Functional Inhibition of Sugar Transport by a Designed Novel Protein

Abstract

Designed ankyrin repeat proteins have been shown to be useful for different applications such as structural, mechanistic, clinical studies, as well as drug development. We have constructed a combinatorial DNA library encoding ankyrin repeat proteins by PCR-based assembly. The ankyrin proteins have both N- and C-terminal caps and five internal repeats (called N5C). The estimated diversity of the library is higher than 1020. Applying circular dichroism spectroscopy, four purified proteins exhibit high melting temperature (> 95 °C). We have tested the constructed library for the selection of specific ankyrin proteins targeting the melibiose transport in Escherichia coli. The melibiose permease is one of few overexpressed proteins during bacterial infection for gaining energy. Using cells with plasmid-encoded constructed library and chromosomally-encoded MelA, the alpha-galactosidase, and MelB, the melibiose permease, we have collected a group of N5C candidates that alter the melibiose fermentation with no inhibition of glucose fermentation, indicating no effect on the downstream utilization pathways. Three candidates show significant inhibition of melibiose transport. Further tests, using sugar transport assay on different genetic backgrounds, MelA activity assay, and protein-protein interaction assay, have been applied to identify specific effect. These preliminary studies suggested that one N5C protein possibly inhibits mel operon. These bio-reagents could be potentially applied to study the sugar transport during bacterial infection and also facilitate transporter proteins crystallography.