conversion of prion protein to protease-resistant forms: A model for the scrapie species barrier. PNAS 92: 3923–3927.

    Article  CAS  Google Scholar 

  22. Saraiva, M.J.M., Birken, S., Costa, P.P., and Goodman, D.S. 1984. Amyloid fibril protein in familial amyloidotic polyneuropathy, Portuguese type: definition of molecular abnormality in transthyretin (prealbumin). J. Clin. Invest. 74: 104–119.

    Article  CAS  Google Scholar 

  23. Chargé, S.B.R., de Koning, E.J.P., and Clark, A. 1995. Effect of pH and insulin on fibrillogenesis of islet amyloid polypeptide in vitro. Biochem. 34: 14588–14593.

    Article  Google Scholar 

  24. Speed, M.A., Wang, D.I.C., and King, J. 1995. Multimeric intermediates in the pathway to the aggregated inclusion body state for P22 tailspike polypeptide chains. Pro. Sci. 4: 900–908.

    Article  CAS  Google Scholar 

  25. Teschke, C.M. and King, J. 1995. In vitro folding of phage P22 coat protein with amino acid substitutions that confer in vivo temperature sensitivity. Biochem. 34: 6815–6826.

    Article  CAS  Google Scholar 

  26. Steinbacher, S., Seckler, R., Miller, S., Steipe, B., Huber, R., and Reinemer, P. 1994. Crystal structure of P22 tailspike protein: Interdigitated subunits in a thermostable trimer. Science 265: 383–386.

    Article  CAS  Google Scholar 

  27. Haase-Pettingell, C. and King, J. 1988. Formation of aggregates from a thermolabile in vivo folding intermediate in P22 tailspike maturation. J. Biol. Chem. 263: 4977–4983.

    CAS  PubMed  Google Scholar 

  28. Friguet, B., Djavadi-Ohaniance, L., Haase-Pettingell, C.A., King, J. and Goldberg, M.E. 1990. Properties of monoclonal antibodies selected for probing the conformation of wild type and mutant forms of the P22 tailspike endorhamnosidase. J. Biol. Chem. 265: 10347–10351.

    CAS  PubMed  Google Scholar 

  29. Teschke, C.M. and King, J. 1993. Folding of the phage P22 coat protein in vitro. Biochem. 32: 10839–10847.

    Article  CAS  Google Scholar 

  30. Gordon, C.L., Sather, S.K., Casjens, S. and King, J. 1994. Selective in vivo rescue by GroEL/ES of thermolabile folding intermediates to phage P22 structural proteins. J. Biol. Chem. 269: 27941–27951.

    CAS  PubMed  Google Scholar 

  31. Goldenberg, D. and King, J. 1982. Trimeric intermediate in the in vivo folding and subunit assembly of the tail spike endorhamnosidase of bacteriophage P22. PNAS 79: 3403–3407.

    Article  CAS  Google Scholar 

  32. Goldenberg, D., Smith, D.H., and King, J. 1983. Genetic analysis of the folding pathway for the tail spike protein of phage P22. Proc. Natl. Acad. Sci. USA 80: 7060–7064.

    Article  CAS  Google Scholar 

  33. Brems, D.N., Plaisted, S.M., Kauffman, E.W. and Havel, H.A. 1986. Characterization of an associated equilibrium folding intermediate of bovine growth hormone. Biochem. 25: 6539–6543.

    Article  CAS  Google Scholar 

  34. Chen, C.-C., King, J., and Wang, D.I.C. 1995. Molecular thermodynamic model for helix-helix docking and protein aggregation. AIChE J. 41: 1015–1024.

    Article  CAS  Google Scholar 

  35. Jaenicke, R. and Rudolph, R. 1986. Refolding and association of oligomeric proteins. Methods Enzymol. 131: 218–250.

    Article  CAS  Google Scholar 

  36. Brunschier, R., Danner, M., and Seckler, R. 1993. Interactions of phage P22 tail-spike protein with GroE molecular chaperones during refolding in vitro. J. Biol. Chem. 268: 2767–2772.

    CAS  PubMed  Google Scholar 

  37. King, J., Haase-Pettingell, C., Robinson, A.S., Speed, M., and Mitraki, A. 1996. Thermostable folding intermediates: inclusion body precursors and chaperonin substrates. FASEB J. 10: 57–66.

    Article  CAS  Google Scholar 

  38. King, J. and Yu, M.-H. 1986. Mutational analysis of protein folding pathways. Methods Enzymol. 131: 250–266.

    Article  CAS  Google Scholar 

  39. Shea, R.G. 1971. Genetic identification of phage P22 antigens and their structural location. PhD diss., Department of Biology, Massachusetts Institute of Technology, Cambridge, MA.

  40. King, J., Griffin-Shea, R., and Fuller, M.T. 1980. Scaffolding proteins and the genetic control of virus shell assembly. Quart. Rev. Biol. 55: 369–393.

    Article  CAS  Google Scholar 

  41. Sather, S. and King, J. 1994. Intracellular trapping of a cytoplasmic folding intermediate of the phage P22 tailspike using iodoacetamide. J. Biol. Chem. 269: 25268–25276.

    CAS  PubMed  Google Scholar 

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Author notes

  1. Margaret A. Speed

    Present address: Amgen, Inc., 1840 DeHavilland Dr., Thousand Oaks, CA, 91320

  2. Jonathan King: e-mail: jaking@mit.edu

Authors and Affiliations

  1. Biotechnology Process Engineering Center, Massachusetts Institute of Technology, Cambridge, MA, 02139

    Daniel I. C. Wang & Jonathan King

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  1. Margaret A. Speed
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  2. Daniel I. C. Wang
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Speed, M., Wang, D. & King, J. Specific aggregation of partially folded polypeptide chains: The molecular basis of inclusion body composition. Nat Biotechnol 14, 1283–1287 (1996). https://doi.org/10.1038/nbt1096-1283

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  • DOI: https://doi.org/10.1038/nbt1096-1283

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