Isolation of mimotopes to the anti-human VEGF antibody Bevacizumab by mRNA display using random peptide libraries and the vaccination of a rabbit
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Abstract
An mRNA display system using synthetic DNA cod-ing for random 10- and 20-amino-acid peptide li-braries was employed to isolate mimotopes that could substitute for the anti-human VEGF antibody Bevacizumab. After six rounds of affinity selection, three clones that bound to the antibody were isolated. Their random-peptide portions were chemically syn-thesized, and further characterized. All of the pep-tides showed clear specific binding to the antibody. Two of them were further conjugated with Keyhole limpet hemocyanin (KLH) to immunize a rabbit. Af-ter five immunizations biweekly, antibodies to the peptides were purified with a column conjugated with the peptides. The purified antibodies reacted specifically to the antibody’s original antigen, human VEGF. mRNA displays could be useful for the isola-tion of mimotopes for vaccines to substitute for ther-apeutic antibodies.
- Lipovsek, D. and Pluckthun, A. (2004) In-vitro protein evolution by ribosome display and mRNA display. Journal of Immunological Methods, 290, 51-67. doi:10.1016/j.jim.2004.04.008
- Shibui, T., Kobayashi, T. and Shiratori, M. (2008) Isolation of cross-reacting antigen candidates by mRNA- display using a mixed cDNA library. Biotechnology Letters, 30, 2037-2043. doi:10.1007/s10529-008-9803-5
- Shimizu, Y., et al. (2001) Cell-free translation recons- tituted with purified components. Nature Biotechnology, 19, 751-755. doi:10.1038/90802
- Kim, D.M. et al. (1996) A highly efficient cell-free protein synthesis system from Escherichia coli. European Journal of Biochemistry, 239, 881-886. doi:10.1111/j.1432-1033.1996.0881u.x
- Koga, H., Misawa, S. and Shibui, T. (2009) A wheat embryo cell-free protein synthesis system not requiring an exogenous supply of GTP. Biotechnology Progress, 25, 1322-1327. doi:10.1002/btpr.230
- Madin, K., et al. (2000) A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: plants apparently contain a suicide system directed at ribosomes. Proceedings of the National Academy of Sciences of the United States of America, 97, 559-564. doi:10.1073/pnas.97.2.559
- Smith, G.P. and Scott, J.K. (1993) Libraries of peptides and proteins displayed on filamentous phage. Methods in Enzymology, 217, 228-257. doi:10.1016/0076-6879(93)17065-D
- Furukawa, H., et al. (2006) Development of novel yeast cell surface display system for homo-oligomeric protein by coexpression of native and anchored subunits. Biotechnology Progress, 22, 994-997. doi:10.1021/bp0601342
- Nemoto, N., et al. (1997) In vitro virus: Bonding of mRNA bearing puromycin at the 3'-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro. FEBS Letters, 414, 405-408. doi:10.1016/S0014-5793(97)01026-0
- Shibui, T., et al. (2009) In vitro selection of scFv and its production: An application of mRNA display and wheat embryo cell-free and E. coli cell production system. Applied Microbiology and Biotechnology, 84, 725-732. doi:10.1007/s00253-009-2010-z
- Miyamoto-Sato, E., et al. (2000) Specific bonding of puromycin to full-length protein at the C-terminus. Nucleic Acids Research, 28, 1176-1182. doi:10.1093/nar/28.5.1176
- Chen, H.X., Gore-Langton, R.E. and Cheson, B.D. (2001) Clinical trials referral resource: Current clinical trials of the anti-VEGF monoclonal antibody bevacizumab. On- cology (Williston Park), 15, 1023-1026.