A generic strategy for subcloning antibody variable regions from the scFv phage display vector pCANTAB 5 E into pASK85 permits the economical production of Fab fragments and leads to improved recombinant immunoglobulin stability
A generic strategy for subcloning antibody variable regions from the scFv phage display vector pCANTAB 5 E into pASK85 permits the economical production of Fab fragments and leads to improved recombinant immunoglobulin stability
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DOI：
10.1016/s0956-5663(01)00292-5
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发表时间：
2002-04-01
影响因子：
12.6
通讯作者：
Hock, B
中科院分区：
文献类型：
--
作者：
Kramer, K;Fiedler, M;Hock, B
除了免疫传感器的决定性灵敏度和特异性之外，所采用的抗体基本上还有助于其他关键因素，如传感器芯片的制造成本和传感器稳定性。重组抗体片段的生产方案已经针对生物传感器开发的这些特定问题进行了优化。噬菌粒载体pCANTAB 5 E广泛用于从相应文库中筛选抗体片段.然而，选择的单链F-v（scFv）片段的大规模生产基本上受到诱导剂IPTG和抗E标签抗体的高成本的限制。重组蛋白的纯化需要大量的后者。建立了一种通用的策略，用于将来自pCANTAB 5 E的scFv可变区亚克隆到质粒pASK 85中以表达F-ab片段。pASK 85具有鼠恒定结构域的编码序列，包括在恒定重链结构域的羧基末端的His（6）标签。抗s-三嗪抗体K47 H在本研究中充当模型系统。在高密度发酵罐中加入无水四环素诱导F-ab片段的生物合成，然后用固定化金属亲和层析（IMAC）从周质提取物中一次性纯化F-ab片段。采用标准发酵方案获得100 μ g/l × OD 550的纯化F-ab片段的产量。当通过酶免疫测定法测定时，F-ab的灵敏度和交叉反应性与亲本scFv相当。然而，F-ab片段表现出显著改善的长期稳定性。(C)2002 Eisevier Science B. V.保留所有权利。
Apart from the decisive sensitivity and specificity of immunosensors, the employed antibodies essentially contribute to additional key factors like fabrication costs for sensor chips and sensor stability. A production scheme for recombinant antibody fragments has been optimised with respect to these particular issues of biosensor development. The phagemid vector pCANTAB 5 E is widely used for the selection of antibody fragments from corresponding libraries. However, large-scale production of the selected singic-chain F-v (scFv) fragments is substantially restricted by the high cost for the inducer IPTG and the anti-E-tag antibody. The latter is needed in significant amounts for the purification of the recombinant protein. A generic strategy, was established for subcloning scFv variable regions from pCANTAB 5 E into the plasmid pASK85 for the expression of F-ab fragments. pASK85 bears coding sequences for murine constant domains including a His(6) tag at the carboxyl-terminal end of the constant heavy chain domain. The anti-s-triazine antibody K47H served as a model system in this study. Biosynthesis of the F ab fragment in a high cell density fermenter was induced by addition of anhydrotetracycline, The F-ab fragment was subsequently purified from the periplasmic extract in a singe stop by immobilized metal affinity chromatography (IMAC). A yield of 100 mug/l x OD550 purified F-ab fragment was obtained employing a standard fermentation scheme. The sensitivity and cross-reactivity of the F-ab was comparable to the parent scFv when assayed by enzyme immunoassay. However, the F-ab fragment exhibited significantly improved long-term stability. (C) 2002 Eisevier Science B.V. All rights reserved.