ENGINEERING THE ASLOV2 DOMAIN FOR OPTICALLY CONTROLLED PROTEIN CONJUGATION
Date
2019-05
Authors
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Publisher
University of Delaware
Abstract
The ability to specifically conjugate proteins together has been a useful tool in cellular monitoring and controlling expression. The SpyTag:SpyCatcher systems has been developed as a robust, irreversible peptide tagging system used in vitro and in vivo. The Sortase A transpeptidation system, derived from Gram-positive bacteria, is a reversible, enzyme mediated conjugation system with applications in purification and tagging. We can imagine that if we can control the conjugation event in time and space, the system could become more versatile with a metaphorical “on/off” switch.
Learning from photoreceptors found in nature, constructs were generated to control protein conjugation events. Blue light dependence was introduced via the light-oxygen-voltage 2 domain from Avena sativa (AsLOV2). When irradiated with blue light the domain undergoes a dramatic conformational change that renders the C-terminus “undocked”. Blue light dependent conjugation was tested by genetically fusing the AsLOV2 domain to a reactive partner of either the SpyTag:SpyCatcher system or Sortase A system. Fusion of SpyTag to LOV showed the best results for the irreversible system. After 5 hours, the conjugation reaction was 3-fold more complete with blue light irradiation. The Sortase A system has been generated and expressed but has not been tested. If deemed feasible, application of these types of systems include but are not limited to intracellular protein localization and the dynamic decoration of hydrogels.
Description
Keywords
Chemical engineering, Protein conjugation, ASLOV2 domain