Abstract
Complete immunoglobulin molecules cannot be produced in Escherichia coli, but different strategies have been developed to express antibody fragments like single-chain antibodies (scFvs), disulfide stabilized antibodies (dsFvs), Fab fragments, bispecific antibodies (bsAbs) or various immunotoxins (overview in Breitling and Dübel, 1999). First, antibody fragments can be deposed as cytoplasmic inclusion bodies followed by refolding in vitro. This application is usually employed if the functional molecule is expected to kill the producing cell, e. g. for immunotoxins. Due to the general low folding efficiency, an alternative strategy has been developed which better imitates the folding conditions of antibodies in eucaryotic cells. The secretion of the antibody fragments into the periplasmic space of E. coli permits their production as soluble and functional proteins with correctly formed intramolecular disulfide bonds. The yields are usually lower than those of inclusion body refolding, however, it is the method of choice for a rapid assessment of antigen binding activity and specificity.
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References
Breitling, F. and Dübel, S. (1999) Recombinant Antibodies. John Wiley and Sons, New York.
Breitling, F. and Little, M. (1986) C-terminal regions on the surface of tubulin and microtubules. Epitope locations of Yol 1/34, DM 1 A and DM 1 B. J. Mol. Biol. 189, 367–370.
Dübel, S., Breitling, F., Klewinghaus, I. and Little, M. (1992) Regulated secretion and purification of recombinant antibodies in E. coli. Cell Biophysics 21, 69–80.
Dübel, S., Breitling, F., Fuchs, P., Klewinghaus, I. and Little, M. (1993) A family of vectors for surface display and production of antibodies. Gene 128, 97–101.
Dübel, S., Breitling, F., Kontermann, R., Schmidt, T., Skerra, A. and Little, M. (1995) Bifunctional and multimeric complexes of streptavidin fused to single chain antibodies (scFv). J. Immunol. Meth. 178, 201–209.
Evan, G. I., Lewis, G. K., Ramsay, G. and Bishop, J. M. (1985) Isolation of Monoclonal Antibodies Specific for Human c-myc Proto-Oncogene Product. Mol. Cell. Biol. 5, 3610–3616.
Kilmartin, J.V., Wright, B. and Milstein, C. (1982) Rat monoclonal antitubulin antibodies derieved by using a new nonsecreting rat cell line. J. Cell. Biol. 93, 576–582.
Kontermann, R.E., Liu, Z., Schulze, R.A., Sommer, K.A., Queitsch, I., Dübel, S., Kipriyanov, S.M., Breitling, F. and Bautz, E.K.F. (1995) Characterization of the epitope recognised by a monoclonal antibody directed against the largest subunit of Drosophila RNA polymerase II. Biol. Chem. Hoppe-Seyler 376, 473–481.
Krämer, A., Haars, R., Kabisch, R., Will, H., Bautz, F.A. and Bautz, E.K.F. (1980) Monoclonal Antibody Directed Against RNA Polymerase II of Drosophila melanogaster. Molec. gen. Genet. 180, 193–199.
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227, 680–685.
Lei, S.-P., Lin, H.-C., Wang, S.-S., Callaway, J. and Wilcox, G. (1987) Characterization of the Erwinia carotovora pelB gene and its product pectate lyase. J. Bacteriol. 169, 4379–4383.
Lilius, G., Person, M., Bülow, L., and Mosbach, K. (1991) Metal affinity precipitation of proteins carrying genetically attached polyhistidine tails. Eur. J. Biochem. 198, 499–504.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Towbin, H., Staehelin, T and Gordon, I. (1980) Electrophoretic transfer of protein from polyacrylamid gels to nitrocellulose sheets: procedures and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354.
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Schmiedl, A., Dübel, S. (2001). Functional Minipreps of scFv-Antibody Fragments. In: Kontermann, R., Dübel, S. (eds) Antibody Engineering. Springer Lab Manuals. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-662-04605-0_18
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DOI: https://doi.org/10.1007/978-3-662-04605-0_18
Publisher Name: Springer, Berlin, Heidelberg
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