Neri D, de Lalla C, Petrul H, Neri P, Winter G
Cambridge Centre for Protein Engineering, MRC Centre, UK.
Biotechnology (N Y). 1995 Apr;13(4):373-7. doi: 10.1038/nbt0495-373.
Calmodulin is a highly acidic protein (net charge -24 at pH 8.0 in the absence of calcium) that binds to peptide and organic ligands with high affinity (Ka > 10(9) M-1) in a calcium-dependent manner. We have exploited these properties to develop calmodulin as a versatile tag for antibody fragments. Fusions of calmodulin with single chain Fv fragments (scFv) could be expressed by secretion from bacteria in good yield (5-15 mg/l in shaker flasks), and purified from periplasmic lysates or broth to homogeneity in a single step, either by binding to anion-exchange resin (DEAE-Sephadex), or to an organic ligand of calmodulin (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide-agarose). The antibody fusions could be detected by binding of fluorescently labeled peptide ligands, as illustrated by their use in confocal microscopy, fluorescent activated cell sorting and "band shift" gel electrophoresis. Moreover, the interaction between calmodulin and peptide ligands could provide a means of heterodimerization of proteins, as illustrated by the assembly of an antibody-calmodulin fusion with maltose binding protein tagged with a peptide ligand of calmodulin.
钙调蛋白是一种高度酸性的蛋白质（在无钙条件下，pH 8.0时净电荷为-24），它以钙依赖的方式与肽和有机配体高亲和力结合（Ka>10⁹ M⁻¹）。我们利用这些特性将钙调蛋白开发为一种用于抗体片段的通用标签。钙调蛋白与单链Fv片段（scFv）的融合蛋白可以通过细菌分泌高效表达（摇瓶中产量为5-15 mg/l），并从周质裂解物或培养液中一步纯化至均一，可通过与阴离子交换树脂（DEAE-葡聚糖凝胶）结合，或与钙调蛋白的有机配体（N-(6-氨基己基)-5-氯-1-萘磺酰胺-琼脂糖）结合来实现。抗体融合蛋白可以通过荧光标记的肽配体的结合来检测，如它们在共聚焦显微镜、荧光激活细胞分选和“带移”凝胶电泳中的应用所示。此外，钙调蛋白与肽配体之间的相互作用可以提供一种蛋白质异源二聚化的方法，如抗体-钙调蛋白融合蛋白与标记有钙调蛋白肽配体的麦芽糖结合蛋白的组装所示。
