Single Chain Fab (scFab) Fragment

Overview

Journal BMC Biotechnol

Publisher Biomed Central

Specialty Biotechnology

Date 2007 Mar 10

PMID 17346344

Citations 49

Authors

Michael Hust

Thomas Jostock

Christian Menzel

Bernd Voedisch

Anja Mohr

Mariam Brenneis

Martina I Kirsch

Doris Meier

Stefan Dubel

Affiliations

Soon will be listed here.

Abstract

Background: The connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display.

Results: Here, we demonstrate that the introduction of a polypeptide linker between the fragment difficult (Fd) and the light chain (LC), resulting in the formation of a single chain Fab fragment (scFab), can lead to improved production of functional molecules. We tested the impact of various linker designs and modifications of the constant regions on both phage display efficiency and the yield of soluble antibody fragments. A scFab variant without cysteins (scFabDeltaC) connecting the constant part 1 of the heavy chain (CH1) and the constant part of the light chain (CL) were best suited for phage display and production of soluble antibody fragments. Beside the expression system E. coli, the new antibody format was also expressed in Pichia pastoris. Monovalent and divalent fragments (DiFabodies) as well as multimers were characterised.

Conclusion: A new antibody design offers the generation of bivalent Fab derivates for antibody phage display and production of soluble antibody fragments. This antibody format is of particular value for high throughput proteome binder generation projects, due to the avidity effect and the possible use of common standard sera for detection.

Citing Articles

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A next-generation Fab library platform directly yielding drug-like antibodies with high affinity, diversity, and developability.

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An Engineered Mouse Model That Generates a Diverse Repertoire of Endogenous, High-Affinity Common Light Chain Antibodies.

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Gagne D, Sarker M, Gingras G, Hodgson D, Frahm G, Creskey M PLoS One. 2023; 18(11):e0294406.

PMID: 38019850 PMC: 10686436. DOI: 10.1371/journal.pone.0294406.

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