Research Paper | Published:

Renaturation, Purification and Characterization of Recombinant Fab-Fragments Produced in Escherichia coli

Bio/Technology volume 9, pages 157162 (1991) | Download Citation

Subjects

Abstract

Cytoplasmatic expression of murine antibody chains in Escherichia coli results in the formation of insoluble and inactive protein aggregates (inclusion bodies). By systematic variation of the parameters influencing the folding, formation of disul-fide bonds and association of the constituent polypeptide chains, we have designed a renaturation procedure allowing the production of microbially expressed Fab-fragments at yields up to 40 percent of the total amount of recombinant protein. The strategy of optimization is generally applicable for disulnde containing proteins produced as inclusion bodies in bacteria. The purified recombinant antibody fragments obtained are identical with the native murine Fab in all functional and phys-icochemical parameters tested.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

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

  2. 2.

    , , and 1984. Assembly for functional antibodies from immunoglobulin heavy and light chains synthesised in E. coli. Nucleic Acids Res. 12: 3791–3806.

  3. 3.

    , , , , , , , and 1984. Generation of antibody activity from immunoglobulin chains produced in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 81: 3273–3277.

  4. 4.

    , , , and 1987. Engineering of antibodies with a known three-dimensional structure. Cold Spring Harbor Symp. Quant. Biol. 52: 105–112.

  5. 5.

    , , , , , , , , and 1988. Single-chain antigen-binding proteins. Science 242: 423–426.

  6. 6.

    , and 1988. A functional recombinant immunoglobulin variable domain from polypeptides produced in Escherichia coli, p. 29–34. In: Ginsberg, H., Brown, F., Lerner, R. A., and Chanock, R. M. (Eds.). Vaccines 88, Cold Spring Harbor, NY.

  7. 7.

    , , , , , , , , , and 1988. Protein engineering of antibody binding sites: Recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 85: 5879–5883.

  8. 8.

    , , , , , , , and 1990. Bacterial expression of antibody fragments thai block human rhinovirus infection in cultured cells. J. Biol. Chem. 265: 2292–2295.

  9. 9.

    , , , , and 1987. Cloning and nucleotide sequence of heavy- and light-chain cDNAs from a creatine-kinase-specific monoclonal antibody. Gene 51: 13–19.

  10. 10.

    1987. Folding and association of proteins. Prog. Bio-phys. Mol. Biol. 49: 117–237.

  11. 11.

    1989. Control of protein exit from the endoplas-matic reticulum. Ann. Rev. Cell Biol. 5 1–23.

  12. 12.

    and 1989. Folding proteins, p. 191–223. In: Protein Structure, a Practical Approach. Creighton, T. E. (Ed.). IRL Press, Oxford.

  13. 13.

    1990. Renaturation of recombinant, disulfide-bonded proteins from “inclusion bodies”, p. 149–171. In: Modern Methods in Protein and Nucleic Acid Analysis. Tschesche, H. (Ed.) Walter de Gruyter, Berlin, New York.

  14. 14.

    , and 1990. In preparation.

  15. 15.

    and 1970. Formation of three-dimensional structure in proteins. I. Rapid nonenzymatic reactivation of reduced lysozyme. Biochemistry 9: 5015–5021.

  16. 16.

    , and 1990. Aktivierung von gentechnologisch hergestellten, in Prokaryonten exprimierten Antikörpern. Eur. Patent Application 0364926.

  17. 17.

    and 1979. Refolding of the mixed disulfide of bovine trypsinogen and gluthathione. J. Biol. Chem. 254: 4291–4295.

  18. 18.

    and 1978. The renaturation of reduced chymotrypsinogen A in guanidine HCl: Refolding versus aggregation. J. Biol. Chem. 253: 3453–3458.

  19. 19.

    , and 1987. Process for the activating of gene-technologically produced, heterologous, disulfide bridge-containing eucaryotic proteins after expression in procary-otes. International Patent Application WO 87/02673.

  20. 20.

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

  21. 21.

    , and 1990. A kinetic study of the competition between renaturation and aggregation during the refolding of denatured-reduced egg-white lysozyme. Biochemistry. In press.

  22. 22.

    , , and 1990. Formation of protein inclusion bodies: Kinetic competition between folding and aggregation. Submitted.

  23. 23.

    , and 1982. The yield of reactivation of lactic dehydrogenase after guanidine·HCl denatur-ation is not determined by proline cis-trans isomerization. Eur. J. Biochem. 125: 605–608.

  24. 24.

    , , , , and 1990. Chaperonin-facilitated refolding of ribulosebisphosphate carboxylase and ATP hydrolysis of chaperonin 60 groEL are K+ dependent. Biochemistry 29: 5665–5671.

  25. 25.

    and 1981. Formation of the intrachain disulfide bond in the constant fragment of the immunoglobulin light chain. J. Mol. Biol. 146: 321–340.

  26. 26.

    and 1985. Reacquisition of quarternary structure by fully reduced and denatured seminal ribonuclease. Eur. J. Biochem. 149: 381–387.

  27. 27.

    and 1990. Designer and catalytic antibodies. New England J. Med. 323: 173–178.

  28. 28.

    1988. Active immunoglobulin fragments synthesized in E. coli-from Fab to Scantibodies. Protein Engineering 2: 169–170.

  29. 29.

    and 1990. A comparison of strategies to stabilize immunoglobulin Fv-fragments. Biochemistry 29: 1362–1367.

  30. 30.

    , and 1988. Isolation and analysis of the gene encoding peripheral myelin protein zero. Neuron 1: 73–83.

  31. 31.

    , , 1990. Recombinant peripheral myelin protein po conferes both the adhesion and neural outgrowth promoting function. J. Neuroscience Research. 27: 286–297.

  32. 32.

    1988. Expression von Fd-Fragment des monoklonalen Antikörpers MAK33 in E. coli. Thesis, Universität Stuttgart.

  33. 33.

    1975. 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.

  34. 34.

    1986. Practical considerations in enzyme-immuno-assays illustrated by a model system, p. 15–37. In: Methods of Enzymatic Analysis, 3rd Edition, Bergmeyer, H. U. (Ed.). Verlag Chemie, Weinheim.

  35. 35.

    1974. Glutathion-reductase, p. 494–495. In: Methoden der Enzymatischen Analyse, Verlag Chemie, Weinheim.

  36. 36.

    1970. Clevage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–683.

Download references

Author information

Author notes

    • Rainer Rudolph

    Corresponding author.

Affiliations

  1. Universität Regensburg, Institut für Biophysik and Physikalische Biochemie, Postfach, D-8400 Regensburg, FRG.

    • Johannes Buchner
  2. Boehringer Mannheim GmbH, Biochemical Research Center, Nonnenwald 2, D-8122 Penzberg, FRG.

    • Rainer Rudolph

Authors

  1. Search for Johannes Buchner in:

  2. Search for Rainer Rudolph in:

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nbt0291-157

Further reading