Stollar B D
Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Methods. 1997 Jan;11(1):12-9. doi: 10.1006/meth.1996.0382.
Bacterial production of recombinant Fab or Fv domains of antibodies is an important tool for analyzing structural correlates of antigen binding or idiotype expression. Bacterial products may be Fab or Fv molecules that assemble from separate chains in the periplasm or a single-chain Fv protein. This article describes properties and applications of a plasmid vector used for production of single-chain Fv (scFv). The expression cassette, initially designed for production of the Fv domain of anti-Z-DNA mAb Z22, has a bacterial secretion signal that permits secretion of soluble scFv into growth medium. A single B domain of staphylococcal protein A facilitates affinity purification and generic assay of products independent of antigen-binding activity. Gene segment swapping and directed mutagenesis identified structural features important for Z-DNA binding and revealed the structural similarity of autoantibody and immunization-induced antibody. The H and L regions of mAb Z22 are readily replaced by those of any cloned Ig, a library of V regions, or other proteins. Modified forms of the vector code for production of separate H or L chain V regions, which can associate with each other to form functional Fv complexes. Although there is large variation in the yield with different V regions, most clones provide enough soluble product for antigen-binding assays. Current developments are aimed at increasing the yield consistency to allow production of enough material for three-dimensional structural analysis.
细菌产生抗体的重组Fab或Fv结构域是分析抗原结合或独特型表达的结构关联的重要工具。细菌产物可以是在周质中由分开的链组装而成的Fab或Fv分子，或者是单链Fv蛋白。本文描述了一种用于生产单链Fv（scFv）的质粒载体的特性和应用。该表达盒最初设计用于生产抗Z-DNA单克隆抗体Z22的Fv结构域，具有一个细菌分泌信号，可使可溶性scFv分泌到生长培养基中。葡萄球菌蛋白A的单个B结构域有助于产物的亲和纯化和独立于抗原结合活性的通用检测。基因片段交换和定向诱变确定了对Z-DNA结合重要的结构特征，并揭示了自身抗体和免疫诱导抗体的结构相似性。单克隆抗体Z22的重链和轻链区域很容易被任何克隆的Ig、V区域文库或其他蛋白质的相应区域所取代。载体的修饰形式编码产生单独的重链或轻链V区域，它们可以相互结合形成功能性Fv复合物。尽管不同V区域的产量差异很大，但大多数克隆都能提供足够的可溶性产物用于抗原结合检测。目前的研究进展旨在提高产量的一致性，以便生产出足够的材料用于三维结构分析。
