School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand.
New England Biolabs, Ipswich, MA, 01938, USA.
Appl Microbiol Biotechnol. 2023 Mar;107(5-6):1959-1970. doi: 10.1007/s00253-023-12395-6. Epub 2023 Feb 2.
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.
在微生物系统中高效选择和生产抗体片段仍然是一个具有挑战性的过程。为了优化单链可变片段（scFv）的微生物生产，我们选择了五个模型靶标，1）一种半抗原，玉米赤霉烯酮（ZEN）真菌毒素，以及传染性病原体 2）狂犬病病毒，3）痤疮丙酸杆菌，4）铜绿假单胞菌，和一种癌细胞 5）急性髓系白血病细胞系（HL-60）。scFv 结合物从非免疫的人噬菌体展示 scFv 抗体文库中通过亲和选择获得，并遗传融合到翠绿荧光蛋白（EmGFP）的 N 端。scFv-EmGFP 融合构建体被亚克隆到表达载体中，在 T7 启动子的控制下，C 端带有六组氨酸标签，并在不同的大肠杆菌（E. coli）宿主中表达。这使得能够通过细胞质中明亮的荧光信号检测表达正确 scFv-EmGFP 融合体的细胞，称为荧光体。在测试的三种大肠杆菌宿主中，一种称为 SHuffle B 的工程大肠杆菌 B 菌株似乎是最合适的宿主，它促进细胞质中二硫键的形成。重组荧光体表达良好（2-8mg/L），具有 EmGFP 的荧光特性，并保留与它们的同源靶标的结合能力。它们的特异性结合通过 ELISA、荧光酶联免疫吸附测定（FLISA）、流式细胞术和荧光显微镜成像得到证实。本研究中的荧光体表达平台可以进一步被采用为基于噬菌体展示文库中分离的 scFv 的一步免疫染色技术，用于许多所需的靶标。关键点：•大肠杆菌 SHuffle express T7 是 scFv-EmGFP（荧光体）的合适表达宿主•只有携带 scFv-EmGFP 质粒的克隆才会显示明亮的荧光信号•该平台可用于生产用于多种用途的荧光体。
