Abstract
Many recombinant proteins, particularly proteins with diagnostic and therapeutic potential such as antibodies, lymphokines, receptors, enzymes, and enzyme-inhibitors, are being produced from transformed host cells containing recombinant DNA. The host cells are transformed with an expression vector containing genes encoding for the proteins of interest, and then are cultured under conditions that favor the production of the desired protein. When Escherichia coli is used for the expression of recombinant proteins, the heterologous protein produced often precipitates within the cell to form refractile (inclusion) bodies. For the isolation of the recombinant protein in the native (biologically active) state, it should be separated from cell debris, solubilized with a chaotropic agent such as high concentrations of urea or guanidinium hydrochloride, then refolded by gradual removal of the denaturant (reviewed in ref. 1). In cases where the native state of the protein is dependent on the formation of disulfide bonds, the protein is reduced while in the denatured state by addition of reducing agents. The formation of disulfide bonds of the protein during the refolding process is facilitated by redox-shuffling induced by the inclusion of reducing and oxidizing agents in the refolding buffer (2).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Lilie H., Schwarz E., and Rudolph R. (1998). Advances in refolding of proteins produced in E. coli. Curr. Opin. Biotechnol. 9, 497–501.
Buchner J., Pastan I., and Brinkmann U. (1992). A method for increasing the yield of properly folded recombinant fusion proteins: single-chain immunotoxins from renaturation of bacterial inclusion bodies. Anal. Biochem. 205, 263–270.
Skerra A. (1993). Bacterial expression of immunoglobulin fragments. Curr. Opin. Immunol. 5, 256–262.
Kreitman R. J. and Pastan I. (1997). Immunotoxins for targeting cancer and autoimmune disease, in Antibody Therapeutics (Harris W. J. and Adair J. R., eds.) CRC Press Boca Raton, FL, pp. 33–52.
Yarranton G. (1997). Antibodies as carriers for drugs and radioisotopes, in Antibody Therapeutics (Harris W. J. and Adair J. R., eds.) CRC Press Boca Raton, FL, pp. 53–72.
Benhar I. and Pastan I. (1997). Tumor targeting by antibody-drug conjugates, in Antibody Therapeutics (Harris W. J. and Adair J. R., eds) CRC Press Boca Raton, FL, pp. 73–85.
Kiefhaber T., Rudolph R., Kohler H. H., and Buchner J. (1991). Protein aggregation in vitro and in vivo: a quantitative model of the kinetic competition between folding andaggregation. Biotechnology (NY) 9, 825–829.
Guise A. D., West S. M., and Chaudhuri J. B. (1996). Protein folding in vivo and renaturation of recombinant proteins from inclusion bodies. Mol. Biotechnol. 6, 53–64.
Fenton W. A., and Horwich A. L. (1997). GroEL-mediated protein folding. Protein Sci. 6, 743–760.
Creighton T. E. (1985). Folding of proteins adsorbed reversibly to ion-exchange resins, in UCLA Symposia on Molecular and Cellular Biology New Series, vol. 39 (Oxender D. L., ed.), Liss, Inc. New York, pp. 249–258.
Glansbeek H. L., van Beuningen H. M., Vitters E. L., van der Kraan P. M., and van den Berg W. B. (1998). Expression of recombinant human soluble type II transforming growth factor-beta receptor in Pichia pastoris and Escherichia coli: two powerful systems to express a potent inhibitor of transforming growth factor-beta. Protein Exp. Purif. 12, 201–207.
Stempfer G., Holl-Neugebauer B., and Rudolph R. (1996). Improved refolding of an immobilized fusion protein. Nat. Biotechnol. 14, 329–34.
Garel J-R. (1992). Large multidomain and multisubunit proteins, in Protein Folding (Creighton T. E., ed.), W. H. Freeman and Company NY, pp. 405–454.
Bayer E. A., Morag E., and Lamed R. (1994). The cellulosome: a treasuretrove for biotechnology. Trends Biotechnol. 12, 379–386.
Ong E., Greenwood J. M., Gilkes N. R., Kilburn D. G., Miller R. C. J., and Warren R. A. J. (1989). The cellulose-binding domains of cellulases: tools for biotechnology. Trends Biotechnol. 7, 239–243.
Assouline Z., Shen H., Kilburn D. G., and Warren R. A. J. (1993). Production and properties of a Factor-X-cellulose binding domain fusion protein. Protein Eng. 6, 787–792.
Ramirez C., Fung J., Miller R. C. J., Warren R. A. J., and Kilburn D. G. (1993). A bifunctional affinity linker to couple antibodies to cellulose. Bio/Technology 11, 1570–1573.
Shoseyov O. and Karmely Y. (1995) Kits and methods of detection using cellulose binding domain fusion proteins. US Patent Application No. 460, 458.
Morag E., Bayer E. A., and Lamed R. (1992). Affinity digestion for the near-total recovery of purified cellulosome from Clostridium thermocellum. Enzyme Microb. Technol. 14, 289–292.
Berdichevsky Y., Ben-Zeev E., Lamed R., and Benhar I. (1999). Phage display of a cellulose binding domain from Clostridium thermocellum and its application as a tool for antibody engineering. J. Immunol. Methods 228, 151–162.
Berdichevsky Y., Lamed R., Frenkel D., Gophna U., Bayer E. A., Yaron S., et al. (1999). Matrix-assisted refolding of single-chain Fv-cellulose binding domain fusion proteins. Protein Expr. Purif. 17, 249–259.
Tsumoto K., Shinoki K., Kondo H., Uchikawa M., Juji T., and Kumagai I. (1998). Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent-application to a human single-chain Fv fragment. J. Immunol. Methods 219, 119–129.
Brizzard B. L., Chubet R. G., and Vizard D. L. (1994). Immunoaffinity purification of FLAG epitope-tagged bacterial alkaline phosphatase using a novel monoclonal antibody and peptide elution. Biotechniques 16, 730–735.
Sambrook J. L., Fritsch E. F., and Maniatis T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. 1989. Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY.
Hoogenboom H. R. and Winter G (1992). By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro. J. Mol. Biol. 227, 381–388
Benhar I., Tamarkin A., Marash L., Berdichevsky Y., Yaron S., Shoham Y., et al. (2001). Phage display of cellulose binding domains for biotechnological application, in Glycosyl Hydrolases for Biomass Conversion. ACS Symposium Series 769 (Himmel M.E., Baker J. O., and Saddler J. N., eds.), American Chemical Society Washington, DC, pp. 168–189.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2003 Humana Press Inc.
About this protocol
Cite this protocol
Benhar, I., Berdichevsky, Y. (2003). Large Scale Production of Recombinant Antibodies by Utilizing Cellulose-Binding Domains. In: Welschof, M., Krauss, J. (eds) Recombinant Antibodies for Cancer Therapy. Methods in Molecular Biology™, vol 207. Humana Press. https://doi.org/10.1385/1-59259-334-8:443
Download citation
DOI: https://doi.org/10.1385/1-59259-334-8:443
Publisher Name: Humana Press
Print ISBN: 978-0-89603-918-6
Online ISBN: 978-1-59259-334-7
eBook Packages: Springer Protocols