Selection of genetically encoded fluorescent single domain antibodies engineered for efficient expression in Escherichia coli

Genetically encoded fluorescent antibodies are desirable for many applications in biotechnology and proteomics. However, the efficient production of single chain antibodies fused to fluorescent proteins like green fluorescent protein is still a major challenge. This is due to the opposite redox requirements of recombinant antibodies and fluorescent proteins for proper folding. To overcome this obstacle, we designed a novel strategy to directly select functional fluorescent antibodies (fluobodies) engineered for efficient cytosolic expression in Escherichia coli, starting from a llama heavy chain antibody (VHH) library. We used this strategy to produce several VHH fluobodies directed against microtubules. After biochemical characterization in vitro, we used a selected fluobody as a convenient biomarker of the microtubule cytoskeleton in eukaryotic cells and engineered its properties. This work provides a reliable approach for the production of fluobodies in wild- type E. coli and furthermore suggests that fusions of recombinant VHH with other cytosolic proteins are now possible, creating new opportunities for biotechnology and biomedical research.