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

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.

Fusion with an albumin-binding domain improves pharmacokinetics of an αvß3-integrin binding fibronectin scaffold protein.

Fusion with an albumin-binding domain (ABD) of streptococcal protein G represents a popular approach for half-life extension of small protein therapeutics in the organism. To increase the circulation time of engineered αvß3-integrin-binding protein (JCL) based on the 10th human fibronectin type III domain (10 Fn3), we have constructed several fusions with ABD with different orientations of the partner proteins and linker length. The recombinant proteins were expressed in Escherichia coli cells and purified by nickel-affinity chromatography. All fusion proteins bound human serum albumin (HSA) in ELISA assay; however, fusions with longer linkers demonstrated better performance. Interaction of ABD-L15 -JCL and JCL-L14 -ABD with HSA was confirmed by analytical size exclusion chromatography and pull-down assays. Surprisingly, the thermal stability of ABD-L15 -JCL was dramatically decreased in comparison with JCL and JCL-L14 -ABD proteins. Pharmacokinetic studies revealed that JCL-L14 -ABD circulated in murine blood about 10 times longer than ABD-L15 -JCL and 960 times longer than JCL. Biodistribution studies of JCL-L14 -ABD in mice revealed its increased level in blood and a decreased accumulation in liver and kidneys in comparison with JCL. Obtained results demonstrate the utility of the fusion with ABD for half-life extension of the binding proteins based on 10 Fn3.

Fusion with the cold-active esterase facilitates autotransporter-based surface display of the 10th human fibronectin domain in Escherichia coli.

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.