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Rapid Selection of High-Affinity Antibody scFv Fragments Using Ribosome Display.


Dreier, Birgit; Plückthun, Andreas (2018). Rapid Selection of High-Affinity Antibody scFv Fragments Using Ribosome Display. In: Nevoltris, Damien; Chames, Patrick. Antibody Engineering. Heidelberg: Springer, 235-268.

Abstract

Ribosome display has proven to be a powerful in vitro selection and evolution method for generating high-affinity binders from libraries of folded proteins. It works entirely in vitro, and this has two important consequences. First, since no transformation of any cells is required, libraries with much greater diversity can be handled than with most other techniques. Second, since a library does not have to be cloned and transformed, it is very convenient to introduce random errors in the library by PCR-based methods and select improved binders. Thus, a true directed evolution, an iteration between randomization and selection over several generations, can be conveniently carried out, e.g., for affinity maturation, either on a given clone or on the whole library. Ribosome display has been successfully applied to antibody single-chain Fv fragments (scFv), which can be selected not only for specificity but also for stability and catalytic activity. High-affinity binders with new target specificity can be obtained from highly diverse libraries in only a few selection rounds. In this protocol, the selection from the library and the process of affinity maturation and off-rate selection are explained in detail.

Abstract

Ribosome display has proven to be a powerful in vitro selection and evolution method for generating high-affinity binders from libraries of folded proteins. It works entirely in vitro, and this has two important consequences. First, since no transformation of any cells is required, libraries with much greater diversity can be handled than with most other techniques. Second, since a library does not have to be cloned and transformed, it is very convenient to introduce random errors in the library by PCR-based methods and select improved binders. Thus, a true directed evolution, an iteration between randomization and selection over several generations, can be conveniently carried out, e.g., for affinity maturation, either on a given clone or on the whole library. Ribosome display has been successfully applied to antibody single-chain Fv fragments (scFv), which can be selected not only for specificity but also for stability and catalytic activity. High-affinity binders with new target specificity can be obtained from highly diverse libraries in only a few selection rounds. In this protocol, the selection from the library and the process of affinity maturation and off-rate selection are explained in detail.

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Additional indexing

Item Type:Book Section, refereed, further contribution
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2018
Deposited On:05 Dec 2018 15:14
Last Modified:24 Sep 2019 23:55
Publisher:Springer
ISSN:1064-3745
OA Status:Closed
Publisher DOI:https://doi.org/10.1007/978-1-4939-8648-4_13
PubMed ID:30196501

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