cloning, E. coli expression and purification of scFv antibody fragments
of diagnostic/therapeutic interest
Vlastimil KRAL1, Milan Fabry1, Magda Horejsi1, Jan Zavada2, Juraj Sedlacek1
1 Department of Gene Manipulation., Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 37 Prague 6, Czech Republic
2 Department of Cellular and Viral Genetics, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 37 Prague 6, Czech Republic
Fv fragments are the smallest antibody molecules that still retain the entire antigen‑binding site. In single-chain Fv fragments (scFv), variable domains are joined by a flexible linker. Such scFv constructs, which generally retain full binding capacities towards the antigen, are the topic of very active research. First, they are of interest for structural studies because they usually yield crystals diffracting to higher resolution than the corresponding Fab fragments. Second, since they can be expressed in bacteria, they are also well suited for binding, mutagenesis and protein engineering studies [for review, see e.g.1].
In this work we describe molecular cloning, expression, purification and properties of two scFvs of potential diagnostic and immunotherapeutic use, scFv M75 and scFv TU‑20.
Monoclonal antibody (mAb) M75 recognizes cell surface protein MN/CA IX strongly associated with several types of human carcinomas . Radioactively labeled humanized scFv M75 could be used for tumor immunodetection and possibly for therapy. Monoclonal antibody TU-20 was raised against beta-III-tubulin, a specific neuronal marker in normal and neoplastic tissues. The antibody TU-20 fragments could thus be useful tools for probing beta-III-tubulin functions in neurons, as well as for immunohistochemical characterization of tumors of neuronal origin .
Coding sequences for light (VL) and heavy (VH) variable domains were obtained from total RNA, isolated from hybridoma cells, by RT-PCR using suitable pairs of primers. Single‑chain Fv genes in the form VL-linker-VH-myc tag were then assembled and cloned into T7 promoter‑driven expression plasmids. Bacterial strain E. coli BL21(DE3) was used for protein expression. The recombinant protein products accumulated in inclusion bodies as insoluble aggregates. To obtain refolded active proteins from inclusion bodies, several protocols were adapted to find optimal conditions for each scFv species.
Purification procedure comprising several conventional chromatography steps (ionex chromatography, gel filtration) yielded scFv proteins in amount and purity necessary for functional characterization. While scFv TU-20 in ELISA assay exhibits specificity and binding activity comparable to parental mAb TU-20, in case of scFv M75, ELISA assay was negative. The reasons for the lack of binding activity are under investigation.
2. J. Zavada, Z. Zavadova, J. Pastorek, Z.Biesova, J. Jezek, & J. Velek,
Br. J. Cancer, 82 (2000) 1808-1813
3. E. Draberova, Z. Lukas, D. Ivanyi, V. Viklicky & P. Draber,
Histochem Cell Biol.,109 (1998) 231-239.