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Expression of catalytic antibodies in eukaryotic systems
Authors:A V Zakharov  I V Smirnov  M V Serebryakova  M A Dronina  A V Kaznacheeva  I N Kurkova  A A Belogurov  A Friboulet  N A Ponomarenko  A G Gabibov  T V Bobik
Institution:1. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia
2. Chemical Department, Moscow State University, Moscow, 119991, Russia
3. Research Institute of Physicochemical Medicine, Moscow, 119992, Russia
4. Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334, Russia
5. Université de Technologie de Compiègne, UMR 6022 CNRS, BP 20529, 60206, Compiègne, Cedex, France
Abstract:Expression of recombinant antibodies in mammalian cells is one of the key problems in immuno-biotechnology. Alternatively, expression of a broad panel of antibodies and of their fragments may be effectively performed in yeast cells. We obtained expression strains of the methylotrophic yeast Pichia pastoris producing single-chain human catalytic antibody A17 (A.17scFv), Fab-fragment (A.17Fab), and full-size light chain (A.17Lch). These antibodies were characterized in terms of functional activity. The capacity to specifically bind and transform organophosphorus compounds has been demonstrated for A.17scFv and A.17Fab. The loss of activity of the antibody light chain when expressed alone indicates that the active site is formed by both heavy and light chains of the antibody. We determined the reversible constant K d and the first order constant (k 2) of the reaction of the covalent modification of A.17scFv and A.17Fab by irreversible inhibitor of the serine proteases p-nitrophenyl 8-methyl-8-azobicyclo3.2.1]phosphonate (phosphonate X). Calculated values indicate that activity of the antibodies expressed in yeast is similar to the full-size antibody A17 and to the single-chain antibody A.17 expressed in CHO and E. coli cells, respectively.
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