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Recombinant Fragments of Antibodies
Král, Vlastimil ; Sedláček, Juraj (advisor) ; Pěknicová, Jana (referee) ; Hašek, Jindřich (referee)
6. CoNct usloN The aim of the thesis was to establish, in the national conditions, technology of preparation of antibody reconrbinant fragrnents and to verífo the cornplete procedure using several model monoclonal antibodies with a potential diagnostic and therapeutic use. For tfuee antibodies' mAb TU-20, M75 and MEM97, recombinant |Ťagments in various formats (scFv fragments monovalent and bivalent, i.e. diabody, and intrabody for intracellular expression) were constructed and for their heterologous expression, vectors allowing expression in E. coli (as cytoplasmic inclusions, periplasmic inclusions and in soluble form) and in Drosophila 32 cells (expression of glycosylated forms of scFv fragments into the medium) were used. In case of proteins expressed in irrsoluble form, especially scFv F|1.2.32, renaturation procedures to obtain active scFv fragments were developed and optimized. The efnect of the length of the linker -(GlyrSer).- (where x is I to 4) connecting the variable domains of the light and heavy chain on the formation of different multimeric forms of scFv rvas studied. For obtaining solell monomeric scFv fragment, the length of 20 amino acid residues tumsd out optimal. Fragnrents rvith a linker l5 residues long. formed a mixture of monomers, dimers and trimers. the proportion of rvhich rvas...
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