Department of Analytical and Biophysical Chemistry.
Department of Pharmaceutical Microbiology, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan.
J Biochem. 2020 Sep 1;168(3):257-263. doi: 10.1093/jb/mvaa042.
Single-chain Fv (scFv) is a recombinant antibody in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. Compared with monoclonal antibodies, scFvs have the advantages of low-cost production using Escherichia coli and easy genetic manipulation. ScFvs are, therefore, regarded as useful modules for producing next-generation medical antibodies. The practical use of scFvs has been limited due to their aggregation propensity mediated by interchain VH-VL interactions. To overcome this problem, we recently reported a cyclic scFv whose N-terminus and C-terminus were connected by sortase A-mediated ligation. Preparation of cyclic scFv is, however, a time-consuming process. To accelerate the application study of cyclic scFv, we developed a method to produce cyclic scFv by the combined use of a protein ligation technique based on protein trans-splicing reaction (PTS) by split intein and a chaperone co-expression system. This method allows for the preparation of active cyclic scFv from the cytoplasm of E. coli. The present method was applied to the production of cyclic 73MuL9-scFv, a GA-pyridine antibody, as a kind of advanced glycation end-product. These findings are expected to evoke further application study of cyclic scFv.
单链抗体 (scFv) 是一种重组抗体，其中重链 (VH) 和轻链 (VL) 的可变区通过短的柔性多肽接头连接。与单克隆抗体相比，scFvs 具有使用大肠杆菌低成本生产和易于遗传操作的优点。因此，scFvs 被认为是生产下一代医疗抗体的有用模块。由于链间 VH-VL 相互作用介导的聚集倾向，scFvs 的实际应用受到限制。为了克服这个问题，我们最近报道了一种由 sortase A 介导的连接连接的环 scFv。然而，环 scFv 的制备是一个耗时的过程。为了加速环 scFv 的应用研究，我们开发了一种方法，通过基于分裂内含肽的蛋白转位反应 (PTS) 的蛋白连接技术和伴侣蛋白共表达系统的联合使用来制备环 scFv。该方法允许从大肠杆菌的细胞质中制备活性环 scFv。该方法应用于 GA-吡啶抗体 73MuL9-scFv 的制备，作为一种高级糖基化终产物。这些发现有望引发对环 scFv 的进一步应用研究。
