Abstract
The periplasmic expression in E. coli has become the standard technology for preparing functional antibody fragments in a rapid way (Skerra and Plückthun 1988, Plückthun et al. 1996). The consequences of choosing Fab or scFv fragments, the properties of suitable expression vectors or the influence of the E. coli strain have been extensively summarized elsewhere (Plückthun et al. 1996). Even when paying attention to all these components and experimental conditions, it has become clear that the yield of recombinant antibody fragments is variable and these variations are a direct consequence of their primary sequences (Wörn and Plückthun 2001). The periplasmic folding is the yield limiting step and is most strongly influenced by the sequence.
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References
Bothmann H, Plückthun A (1998) Selection for a periplasmic factor improving phage display and functional periplasmic expression. Nature Biotechnol 16:376–380
Bothmann H, Plückthun A(2000) The periplasmic E. coli peptidylprolyl cis, trans-isomerase FkpA (I): increased functional expression of antibody fragments with and without cis-prolines. J Biol Chem 275:17100–17105
de Cock H, Schäfer U, Poteger M, Demel R, Müller M, Tommassen J (1999) Affinity of the periplasmic chaperone Skp of Escherichia coli for phospholipids, lipopolysaccharides and non-native outer membrane proteins. Eur J Biochem 259:96–103
Hayhurst A, Harris WJ (1999) Escherichia coli Skp chaperone coexpression improves solubility and phage display of single-chain antibody fragments
Jung S, Plückthun A (1997) Improving in vivo folding and stability of a single-chain Fv antibody fragment by loop grafting. Prot Eng 10:959–966
Jung S, Honegger A, Plückthun A (1999) Selection for improved protein stability by phage display. J Mol Biol 294:163–180
Knappik A, Plückthun A (1995) Engineered turns of a recombinant antibody improve its in vivo folding. Prot Eng 8:81–89
Krebber A, Bornhauser S, Burmester J, Honegger A, Willuda J, Bosshard HR, Plückthun A (1997) Reliable cloning of functional antibody domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J Immunol Meth 201:35–55
Missiakas D, Betton JM, Raina S (1996) New components of protein folding in extracytoplasmic compartments of Escherichia coli SurA, FkpA and Skp/OmpH. Mol Microbiol 21:871–884
Nieba L, Honegger A, Krebber C, Plückthun A (1997) Disrupting the hydrophobic patches at the antibody variable/constant domain interface: improved in vivo folding and physical characterization of an engineered scFv fragment. Prot Eng 10:435–444
Plückthun A, Krebber A, Krebber C, Horn U, Knüpfer U, Wenderoth R, Nieba L, Proba K, Riesenberg D (1996) Producing antibodies in Escherichia coli: From PCR to fermentation. In: McCafferty J, Hoogenboom H, Chiswell D (eds) Antibody Engineering. A practical approach, Oxford University Press, Oxford, pp 203–252
Proba K, Wörn A, Honegger A, Plückthun A (1998) Antibody scFv fragments without disulfide bonds made by molecular evolution. J Mol Biol 275:245–253
Ramm K, Plückthun A (2000) The periplasmic E. coli peptidyl-prolyl isomerase FkpA (II): isomerase-independent chaperone activity in vitro. J Biol Chem 275:17106–17113
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York
Schäfer U, Beck K, Müller M (1999) Skp, a molecular chaperone of Gram-negative bacteria, is required for the formation of soluble periplasmic intermediates or outer membrane proteins. J Biol Chem 274:24567–24574
Skerra A, Plückthun A (1988) Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science 263:14315–14322
Thorpe SJ, Kerr MA (1994) Common immunological techniques: ELISA, blotting, immunohistochemistry and immunocytochemistry. In: Kerr MA, Thorpe R (eds) Immunochemistry. Labfax, BIOS Scientific Publishers Limited, Oxford, pp 175–209
Willuda J, Honegger A, Waibel R, Schubiger PA, Stahel R, Zangenmeister-Wittke U, Plückthun A (1999) High thermal stability is essential for tumor targeting of antibody fragments: Engineering of a humanized anti-epithelial glycoprotein-2 (epithelial cell adhesion molecule) single-chain Fv fragment. Cancer Research 59:5758–5767
Wörn A, Plückthun A (2001) Stability engineering of antibody single-chain Fv fragments. J Mol Biol, 305: 989–1010
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© 2001 Springer-Verlag Berlin Heidelberg
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Bothmann, H., Plückthun, A. (2001). Improving Expression of scFv Fragments by Coexpression of Periplasmic Chaperones. In: Kontermann, R., Dübel, S. (eds) Antibody Engineering. Springer Lab Manuals. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-662-04605-0_23
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DOI: https://doi.org/10.1007/978-3-662-04605-0_23
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