Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan.
Biotechnol Bioeng. 2019 Feb;116(2):239-249. doi: 10.1002/bit.26857. Epub 2018 Dec 7.
In the cell surface display system, the distance of a surface-displayed molecule from the cell surface should influence its functionality due to the interference by other surface structures. For the purpose of developing this distance-variable surface display system, we utilized a long fibrous adhesin, Acinetobacter trimeric autotransporter adhesin (AtaA) of the strain Tol 5. We constructed His-tagged full-length and shorter AtaA fibers designed by N-terminal deletion and expressed them in the ΔataA mutant. Immunoelectron microscopy clearly showed that they formed fibers on the cell surface and the His-tag was displayed on the fiber tip located at fixed distances from the cell surface. N-terminal deletion of AtaA shortened the distance between the His-tag and the cell surface, as designed. Time-course analyses of the cell-to-Ni-Sepharose beads binding revealed that cells producing the longer fibers bound more rapidly to the beads. The His-tagged AtaA derivatives were also displayed on Escherichia coli cells, and a similar tendency was shown; the His-tag on the longer fiber was more functional than that on the shorter one. Thus, we developed an on-fiber display system of a functional peptide using a long trimeric autotransporter adhesin (TAA) fiber, which can vary the distance between the displayed molecule and the cell surface.
在细胞表面展示系统中，由于其他表面结构的干扰，表面展示分子与细胞表面的距离会影响其功能。为了开发这种距离可变的表面展示系统，我们利用了一种长纤维黏附素，即托氏不动杆菌三聚体自转运黏附素（AtaA）。我们构建了带有 His 标签的全长和较短的 AtaA 纤维，通过 N 端缺失设计，并在ΔataA 突变体中表达。免疫电子显微镜清楚地显示，它们在细胞表面形成纤维，His 标签显示在纤维尖端，位于与细胞表面固定距离处。AtaA 的 N 端缺失如设计的那样缩短了 His 标签与细胞表面之间的距离。细胞与 Ni-Sepharose 珠结合的时间过程分析表明，产生较长纤维的细胞更快地与珠结合。His 标签化的 AtaA 衍生物也在大肠杆菌细胞上展示，表现出类似的趋势；较长纤维上的 His 标签比较短纤维上的更具功能。因此，我们使用长的三聚体自转运黏附素（TAA）纤维开发了一种功能性肽的纤维上展示系统，该系统可以改变展示分子与细胞表面的距离。
