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Construction of Artificial TNF-Binding Proteins Based on the 10th Human Fibronectin Type III Domain Using Bacterial Display

L. N. Shingarova1,a*, L. E. Petrovskaya1, A. V. Zlobinov1,2, S. Sh. Gapizov1,2, E. A. Kryukova1, K. R. Birikh1, E. F. Boldyreva1, S. A. Yakimov1, D. A. Dolgikh1,2, and M. P. Kirpichnikov1,2

1Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia

2Lomonosov Moscow State University, Faculty of Biology, 119234 Moscow, Russia

* To whom correspondence should be addressed.

Received December 22, 2017; Revision received February 21, 2018
Construction of antibody mimetics on the base of alternative scaffold proteins is a promising strategy for obtaining new products for medicine and biotechnology. The aim of our work was to optimize the cell display system for the 10th human fibronectin type III domain (10Fn3) scaffold protein based on the AT877 autotransporter from Psychrobacter cryohalolentis K5T and to construct new artificial TNF-binding proteins. We obtained a 10Fn3 gene combinatorial library and screened it using the bacterial display method. After expression of the selected 10Fn3 variants in Escherichia coli cells and analysis of their TNF-binding activity, we identified proteins that display high affinity for TNF and characterized their properties.
KEY WORDS: tumor necrosis factor, TNF-binding proteins, 10th human fibronectin type III domain, bacterial display, autotransporter from Psychrobacter cryohalolentis K5T

DOI: 10.1134/S0006297918060081