Production and applications of fluorobody from redox-engineered Escherichia coli

Abstract

Efficient selection and production of antibody fragments in microbial systems remain to be a challenging process. To optimize microbial production of single-chain variable fragments (scFvs), we have chosen five model targets, 1) a hapten, Zearalenone (ZEN) mycotoxin, along with infectious agents 2) rabies virus, 3) Propionibacterium acnes, 4) Pseudomonas aeruginosa, and a cancer cell 5) acute myeloid leukemia cell line (HL-60). The scFv binders were affinity selected from a non-immunized human phage display scFv antibody library and genetically fused to the N-terminus of emerald green fluorescent protein (EmGFP). The scFv-EmGFP fusion constructs were subcloned into an expression vector, under the control of T7 promoter, C-terminally tagged with hexa-histidine and expressed in different Escherichia coli (E. coli) hosts. This enabled the detection of cells that expressed the correct scFv-EmGFP fusion, termed fluorobody, via bright fluorescent signal in the cytoplasm. Among the three E. coli hosts tested, an engineered E. coli B strain called SHuffle B that promotes disulfide bond formation in the cytoplasm appeared to be the most appropriate host. The recombinant fluorobodies were well expressed (2-8 mg/L), possessed the fluorescence property of EmGFP, and retained the ability to bind to their cognate targets. Their specific bindings were demonstrated by ELISA, fluorescence-linked immunosorbent assay (FLISA), flow cytometry, and fluorescent microscope imaging. The fluorobody expression platform in this study could be further adopted as a one-step immunostaining technique based on scFv, isolated from phage display library to numerous desired targets. KEY POINTS: • E. coli SHuffle express T7 is a suitable expression host for scFv-EmGFP (fluorobody) • Only the clones harboring scFv-EmGFP plasmid will show bright fluorescent signal • This platform can be used to produce fluorobodies for numerous purposes.

Keywords: E. coli SHuffle; Emerald green fluorescent protein (EmGFP); Fluorobody; Fusion; Immunofluorescence; Single-chain variable fragment (scFv).