Kipriyanov S M, Little M, Kropshofer H, Breitling F, Gotter S, Dübel S
Recombinant Antibody Research Group, Heidelberg, Germany.
Protein Eng. 1996 Feb;9(2):203-11. doi: 10.1093/protein/9.2.203.
In antigen-antibody interactions, the high avidity of antibodies depends on the affinity and number of the individual binding sites. To develop artificial antibodies with multiple valency, we have fused the single-chain antibody Fv fragments to core streptavidin. The resulting fusion protein, termed scFv::strep, was found after expression in Escherichia coli in periplasmic inclusion bodies. After purification of the recombinant product by immobilized metal affinity chromatography, refolding and size-exclusion FPLC, tetrameric complexes resembling those of mature streptavidin were formed. The purified tetrameric scFv::strep complexes demonstrated both antigen- and biotin-binding activity, were stable over a wide range of pH and did not dissociate at high temperatures (up to 70 degrees C). Surface plasmon resonance measurements in a BIAlite system showed that the pure scFv::strep tetramers bound immobilized antigen very tightly and no dissociation was measurable. The association rate constant for scFv::strep tetramers was higher than those for scFv monomers and dimers. This was also reflected in the apparent constants, which was found to be 35 times higher for pure scFv::strep tetramers than monomeric single-chain antibodies. We could also show that most of biotin binding sites were accessible and not blocked by biotinylated E.coli proteins or free biotin from the medium. These sites should therefore facilitate the construction of bispecific multivalent antibodies by the addition of biotinylated ligands.
在抗原 - 抗体相互作用中，抗体的高亲和力取决于各个结合位点的亲和力和数量。为了开发具有多价性的人工抗体，我们将单链抗体Fv片段与核心链霉亲和素融合。在大肠杆菌周质包涵体中表达后，发现了产生的融合蛋白，称为scFv::strep。通过固定化金属亲和色谱、重折叠和尺寸排阻FPLC对重组产物进行纯化后，形成了类似于成熟链霉亲和素的四聚体复合物。纯化的四聚体scFv::strep复合物表现出抗原结合和生物素结合活性，在很宽的pH范围内稳定，并且在高温（高达70摄氏度）下不会解离。在BIAlite系统中进行的表面等离子体共振测量表明，纯scFv::strep四聚体与固定化抗原紧密结合，且未检测到解离。scFv::strep四聚体的结合速率常数高于scFv单体和二聚体。这也反映在表观常数上，发现纯scFv::strep四聚体的表观常数比单体单链抗体高35倍。我们还可以表明，大多数生物素结合位点是可及的，未被生物素化的大肠杆菌蛋白或培养基中的游离生物素阻断。因此，这些位点应有助于通过添加生物素化配体构建双特异性多价抗体。
