Schwalbach G, Sibler A P, Choulier L, Deryckère F, Weiss E
Ecole Supérieure de Biotechnologie de Strasbourg, ULP/UPRES 1329, boulevard Sébastien Brant, Illkirch, 67400, France.
Protein Expr Purif. 2000 Mar;18(2):121-32. doi: 10.1006/prep.1999.1185.
We describe a novel vector-host system suitable for the efficient preparation of fluorescent single-chain antibody Fv fragments (scFv) in Escherichia coli. The previously described pscFv1F4 vector used for the bacterial expression of functional scFv to the E6 protein of human papillomavirus type 16 was modified by appending to its C-terminus the green fluorescent protein (GFP). The expression of the scFv1F4-GFP fusion proteins was monitored by analyzing of the typical GFP fluorescence of the transformed cells under UV illumination. The brightest signal was obtained when scFv1F4 was linked to the cycle 3 GFP variant (GFPuv) and expressed in the cytoplasm of AD494(DE3) bacteria under control of the arabinose promoter. Although the scFv1F4 expressed under these conditions did not contain disulfide bridges, about 1% of the molecules were able to bind antigen. Fluorescence analysis of antigen-coated agarose beads incubated with the cytoplasmic scFv-GFP complexes showed that a similar proportion of fusions retained both E6-binding and green-light-emitting activities. The scFv1F4-GFPuv molecules were purified by affinity chromatography and successfully used to detect viral E6 protein in transfected COS cells by fluorescence microscopy. When an anti-beta-galactosidase scFv, which had previously been adapted to cytoplasmic expression at high levels, was used in this system, it was possible to produce large amounts of functional fluorescent antibody fragments. This indicates that these labeled scFvs may have many applications in fluorescence-based single-step immunoassays.
我们描述了一种新型载体-宿主系统，适用于在大肠杆菌中高效制备荧光单链抗体Fv片段（scFv）。先前描述的用于在细菌中表达针对人乳头瘤病毒16型E6蛋白的功能性scFv的pscFv1F4载体，通过在其C末端附加绿色荧光蛋白（GFP）进行了修饰。通过在紫外光照射下分析转化细胞的典型GFP荧光来监测scFv1F4-GFP融合蛋白的表达。当scFv1F4与第3代GFP变体（GFPuv）连接并在阿拉伯糖启动子控制下在AD494(DE3)细菌的细胞质中表达时，获得了最亮的信号。尽管在这些条件下表达的scFv1F4不含有二硫键，但约1%的分子能够结合抗原。用细胞质scFv-GFP复合物孵育抗原包被的琼脂糖珠的荧光分析表明，类似比例的融合蛋白保留了E6结合和绿色发光活性。scFv1F4-GFPuv分子通过亲和层析纯化，并成功用于通过荧光显微镜检测转染的COS细胞中的病毒E6蛋白。当在此系统中使用先前已适应高水平细胞质表达的抗β-半乳糖苷酶scFv时，有可能产生大量功能性荧光抗体片段。这表明这些标记的scFv在基于荧光的单步免疫分析中可能有许多应用。
