Deletion Variants of Autotransporter from Psychrobacter cryohalolentis Increase Efficiency of ¹⁰FN3 Exposure on the Surface of Escherichia coli Cells

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Lyudmila N. Shingarova^1,a*, Lada E. Petrovskaya¹, Elena A. Kryukova¹, Sultan S. Gapizov^1,2, Elena F. Boldyreva¹, Dmitriy A. Dolgikh^1,2, and Mikhail P. Kirpichnikov^1,2

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

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

^* To whom correspondence should be addressed.

Received June 16, 2022; Revised July 11, 2022; Accepted July 11, 2022

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The autotransporter AT877 from Psychrobacter cryohalolentis belongs to the family of outer membrane proteins containing N-terminal passenger and C-terminal translocator domains that form the basis for the design of display systems on the surface of bacterial cells. It was shown in our previous study that the passenger domain of AT877 can be replaced by the cold-active esterase EstPc or the tenth domain of fibronectin type III (¹⁰Fn3). In order to increase efficiency of the ¹⁰Fn3 surface display in Escherichia coli cells, four deletion variants of the Fn877 hybrid autotransporter were obtained. It was demonstrated that all variants are present in the membrane of bacterial cells and facilitate binding of the antibodies specific against ¹⁰Fn3 on the cell surface. The highest level of binding is provided by the variants Δ239 and Δ310, containing four and seven beta-strands out of twelve that comprise the structure of the translocator domain. Using electrophoresis under semi-native conditions, presence of heat modifiability in the full-size Fn877 and its deletion variants was demonstrated, which indicated preservation of beta structure in their molecules. The obtained results could be used to optimize the bacterial display systems of ¹⁰Fn3, as well as of other heterologous passenger domains.

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KEY WORDS: secretion, autotransporter, deletion variants, 10th human fibronectin type III domain, bacterial display

DOI: 10.1134/S0006297922090061

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