Rau Doris, Kramer Karl, Hock Bertold
Technische Universität München, Center of Life Sciences Weihenstephan, Department of Plant Sciences, Freising, Germany.
Anal Bioanal Chem. 2002 Jan;372(2):261-7. doi: 10.1007/s00216-001-1194-6. Epub 2001 Dec 22.
Fab antibody fragments were constructed by subcloning single chain Fv variable regions from the phagemid vector pCANTAB 5E into the expression vector pASK99. The vector was designed for bacterial secretion of Fab fragments and bears coding sequences for murine constant domains including the Strep-tag II at the carboxyl-terminal end of the constant heavy chain domain. The cloning procedure was carried out with the scFv antibodies IPR-7, IPR-53 and IPR-23. The second and third clone originated from the molecular evolution of the s-triazine selective antibody IPR-7. The Fab fragments were expressed under the transcriptional control of the tetA promoter system. Large-scale production benefits from the anhydrotetracycline-inducible system because of the lower costs for the inducer compared to IPTG and the tightly regulated expression of the recombinant antibody fragments. The Strep-tag purification technology facilitates the isolation of Fab fragments from the E. coli periplasm. The characteristics of functionally expressed Fab fragments were determined by employing a BIAcore 2000 system. The KD of the Fab variant IPR-23 (K(D)= 1.1 2 x 10(-9) M) optimized by molecular evolution was improved by a factor of 24 compared to the Fab IPR-7 (K(D) = 2.73 x 10(-8) M), which was derived from the template scFv antibody IPR-7. The affinity alteration was also reflected in the 22-fold reduction of the IC50 values of the variants Fab IPR-7 (IC50 = 60.5 microg/L) and Fab IPR-23 (IC50=2.7 microg/L) in the corresponding atrazine ELISA.
通过将噬菌体展示载体pCANTAB 5E中的单链Fv可变区亚克隆到表达载体pASK99中构建Fab抗体片段。该载体设计用于细菌分泌Fab片段，并带有鼠源恒定区的编码序列，包括恒定重链结构域羧基末端的链霉亲和素标签II。克隆过程使用scFv抗体IPR - 7、IPR - 53和IPR - 23进行。第二个和第三个克隆源自s - 三嗪选择性抗体IPR - 7的分子进化。Fab片段在tetA启动子系统的转录控制下表达。大规模生产受益于脱水四环素诱导系统，因为与IPTG相比，诱导剂成本更低，且重组抗体片段的表达受到严格调控。链霉亲和素标签纯化技术有助于从大肠杆菌周质中分离Fab片段。通过使用BIAcore 2000系统测定功能表达的Fab片段的特性。通过分子进化优化的Fab变体IPR - 23（K(D)= 1.1 2 x 10(-9) M）的KD与源自模板scFv抗体IPR - 7的Fab IPR - 7（K(D) = 2.73 x 10(-8) M）相比提高了24倍。亲和力的改变也反映在相应的阿特拉津ELISA中Fab变体IPR - 7（IC50 = 60.5 μg/L）和Fab IPR - 23（IC50 = 2.7 μg/L）的IC50值降低了22倍。
