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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Acknowledgements
The work was supported by Russian Academy of Sciences program “Molecular and Cellular Biology”, RF President Grant for Scientific Schools Grant SS-8384.2016.4 and RFBR Grants nos. 16-04-00717a, 15-04-07772a.
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Petrovskaya, L.E., Zlobinov, A.V., Shingarova, L.N. et al. Fusion with the cold-active esterase facilitates autotransporter-based surface display of the 10th human fibronectin domain in Escherichia coli . Extremophiles 22, 141–150 (2018). https://doi.org/10.1007/s00792-017-0990-7
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DOI: https://doi.org/10.1007/s00792-017-0990-7