ENGINEERING AFFIBODIES TO INHIBIT GLUCOSYLTRANSFERASES USING YEAST DISPLAY
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Date
2017-08-31
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Johns Hopkins University
Abstract
Dextransucrases are a type of Glucosyltransferase (Gtf) which have many industrial applications, including oral care and baking, and have been identified as a biologically relevant target to inhibit. There are many advantages to designing a small protein affibody to accomplish this goal compared to using antibodies and small molecule inhibitors, including their inherent stability, expressibility, and engineerability. Affibodies are small protein domains, the original IgG binding surface of which can be varied combinatorially to produce tight binders to diverse protein targets. Yeast display provides a powerful tool to assemble and rapidly screen large libraries for binders against the target of interest, Gtfs. Initial studies showed the wild-type affibody could be successfully expressed and exhibit binding to an IgG antibody in the yeast display system. Following this, a valid approach to designing, assembling, and expressing an affibody variant library in this yeast display system was systematically employed. The assembled library contained an estimated 2.5 x 108 members, and a sampling of this library illustrated the high quality and diversity of these variants. Initial surface display analysis indicated sufficient library expression level, as well as an anticipated disruption to IgG binding.
The affibody variant library was enriched and sorted for Gtf binders using fluorescent activated cell sorting under several different conditions. Upon analysis of the binding populations, a potential Gtf binding motif was uncovered. Moreover, a dominant variant identified was found to be present in the majority of the libraries analyzed, regardless of sorting conditions. This highly represented variant was confirmed as a specific Gtf binder, with a dissociation constant in the range of tens to hundreds nanomolar.
In addition to this validated binder, fifteen variants of interest were expressed and analyzed for Gtf inhibition. Though no Gtf inhibitors were found in these select few variants analyzed, the results from this screening demonstrate great promise to obtain such a molecule. Furthermore, the findings from this library screening could be strategically leveraged in the future using an optimized approach to maximize chances of identifying a Gtf inhibitor.
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Keywords
Glucosyltransferase Inhibitors, Affibodies