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Fusion with the cold-active esterase facilitates autotransporter-based surface display of the 10th human fibronectin domain in Escherichia coli |
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| Authors: | Petrovskaya L. E. Zlobinov A. V. Shingarova L. N. Boldyreva E. F. Gapizov S. Sh. Novototskaya-Vlasova K. A. Rivkina E. M. Dolgikh D. A. Kirpichnikov M. P. |
| Affiliation: | 1.Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya, 16/10, 117997, Moscow, Russian Federation ;2.Department of Biology, M. V. Lomonosov Moscow State University, Leninskie gory, 1, 119234, Moscow, Russian Federation ;3.Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, 142290, Pushchino, Moscow Region, Russian Federation ; |
| Abstract: | Cell surface display is a popular approach for the construction of whole-cell biocatalysts, live vaccines, and screening of combinatorial libraries. To develop a novel surface display system for the popular scaffold protein 10th human fibronectin type III domain (10Fn3) in Escherichia coli cells, we have used an α-helical linker and a C-terminal translocator domain from previously characterized autotransporter from Psychrobacter cryohalolentis K5T. The level of 10Fn3 passenger exposure at the cell surface provided by the hybrid autotransporter Fn877 and its C-terminal variants was low. To improve it, the fusion proteins containing 10Fn3 and the native autotransporter passenger Est877 or the cold-active esterase EstPc in different orientations were constructed and expressed as passenger domains. Using the whole-cell ELISA and activity assays, we have demonstrated that N-terminal position of EstPc in the passenger significantly improves the efficiency of the surface display of 10Fn3 in E. coli cells. |
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