Genomics Research Center, Academia Sinica, Taipei 115, Taiwan, ROC.
Biochem Biophys Res Commun. 2011 Jul 29;411(2):348-53. doi: 10.1016/j.bbrc.2011.06.146. Epub 2011 Jun 28.
Phage-displayed single chain variable fragment (scFv) libraries are powerful tools in antibody engineering. Disulfide-stabilized scFv (sc-dsFv) with an interface disulfide bond is structure-wise more stable than the corresponding scFv. A set of recently discovered signal sequences replacing the wild type (pelB) signal peptidase cleavage site in the c-region has been shown to be effective in rescuing the expression of sc-dsFv libraries on the phage surface. However, the effects of the other regions of the signal sequence on the expression of the sc-dsFv libraries and on the formation of the interface disulfide bond in the phage-displayed sc-dsFv have not been clear. In this work, selected novel signal sequence variants in the h-region were shown to be equally effective in promoting sc-dsFv library expression on the phage surface; the expression level and complexity of the sc-dsFv libraries were comparable to the corresponding scFv libraries produced with the wild-type (pelB) signal sequence. The interface disulfide bond in the phage-displayed sc-dsFv was proven to form to a large extent in the library variant ensemble generated with signal sequence variants in both the h-region and the c-region. The sc-dsFv engineering platform established in this work can be applied to many of the known scFv molecules which are in need of a more stable version for the applications under harsh conditions or for longer shelf-life.
噬菌体展示单链可变片段 (scFv) 文库是抗体工程中的强大工具。具有界面二硫键的稳定化 scFv (sc-dsFv) 在结构上比相应的 scFv 更稳定。最近发现的一组信号序列可以替代 c 区中的野生型 (pelB) 信号肽切割位点，已被证明可有效促进 sc-dsFv 文库在噬菌体表面的表达。然而，信号序列的其他区域对 sc-dsFv 文库的表达以及噬菌体展示的 sc-dsFv 中界面二硫键的形成的影响尚不清楚。在这项工作中，展示了 h 区中选定的新型信号序列变体在促进噬菌体表面 sc-dsFv 文库表达方面同样有效；sc-dsFv 文库的表达水平和复杂性可与使用野生型 (pelB) 信号序列产生的相应 scFv 文库相媲美。证明噬菌体展示的 sc-dsFv 中的界面二硫键在 h 区和 c 区的信号序列变体生成的文库变体集中在很大程度上形成。本工作中建立的 sc-dsFv 工程平台可应用于许多已知的 scFv 分子，这些分子在苛刻条件下或更长的保质期下应用时需要更稳定的版本。
