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Renaturation of a Single–Chain Immunotoxin Facilitated by Chaperones and Protein Disulfide Isomerase

Bio/Technology volume 10, pages 682–685 (1992)Cite this article

Abstract

B3(Fv)–PE38KDEL, a recombinant immunotoxin, forms inclusion bodies when produced in Escherichia coli. In renaturation experiments, nonspecific aggregation of non–native polypeptide chains, and the formation of incorrect disulfide linkages lead to inactive molecules. To prevent these side reactions, we added molecular chaperones and protein disulfide isomerase (PDI) to the refolding buffer. Both DnaK and GroEL/S influenced the reactivation process. GroEL alone inhibited reactivation, but in the presence of ATP, GroEL and GroES significantly increased the yield of active protein. DnaK also increased the yield of properly folded protein and the stimulating effect of DnaK was also observed using immobilized DnaK, which can be used repeatedly without significant loss of activity. PDI, which catalyzes disulfide bridging of proteins, also stimulated reactivation of the immunotoxin. Under optimum conditions, reactivation yields in the presence of PDI were about twice that obtained with nonenzymatic disulfide bond formation. Furthermore, DnaK and PDI were additive when renaturation was performed in the presence of both proteins.

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References

  1. Jaenicke, R. 1987. Folding and association of proteins. Prog. Biophys. Mol. Biol. 49: 117–237.

    Article  CAS  Google Scholar 

  2. Jaenicke, R. 1991. Protein folding: local structures, domains, subunits and assemblies. Biochemistry 30: 3147–3161.

    Article  CAS  Google Scholar 

  3. Rudolph, R. 1990. Renaturation of recombinant, disulfide-bonded proteins from inclusion bodies. p. 149–171. In: Modern Methods in Protein and Nucleic Acid Research. H. Tschesche (Ed.). Walter DeGruyter, Berlin.

    Google Scholar 

  4. Buchner, J. and Rudolph, R. 1991. Renaturation, purification and characterization of recombinant Fab-fragments produced in Escherichia coli. Bio/Technology 9: 157–162.

    CAS  PubMed  Google Scholar 

  5. Ellis, J.R. and van der Vies, S.M. 1991. Molecular chaperones. Ann. Rev. Biochem. 60: 321–348.

    Article  CAS  Google Scholar 

  6. Skowyra, D., Georgopoulos, C. and Zylicz, M. 1990. The E. coli DnaK gene product, the hsp70 homolog can reactivate heat-inactivated RNA polymerase in an ATP hydrolysis-dependent manner. Cell 62: 939–944.

    Article  CAS  Google Scholar 

  7. Goloubinoff, P., Christelle, S.T., Gatenby, A.A. and Lorimer, G.J. 1989. Reconstitution of active dimeric ribulose biphosphate carboxylase from an unfolded state depends on two chaperonine proteins and Mg-ATP. Nature 342: 884–885.

    Article  CAS  Google Scholar 

  8. Buchner, J., Schmidt, M., Fuchs, M., Jaenicke, R., Rudolph, R., Schmid, F.X. and Kiefhaber, T. 1991. GroE facilitates refolding of citrate synthase by suppressing aggregation. Biochemistry 30: 1587–1591.

    Google Scholar 

  9. Martin, J., Mendoza, J.A., Rogers, E., Lorimer, G.H. and Horowitz, P. 1991. Chaperonines facilitate the in vitro folding of monomeric mitochondrial rhodanese. J. Biol. Chem. 266: 13044–13049.

    Google Scholar 

  10. Laminet, A.A., Ziegelhofer, T., Georgopoulos, C. and Pluckthun, A. 1990. The Escherichia coli heat shock proteins GroEL and GroES modulate the folding of the β-lactamase precursor. EMBO J. 9: 2315–2319.

    Article  CAS  Google Scholar 

  11. Holl-Neugebauer, B., Rudolph, R., Schmidt, M. and Buchner, J. 1992. Reconstitution of a heat shock effect in vitro: influence of GroE on the thermal aggregation of α-glucosidase from yeast. Biochemistry In press.

  12. Mendoza, S.A., Rogers, E., Lorimer, G.H. and Horowitz, P.M. 1991. Chaperonine facilitate the in vitro folding of monomeric mitochondrial rhodanese. J. Biol. Chem. 266: 13044–13049.

    CAS  PubMed  Google Scholar 

  13. Gatenby, A.A., Viitanen, P.V. and Lorimer, G.H. 1990. Chaperonine assisted polypeptide folding and assembly: implications for the production of functional proteins in bacteria. Trends Biotechnol. 8: 354–358.

    Article  CAS  Google Scholar 

  14. Buchner, J. and Rudolph, R. 1991. Routes to active proteins from transformed microorganisms. Current Opinion in Biotechnolo. 2: 532–538.

    Article  CAS  Google Scholar 

  15. Bulleid, N.J. and Freedman, R.B. 1990. Defective co-translational formation of disulfide bonds in protein disulfide-isomerase-deficient microsomes. Nature 335: 649–657.

    Article  Google Scholar 

  16. Freedman, R.B., Bulleid, N.J., Hawkins, H.C. and Paver, J.L. 1989. Role of protein disulfide isomerase in the expression of native proteins. Biochem. Soc. Symp. 55: 167–192.

    CAS  Google Scholar 

  17. Buchner, J., Pastan, I. and Brinkmann, U. 1992. Refolding of a single-chain immunotoxin from bacterial inclusion bodies. Anal. Biochem. In press.

  18. Brinkmann, U., Pai, L.H., FitzGerald, D.J., Willingham, M. and Pastan, I. 1991. B3(Fv)-PE38KDEL, a single-chain immunotoxins that causes complete regression of a human carcinoma in mice. Proc. Natl. Acad. Sci. USA 88: 8616–8620.

    Article  CAS  Google Scholar 

  19. Pastan, I., Lovelace, E.T., Gallo, M.G., Rutherford, A.V., Magnani, J.I. and Willingham, M.C. 1991. Characterization of monoclonal antibodies B1 and B3 that react with mucinous adenocarcinomas. Cancer Res. 51: 3781–3787.

    CAS  Google Scholar 

  20. Pai, L.H., FitzGerald, D.J., Willingham, M.C. and Pastan, I. 1991. Anti-tumor activities of immunotoxins made of monoclonal antibody B3 and various forms of Pseudomonas exotoxin. Proc. Natl. Acad. Sci. USA 88: 3358–3362.

    Article  CAS  Google Scholar 

  21. Brinkmann, U., Buchner, J. and Pastan, I. 1992. Independent domain folding of Pseudomonas exotoxin and single-chain immunotoxins: Influence of interdomain connectors. Proc. Natl. Acad. Sci. USA 89: 3075–3079.

    Article  CAS  Google Scholar 

  22. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 241–245.

    Article  Google Scholar 

  23. Studier, F.W. and Moffatt, B.A. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189: 113–130.

    Article  CAS  Google Scholar 

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Author notes

  1. Johannes Buchner

    Present address: Institut fuer Biophysik und Physikalische Biochemie, Universitaet Regensburg, Universitaetsstr. 31, D-8400, Regensburg, Germany

  2. Ira Pastan: Corresponding author.

Authors and Affiliations

  1. Laboratory of Molecular Biology, Division of Cancer Biology, Diagnosis and Centers, National Cancer Institute, National Institutes of Health, 9000 Rockville Pike, 37/4E16, Bethesda, MD, 20892

    Ulrich Brinkmann & Ira Pastan

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  1. Johannes Buchner
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  2. Ulrich Brinkmann
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Buchner, J., Brinkmann, U. & Pastan, I. Renaturation of a Single–Chain Immunotoxin Facilitated by Chaperones and Protein Disulfide Isomerase. Nat Biotechnol 10, 682–685 (1992). https://doi.org/10.1038/nbt0692-682

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