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. 2023:2681:47-60.
doi: 10.1007/978-1-0716-3279-6_4.

Facile One-Step Generation of Camelid VHH and Avian scFv Libraries for Phage Display by Golden Gate Cloning

Christina Bauer  1 , Elke Ciesielski  1 , Lukas Pekar  1 , Simon Krah  1 , Lars Toleikis  1 , Stefan Zielonka  1 , Carolin Sellmann  2
Affiliations

Facile One-Step Generation of Camelid VHH and Avian scFv Libraries for Phage Display by Golden Gate Cloning

Christina Bauer et al. Methods Mol Biol. 2023.

Abstract

Since its development in the 1980s, the Nobel Prize-awarded phage display technology has been one of the most commonly used in vitro selection technologies for the discovery of therapeutic and diagnostic antibodies. Besides the importance of selection strategy, one key component of the successful isolation of highly specific recombinant antibodies is the construction of high-quality phage display libraries. However, previous cloning protocols relied on a tedious multistep process with subsequent cloning steps for the introduction of first heavy and then light chain variable genetic antibody fragments (VH and VL). This resulted in reduced cloning efficiency, higher frequency of missing VH or VL sequences, as well as truncated antibody fragments. With the emergence of Golden Gate Cloning (GGC) for the generation of antibody libraries, the possibility of more facile library cloning has arisen. Here, we describe a streamlined one-step GGC strategy for the generation of camelid heavy chain only variable phage display libraries as well as the simultaneous introduction of heavy chain and light chain variable regions from the chicken into a scFv phage display vector.

Keywords: Antibody engineering; Camelid; Chicken; Golden Gate Cloning; Library generation; Phage display; Protein engineering; Single domain antibody; Type IIs restriction enzyme; VHH; scFv.

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