Abstract
In the past few years, some of the limitations of monoclonal antibodies (MAbs) as therapeutic agents have been addressed by genetic engineering. Such an approach is particularly suitable because of the domain structure of the Ab molecule, where functional domains carrying antigen (Ag)-binding activities (Fabs or Fvs) or effector functions (Fcs) can be exchanged between Abs. Furthermore, genetically truncated versions of Ab can be produced, ranging in size from the smallest Ag-binding unit or Fv, to Fab′ and F(ab′)2s. To stabilize the association of recombinant VH and VL domains, they have been joined in scFv constructs with a short peptide linker (12)). These small scFvs are particularly interesting for clinical applications (3). They are only one half the size of Fabs and thus have lower retention times in nontarget tissues, more rapid blood clearance, and better tumor penetration. They are also less immunogenic and are amenable to fusions with proteins and peptides.
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References
Bird, R. E., Hardman, K. D., Jacobson, J. W., Johnson, S., Kaufman, B. M., Lee, S. M., et al. (1988) Single-chain antigen-binding proteins. Science 242, 423–426.
Huston, J. S., Levinson, D., Mudgett-Hunter, M., Tai, M. S., Novotny, J., Margolies, M. N., et al. (1988) Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. NatlAcad. Sci. USA 85, 5879–5883.
Huston, J. S., McCartney, J., Tai, M. S., Mottola-Hartshorn, C., Jin, D., Warren, R., Keck, P., and Oppermann, H. (1993) Medical applications of single-chain antibodies. Int. Rev. Immunol. 10, 195–217.
Kipriyanov, S. M. and Little, M. (1999) Generation of recombinant antibodies. Mol. Biotechnol. 12, 173–201.
Better, M., Chang, C. P., Robinson, R. R., and Horwitz, A. H. (1988) Escherichia coli secretion of an active chimeric antibody fragment. Science 240, 1041–1043.
Skerra, A. and Plückthun, A. (1988) Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science 240, 1038–1041.
Whitlow, M. and Filpula, D. (1991) Single-chain Fv proteins and their fusion proteins. Methods: Companion Methods Enzymol. 2, 97–105.
Kipriyanov, S. M., Dübel, S., Breitling, F., Kontermann, R. E., and Little, M. (1994) Recombinant single-chain Fv fragments carrying C-terminal cysteine residues: production of bivalent and biotinylated miniantibodies. Mol. Immunol. 31, 1047–1058.
Plückthun, A. (1994) Antibodies from Escherichia coli, in Handbook of Experimental Pharmacology, vol. 113: The Pharmacology of Monoclonal Antibodies(Rosenberg, M. and Moore, G. P., eds.) Springer-Verlag, Berlin, Heidelberg, pp. 269–315.
Hockney, R. C. (1994) Recent developments in heterologous protein production in Escherichia coli. Trends Biotechnol. 12, 456–463.
Knappik, A. and Plückthun, A. (1995) Engineered turns of a recombinant antibody improve its in vivo folding. Protein Eng. 8, 81–89.
Skerra, A. and Plückthun, A. (1991) Secretion and in vivo folding of the Fab fragment of the antibody McPC603 in Escherichia coli: influence of disulphides and cis-prolines. Protein Eng. 4, 971–979.
Kipriyanov, S. M., Moldenhauer, G., and Little, M. (1997) High level production of soluble single chain antibodies in small-scale Escherichia coli cultures. J. Immunol. Methods 200, 69–77.
Kipriyanov, S. M., Moldenhauer, G., Schuhmacher, J., Cochlovius, B., von der Lieth, C. W., Matys, E. R. and Little, M. (1999) Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics. J. Mol. Biol. 293, 41–56.
Skerra, A., Pfitzinger, I., and Plückthun, A. (1991) The functional expression of antibody Fv fragments in Escherichia coli: improved vectors and a generally applicable purification technique. Biotechnology 9, 273–278.
Casey, J. L., Keep, P. A., Chester, K. A., Robson, L., Hawkins, R. E., and Begent, R. H. (1995) Purification of bacterially expressed single chain Fv antibodies for clinical applications using metal chelate chromatography. J. Immunol. Methods 179, 105–116.
Kipriyanov, S. M., Moldenhauer, G., Strauss, G., and Little, M. (1998) Bispecific CD3 x CD 19 diabody for T cell-mediated lysis of malignant human B cells. Int. J. Cancer 77, 763–772.
Müller, K. M., Arndt, K. M., Bauer, K., and Plückthun, A. (1998) Tandem immobilized metal-ion affinity chromatography/immunoaffinity purification of His-tagged proteins: evaluation of two anti-His-tag monoclonal antibodies. Anal. Biochem. 259, 54–61.
Schulze, R. A., Kontermann, R. E., Queitsch, I., Dübel, S., and Bautz, E. K. (1994) Thiophilic adsorption chromatography of recombinant single-chain antibody fragments. Anal. Biochem. 220, 212–214.
Müller, K. M., Arndt, K. M., and Plückthun, A. (1998) A dimeric bispecific miniantibody combines two specificities with avidity. FEBS Let. 432, 45–49.
Horn, U., Strittmatter, W., Krebber, A., Knupfer, U., Kujau, M., Wenderoth, R., et al. (1996) High volumetric yields of functional dimeric miniantibodies in Escherichia coli, using an optimized expression vector and high-cell-density fermentation under non-limited growth conditions. Appl. Microbiol. Biotechnol. 46, 524–532.
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, 248–254.
Le Gall, F., Kipriyanov, S. M., Moldenhauer, G., and Little, M. (1999) Di-, triand tetrameric single chain Fv antibody fragments against human CD 19: effect of valency on cell binding. FEBS Let. 453, 164–168.
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Kipriyanov, S.M. (2002). High-Level Periplasmic Expression and Purification of scFvs. In: O’Brien, P.M., Aitken, R. (eds) Antibody Phage Display. Methods in Molecular Biology™, vol 178. Humana Press. https://doi.org/10.1385/1-59259-240-6:333
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DOI: https://doi.org/10.1385/1-59259-240-6:333
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