Single chain Fab (scFab) fragment.

Michael Hust,Bernd Voedisch,Mariam Brenneis,Doris Meier,Martina I Kirsch,Stefan Dübel,Christian Menzel,Anja Mohr,Thomas Jostock

doi:10.1186/1472-6750-7-14

Abstract

BackgroundThe 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.ResultsHere, 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 (scFabΔC) 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.ConclusionA 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.

Highlights

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 was a breakthrough for the functional production of antibody fragments in Escherichia coli
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 fragment antigen binding (Fab) fragment, can lead to improved production of functional molecules
Beside the expression system E. coli, the new antibody format was expressed in Pichia pastoris

Summary

Introduction

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. The linkage of the variable regions by a 15– 25 amino acid linker of both Fv chains improved the expression of antibody fragments in E. coli [2,3] These so called single chain fragment variable (scFv) have the tendency to form aggregates and are relatively unstable over longer periods of time [4]. In rare cases have scFvs with a higher affinity than the associated Fab been found [6] Because they are double the molecular size, and require the production and connection of two different polypeptides with a disulphide bond, folding and assembly of Fab fragments in the periplasm of E. coli is less efficient than for scFvs [7]. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display

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Results

Discussion

Conclusion