Abstract
BackgroundThe isolation of recombinant antibody fragments from displayed libraries represents a powerful alternative to the generation of IgGs using hybridoma technology. The selected antibody fragments can then be easily engineered into (multi)-tagged constructs of variable mass and complexity as well as reconstituted into Camelidae IgG-like molecules when expressed fused to Fc domains. Nevertheless, all antibody constructs depend on an oxidizing environment for correct folding and consequently still belong to the proteins difficult to express in bacteria. In such organisms they are mostly produced at low yields in the periplasmic space.ResultsWe demonstrate that fusion constructs of recombinant antibodies in combination with multiple tags can be produced at high yields and totally functional in the cytoplasm of bacteria expressing sulfhydryl oxidase. The method was applied to structurally demanding molecules such as VHHs fused to SNAP and Fc domains and was validated using the antibody-derived reagents in a variety of immune techniques (FACS, ELISA, WB, IP, SPR, and IF).ConclusionsThe collected data demonstrate the feasibility of a method that establishes a totally new approach for producing rapidly and inexpensively functional Camelidae IgG-like monoclonal antibodies and antibody-based reagents containing multiple disulfide bonds and suitable for both basic research and clinical applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0140-1) contains supplementary material, which is available to authorized users.
Highlights
The isolation of recombinant antibody fragments from displayed libraries represents a powerful alternative to the generation of IgGs using hybridoma technology
Such fusion constructs are often easy to produce in bacteria and we designed a collection of vectors for the parallel expression of applicationfriendly VHHs production in both bacterial periplasm and cytoplasm (Additional file 1: Figure S1)
SNAP/CLIP and GFP were poorly folded and were prone to aggregate when expressed in the periplasm whereas the presence of trans disulfide bonds in structurally complex proteins such as alkaline phosphatase, peroxidases, and enzymatic toxins seemed to be compatible only with periplasmic expression
Summary
The isolation of recombinant antibody fragments from displayed libraries represents a powerful alternative to the generation of IgGs using hybridoma technology. We demonstrate that several VHHbased constructs as complex as the IgG-like reconstituted VHH-Fc antibodies can be produced in bacterial cytoplasm at elevated yields and preserve their complete functionality. This opportunity represents a time and cost effective alternative to the conventional expression of IgG antibodies from hybridoma cells. It allows the production of fusion molecules such as the VHH-SNAP or VHH-GFP constructs that are difficult to obtain in oxidizing environments
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