RESUMO
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.
Assuntos
Proteínas de Transporte/metabolismo , Escherichia coli/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Domínio de Fibronectina Tipo III , Humanos , Plasmídeos/genética , Plasmídeos/metabolismo , Engenharia de Proteínas , Psychrobacter/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Fatores de Necrose Tumoral/química , Fatores de Necrose Tumoral/metabolismoRESUMO
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.