Production and applications of fluorobody from redox-engineered Escherichia coli.
Witsanu SrilaThae Thae MinThitima SumphanapaiKuntalee RangnoiMehmet BerkmenMontarop YamabhaiPublished in: Applied microbiology and biotechnology (2023)
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.
Keyphrases
- escherichia coli
- pseudomonas aeruginosa
- poor prognosis
- quantum dots
- living cells
- biofilm formation
- flow cytometry
- label free
- acute myeloid leukemia
- high throughput
- binding protein
- microbial community
- single molecule
- klebsiella pneumoniae
- cystic fibrosis
- dna methylation
- induced apoptosis
- endothelial cells
- high resolution
- gene expression
- staphylococcus aureus
- fluorescent probe
- crispr cas
- cell proliferation
- acute lymphoblastic leukemia
- small molecule
- photodynamic therapy
- fluorescence imaging
- sensitive detection