Summary
The isolation of recombinant antibodies by phage display naturally leads to experiments to evaluate their biological and immunological properties. Although crude preparations may have their value in initial studies, the need often exists for highly purified protein that can be tested in vivo. This chapter describes methods to generate high yields of scFv from bacterial cultures and to purify protein to the degree of homogeneity required for the most exacting analysis.
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
Bird, R. E., Hardman, K. D., Jacobson, J. W., Johnson, S., Kaufman, B. M., Lee, S. M., Lee, T., Pope, S. H., Riordan, G. S., and Whitlow, M. (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., Ridge, R. J., Bruccoleri, R. E., Haber, E., Crea, R., and Oppermann, H. (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. USA 85, 5879–5883
Huston, J. S., McCartney, J., Tai, M. S., Mottola Hartshorn, C., Jin, D., Warren, F., Keck, P., and Oppermann, H. (1993) Medical applications of single-chain antibodies. Int. Rev. Immunol. 10, 195–217
Huhalov, A., and Chester, K. A. (2004) Engineered single chain antibody fragments for radioimmunotherapy. Q. J. Nucl. Med. Mol. Imaging 48, 279–288
Bhatti, M., Yahioglu, G., Milgrom, L. R., Garcia-Maya, M., Chester, K. A., and Deonarain, M. P. (2008) Targeted photodynamic therapy with multiply-loaded recombinant antibody fragments. Int. J. Cancer 122, 1155–1163
Villa, A., Trachsel, E., Kaspar, M., Schliemann, C., Sommavilla, R., Rybak, J. N., Rosli, C., Borsi, L., and Neri, D. (2008) A high-affinity human monoclonal antibody specific to the alternatively spliced EDA domain of fibronectin efficiently targets tumor neo-vasculature in vivo. Int. J. Cancer 122, 2405–2413
Balza, E., Mortara, L., Sassi, F., Monteghirfo, S., Carnemolla, B., Castellani, P., Neri, D., Accolla, R. S., Zardi, L., and Borsi, L. (2006) Targeted delivery of tumor necrosis factor-alpha to tumor vessels induces a therapeutic T cell-mediated immune response that protects the host against syngeneic tumors of different histologic origin. Clin. Cancer Res. 12, 2575–2582
Borsi, L., Balza, E., Carnemolla, B., Sassi, F., Castellani, P., Berndt, A., Kosmehl, H., Biro, A., Siri, A., Orecchia, P., Grassi, J., Neri, D., and Zardi, L. (2003) Selective targeted delivery of TNF-alpha to tumor blood vessels. Blood 102, 4384–4392
Halin, C., Niesner, U., Villani, M. E., Zardi, L., and Neri, D. (2002) Tumor-targeting properties of antibody-vascular endothelial growth factor fusion proteins. Int. J. Cancer 102, 109–116
Kaspar, M., Trachsel, E., and Neri, D. (2007) The antibody-mediated targeted delivery of interleukin-15 and GM-CSF to the tumor neovasculature inhibits tumor growth and metastasis. Cancer Res. 67, 4940–4948
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
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
Whitlow, M., and Filpula, D. (1991) Single-chain Fv proteins and their fusion proteins. Methods: A Companion to Methods Enzymol. 2, 97–105
Hockney, R. C. (1994) Recent developments in heterologous protein production in Escherichia coli. Trends Biotechnol. 12, 456–463
Plückthun, A. (1994) Antibodies from Escherichia coli. In The Pharmacology of Monoclonal Antibodies (Rosenberg, M., and Moore, G. P., eds.) Vol. 113 pp.269–315, Springer, Berlin, Heidelberg
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
Bothmann, H., and Plückthun, A. (1998) Selection for a periplasmic factor improving phage display and functional periplasmic expression. Nat. Biotechnol. 16, 376–380
Bothmann, H., and Plückthun, A. (2000) The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA. I. Increased functional expression of antibody fragments with and without cis-prolines. J. Biol. Chem. 275, 17100–17105
Ramm, K., and Pluckthun, A. (2000) The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA. II. Isomerase-independent chaperone activity in vitro. J. Biol. Chem. 275, 17106–17113
Ramm, K., and Plückthun, A. (2001) High enzymatic activity and chaperone function are mechanistically related features of the dimeric E. coli peptidyl-prolyl-isomerase FkpA. J. Mol. Biol. 310, 485–498
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 CD19 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
Müller, K. M., Arndt, K. M., and Plückthun, A. (1998) A dimeric bispecific miniantibody combines two specificities with avidity. FEBS Lett. 432, 45–49
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
Horn, U., Strittmatter, W., Krebber, A., Knupfer, U., Kujau, M., Wenderoth, R., Müller, K., Matzku, S., Plückthun, A., and Riesenberg, D. (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
Le Gall, F., Reusch, U., Moldenhauer, G., Little, M., and Kipriyanov, S. M. (2004) Immunosuppressive properties of anti-CD3 single-chain Fv and diabody. J. Immunol. Methods 285, 111–127
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-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding. FEBS Lett. 453, 164–168
Kipriyanov, S. M., Dübel, S., Breitling, F., Kontermann, R. E., Heymann, S., and Little, M. (1995) Bacterial expression and refolding of single-chain Fv fragments with C-terminal cysteines. Cell Biophys. 26, 187–204
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Kipriyanov, S.M. (2009). High-Level Periplasmic Expression and Purificationof scFvs. In: Aitken, R. (eds) Antibody Phage Display. Methods in Molecular Biology, vol 562. Humana Press. https://doi.org/10.1007/978-1-60327-302-2_16
Download citation
DOI: https://doi.org/10.1007/978-1-60327-302-2_16
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-301-5
Online ISBN: 978-1-60327-302-2
eBook Packages: Springer Protocols