Schiweck W, Buxbaum B, Schätzlein C, Neiss H G, Skerra A
Institut für Biochemie, Technische Hochschule, Darmstadt, Germany.
FEBS Lett. 1997 Sep 1;414(1):33-8. doi: 10.1016/s0014-5793(97)00983-6.
The cDNAs for the two variable domains of the antibody 9E10 were cloned from the hybridoma cell line. A chimeric 9E10 Fab fragment was produced in E. coli under control of the tightly controlled tetracycline promoter. The functional Fab fragment was isolated in a single step via a His6-tag, which also served for its recognition by a nickel chelate-alkaline phosphatase conjugate. Thus, the recombinant Fab fragment permitted the immunochemical detection of the myc tag in a sandwich ELISA. The dissociation constant for the interaction with the myc tag peptide was determined as 80 +/- 5 nM by fluorescence titration. In an attempt to produce the smaller 9E10 Fv fragment it was found that its V(H) domain alone can be readily isolated from E. coli as a soluble protein. This unusual behaviour may be explained by the 18 amino acid-long CDR-H3 and could be of value in the design of 'single domain' antibodies.
从杂交瘤细胞系中克隆出抗体9E10两个可变结构域的cDNA。在严格调控的四环素启动子控制下，在大肠杆菌中产生了嵌合的9E10 Fab片段。通过His6标签一步分离出功能性Fab片段，该标签也用于通过镍螯合碱性磷酸酶偶联物对其进行识别。因此，重组Fab片段允许在夹心ELISA中对myc标签进行免疫化学检测。通过荧光滴定法测定与myc标签肽相互作用的解离常数为80±5 nM。在尝试产生较小的9E10 Fv片段时，发现其单独的V(H)结构域可以很容易地作为可溶性蛋白从大肠杆菌中分离出来。这种不寻常的行为可能由18个氨基酸长的CDR-H3来解释，并且在“单结构域”抗体的设计中可能具有价值。
