ASSEMBLY OF A FUNCTIONAL IMMUNOGLOBULIN-FV FRAGMENT IN ESCHERICHIA-COLI
ASSEMBLY OF A FUNCTIONAL IMMUNOGLOBULIN-FV FRAGMENT IN ESCHERICHIA-COLI
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DOI：
10.1126/science.3285470
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发表时间：
1988-05-20
期刊：
影响因子：
56.9
通讯作者：
PLUCKTHUN, A
中科院分区：
文献类型：
--
作者：
SKERRA, A;PLUCKTHUN, A
开发了一种表达系统，该系统允许在大肠杆菌中生产完全功能的抗体抗原结合片段。磷酸胆碱结合抗体McPC603的可变区被一起分泌到周质空间，在那里蛋白质折叠以及异源二聚体结合正确发生。因此，Fv片段在大肠杆菌中的组装途径与整个抗体在真核细胞中的组装途径相似。McPC603的Fv片段用抗原亲和层析柱一步纯化为均一。氨基末端蛋白测序证实了两种信号序列的正确处理。平衡透析实验证明重组Fv片段具有一定的功能。这些实验表明，Fv片段的亲和常数与天然抗体McPC603的亲和常数相同，Fv片段中存在一个磷胆碱结合部位，制备的Fv片段中不存在失活蛋白。这种表达系统应该会促进未来针对抗体的蛋白质工程实验。
An expression system was developed that allows the production of a completely functional antigen-binding fragment of an antibody in Escherichia coli. The variable domains of the phosphorylcholine-binding antibody McPC603 were secreted together into the periplasmic space, where protein folding as well as heterodimer association occurred correctly. Thus, the assembly pathway for the Fv fragment in E. coli is similar to that of a whole antibody in the eukaryotic cell. The Fv fragment of McPC603 was purified to homogeneity with an antigen-affinity column in a single step. The correct processing of both signal sequences was confirmed by amino-terminal protein sequencing. The functionality of the recombinant Fv fragment was demonstrated by equilibrium dialysis. These experiments showed that the affinity constant of the Fv fragment is identical to that of the native antibody McPC603, that there is one binding site for phosphorylcholine in the Fv fragment, and that there is no inactive protein in the preparation. This expression system should facilitate future protein engineering experiments on antibodies.