Abstract
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
Graphical abstract
Funder
Thailand Science Research and Innovation
Royal Golden Jubilee (RGJ) Ph.D. Programme
National Research Council of Thailand
Publisher
Springer Science and Business Media LLC
Subject
Applied Microbiology and Biotechnology,General Medicine,Biotechnology
Cited by
1 articles.
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