Research Article | Published:

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

Nature Biotechnology volume 14, pages 12831287 (1996) | Download Citation

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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. 1.

    1986. The purification of eukaryotic polypeptides synthesized in Escherichia coli . Biochem. J. 240: 1–12.

  2. 2.

    and 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.

  3. 3.

    , , and 1995. Defective protein folding as a basis of human disease. TIBS 20: 456–459.

  4. 4.

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

  5. 5.

    and 1989. Protein folding intermediates and inclusion body formation. Bio/Technology 7: 690–697.

  6. 6.

    , and 1979. Reconstitution of lactic dehydrogenase. Noncovalent aggregation vs. reactivation. 1. Physical properties and kinetics of aggregation. Biochem. 18: 5567–5571.

  7. 7.

    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.

  8. 8.

    , , , and 1988. Stabilization of an associated folding intermediate of bovine growth hormone by site-directed mutagenesis. PNAS USA 85: 3367–3371.

  9. 9.

    , , , , , and 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.

  10. 10.

    , , , and 1987. Quasi-irreversibility in the unfolding-refolding transition of phosphoglycerate kinase induced by guanidine hydrochloride. Eur. J. Biochem. 163: 29–34.

  11. 11.

    , , , , and 1991. Global suppression of protein folding defects and inclusion body formation. Science 253: 54–58.

  12. 12.

    , , and 1993. Temperature-sensitive mutations and second-site suppressor substitutions affect folding of the P22 tailspike protein in vitro. J. Biol. Chem. 268: 20071–20075.

  13. 13.

    and 1993. Mechanism of phage P22 tailspike protein folding mutations. Protein Science 2: 1869–1881.

  14. 14.

    and 1994. Inclusion body formation by interteukin-1 β depends on the thermal sensitivity of a folding intermediate. FEES Letters 350: 245–248.

  15. 15.

    , , and 1992. Polyethylene glycol enhanced refolding of bovine carbonic anhydrase B: reaction stoichiometry and refolding model. J. Biol. Chem. 19: 13327–13334.

  16. 16.

    , , , , and 1995. Specificify in chaperonin-mediated protein folding. Nature 375: 250–253.

  17. 17.

    , , and 1991. A kinetic study of the competition between renaturation and aggregation during the refolding of denatured-reduced egg white lysozyme. Biochem. 30: 2790–2797.

  18. 18.

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

  19. 19.

    , , and 1993. Aggregation and denaturation of apomyoglobin in aqueous urea solutions. Biochem. 32: 3877–3886.

  20. 20.

    and 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.

  21. 21.

    , , , , , and 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.

  22. 22.

    , , , and 1984. Amyloid fibril protein in familial amyloidotic polyneuropathy, Portuguese type: definition of molecular abnormality in transthyretin (prealbumin). J. Clin. Invest. 74: 104–119.

  23. 23.

    , , and 1995. Effect of pH and insulin on fibrillogenesis of islet amyloid polypeptide in vitro. Biochem. 34: 14588–14593.

  24. 24.

    , , and 1995. Multimeric intermediates in the pathway to the aggregated inclusion body state for P22 tailspike polypeptide chains. Pro. Sci. 4: 900–908.

  25. 25.

    and 1995. In vitro folding of phage P22 coat protein with amino acid substitutions that confer in vivo temperature sensitivity. Biochem. 34: 6815–6826.

  26. 26.

    , , , , , and 1994. Crystal structure of P22 tailspike protein: Interdigitated subunits in a thermostable trimer. Science 265: 383–386.

  27. 27.

    and 1988. Formation of aggregates from a thermolabile in vivo folding intermediate in P22 tailspike maturation. J. Biol. Chem. 263: 4977–4983.

  28. 28.

    , , , and 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.

  29. 29.

    and 1993. Folding of the phage P22 coat protein in vitro. Biochem. 32: 10839–10847.

  30. 30.

    , , and 1994. Selective in vivo rescue by GroEL/ES of thermolabile folding intermediates to phage P22 structural proteins. J. Biol. Chem. 269: 27941–27951.

  31. 31.

    and 1982. Trimeric intermediate in the in vivo folding and subunit assembly of the tail spike endorhamnosidase of bacteriophage P22. PNAS 79: 3403–3407.

  32. 32.

    , , and 1983. Genetic analysis of the folding pathway for the tail spike protein of phage P22. Proc. Natl. Acad. Sci. USA 80: 7060–7064.

  33. 33.

    , , and 1986. Characterization of an associated equilibrium folding intermediate of bovine growth hormone. Biochem. 25: 6539–6543.

  34. 34.

    , , and 1995. Molecular thermodynamic model for helix-helix docking and protein aggregation. AIChE J. 41: 1015–1024.

  35. 35.

    and 1986. Refolding and association of oligomeric proteins. Methods Enzymol. 131: 218–250.

  36. 36.

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

  37. 37.

    , , , , and 1996. Thermostable folding intermediates: inclusion body precursors and chaperonin substrates. FASEB J. 10: 57–66.

  38. 38.

    and 1986. Mutational analysis of protein folding pathways. Methods Enzymol. 131: 250–266.

  39. 39.

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

  40. 40.

    , , and 1980. Scaffolding proteins and the genetic control of virus shell assembly. Quart. Rev. Biol. 55: 369–393.

  41. 41.

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

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Author information

Author notes

    • Margaret A. Speed

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

    • Jonathan King

    e-mail: jaking@mit.edu

Affiliations

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

    • Daniel I. C. Wang
    •  & Jonathan King

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DOI

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