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A One-Step Process for the Construction of Phage Display scFv and VHH Libraries

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Abstract

In this work we present a one-step cloning approach for the establishment of antibody phage display libraries relying on type IIs restriction enzymes. We show that single chain variable fragment (scFv) libraries with adequate qualities can readily be cloned in a ‘scar-less’ manner and that the isolation of antigen-specific antibodies from immunized chickens is feasible within three selection rounds. Moreover, we demonstrate the general applicability of this method by rapidly constructing and panning VHH single domain antibody phage display libraries from immunized Llama repertoires.

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Abbreviations

BLI:

Bio-layer interferometry

BSA:

Bovine serum albumin

CDR:

Complementarity-determining region

ECD:

Extracellular domain

EGFR:

Epidermal Growth Factor Receptor

ELISA:

Enzyme-linked immunosorbent assay

Fab:

Antigen-binding fragment

GGC:

Golden Gate Cloning

IPTG:

Isopropyl-beta-d-thiogalacto pyranoside

MTP:

Microtiter plate

PBS:

Phosphate-buffered saline

RT:

Room temperature

ScFv:

Single chain variable fragment

TMB:

3,3′,5,5′-Tetramethylbenzidne

VH:

Variable domain of the heavy chain

VHH:

Variable domain of the heavy chain of a heavy chain only antibody

VL:

Variable domain of the light chain

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Acknowledgements

We are grateful to Prof. Harald Kolmar for providing RNA and cDNA from EGFR-immunized chicken.

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Author notes

  1. Carolin Sellmann and Lukas Pekar contributed equally to this work.

Authors and Affiliations

  1. Protein Engineering and Antibody Technologies, Merck Healthcare KGaA, Frankfurter Strasse 250, 64293, Darmstadt, Germany

    Carolin Sellmann, Lukas Pekar, Christina Bauer, Elke Ciesielski, Simon Krah, Stefan Becker, Lars Toleikis & Stefan Zielonka

  2. YUMAB GmbH, Inhoffenstraße 7, 38124, Braunschweig, Germany

    Jonas Kügler, André Frenzel & Michael Hust

  3. Chemical and Pharmaceutical Development, Merck Healthcare KGaA, Frankfurter Strasse 250, 64293, Darmstadt, Germany

    Bernhard Valldorf

  4. Institut für Biochemie, Biotechnologie Und Bioinformatik, Technische Universität Braunschweig, Spielmannstr. 7, 38106, Braunschweig, Germany

    Michael Hust

Authors
  1. Carolin Sellmann
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  2. Lukas Pekar
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Contributions

SZ, CS and LP designed the experiments. LP, CB, EC, BV, LT, SB and SK performed in silico analysis and experiments. JK, AF and MH gave scientific advice and guidance on overall strategy. CS, LP and SZ wrote the manuscript.

Corresponding author

Correspondence to Stefan Zielonka.

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All authors are either affiliated with Merck Healthcare KGaA or Yumab GmbH.

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Sellmann, C., Pekar, L., Bauer, C. et al. A One-Step Process for the Construction of Phage Display scFv and VHH Libraries. Mol Biotechnol 62, 228–239 (2020). https://doi.org/10.1007/s12033-020-00236-0

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