Antibody fragments have gained strong attention in the last years and presumably represent one of the most promising molecules to construct new types of therapeutic proteins. Contrary to complete IgG, antibody fragments like scFv are not very stable and are frequently expressed at a very low level. This has prompted the development of new phage-displayed antibody libraries based on optimized scFv frameworks. In this protocol, we will present the construction of such a library based on a single scFv framework with randomized CDR3 sequences. Many variations of this basic protocol are possible and discussed.
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References
Adams GP, Weiner LM (2005) Monoclonal antibody therapy of cancer. Nat Biotechnol 23:1147–1157
Demarest SJ, Glaser SM (2008) Antibody therapeutics, antibody engineering, and the merits of protein stability. Curr Opin Drug Discov Devel 11:675–687
Donini M, Morea V, Desiderio A, Pashkoulov D, Villani ME, Tramontano A, Benvenuto E (2003) Engineering stable cytoplasmic intrabodies with designed specificity. J Mol Biol 330:323–332
Holliger P, Hudson PJ (2005) Engineered antibody fragments and the rise of single domains. Nat Biotechnol 23:1126–1136
Jung S, Pluckthun A (1997) Improving in vivo folding and stability of a single-chain Fv antibody fragment by loop grafting. Protein Eng 10:959–966
Knappik A, Ge L, Honegger A, Pack P, Fischer M, Wellnhofer G, Hoess A, Wolle J, Pluckthun A, Virnekas B (2000) Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol 296:57–86
Martineau P, Jones P, Winter G (1998) Expression of an antibody fragment at high levels in the bacterial cytoplasm. J Mol Biol 280:117–127
Philibert P, Stoessel A, Wang W, Sibler A, Bec N, Larroque C, Saven JG, Courtete J, Weiss E, Martineau P (2007) A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm. BMC Biotechnol 7:81
Sidhu SS, Lowman HB, Cunningham BC, Wells JA (2000) Phage display for selection of novel binding peptides. Methods Enzymol 328:333–363
Silacci M, Brack S, Schirru G, Marlind J, Ettorre A, Merlo A, Viti F, Neri D (2005) Design, construction, and characterization of a large synthetic human antibody phage display library. Proteomics 5:2340–2350
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Martineau, P. (2010). Synthetic Antibody Libraries. In: Kontermann, R., Dübel, S. (eds) Antibody Engineering. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-01144-3_6
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DOI: https://doi.org/10.1007/978-3-642-01144-3_6
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