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

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L. N. Shingarova^1,a*, L. E. Petrovskaya¹, A. V. Zlobinov^1,2, S. Sh. Gapizov^1,2, E. A. Kryukova¹, K. R. Birikh¹, E. F. Boldyreva¹, S. A. Yakimov¹, D. A. Dolgikh^1,2, and M. P. Kirpichnikov^1,2

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

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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 K5^T 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.

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KEY WORDS: tumor necrosis factor, TNF-binding proteins, 10th human fibronectin type III domain, bacterial display, autotransporter from Psychrobacter cryohalolentis K5^T

DOI: 10.1134/S0006297918060081

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