University of Kentucky

Poster Title

Utilization of a Calmodulin Lysine Methyltransferase Co-expression System for the Generation of a Combinatorial Library of Post-translationally Modified Proteins

Institution

University of Kentucky

Abstract

Modified proteins are increasingly being utilized as tools for Kentucky's and the nation's growing biotechnology and biopharmaceutical industry. By successfully incorporating sequence diversity into proteins, combinatorial libraries have been a staple technology used in protein engineering, directed evolution, and synthetic biology for generating proteins with novel specificities and activities. However, these approaches mostly overlook the incorporations of post-translational modifications, which nature extensively uses for modulating protein activities in vivo. Therefore, we hypothesize that post translational modifications of libraries will alter specificity toward the initial step of incorporating post- translational modifications into combinatorial libraries. In order to do so, we present a bacterial co-expression system, utilizing a recently characterized calmodulin methyltransferase (CaM KMT), to trimethylate a combinatorial library of the calmodulin central linker region. We show that this system is robust, with the successful overexpression and post- translational modification performed in E. coli. Furthermore we show that trimethylation differentially affected the conformational dynamics of the protein upon the binding of calcium, and the thermal stability of the apoprotein. Collectively, these data support that when applied to an appropriately designed protein library scaffold, CaM KMT is able to produce a post-translationally modified library of protein sequences, thus providing a powerful tool for future protein library designs and constructions. Thus, could prove essential in Kentucky's emerging the biotechnology industry.

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Utilization of a Calmodulin Lysine Methyltransferase Co-expression System for the Generation of a Combinatorial Library of Post-translationally Modified Proteins

Modified proteins are increasingly being utilized as tools for Kentucky's and the nation's growing biotechnology and biopharmaceutical industry. By successfully incorporating sequence diversity into proteins, combinatorial libraries have been a staple technology used in protein engineering, directed evolution, and synthetic biology for generating proteins with novel specificities and activities. However, these approaches mostly overlook the incorporations of post-translational modifications, which nature extensively uses for modulating protein activities in vivo. Therefore, we hypothesize that post translational modifications of libraries will alter specificity toward the initial step of incorporating post- translational modifications into combinatorial libraries. In order to do so, we present a bacterial co-expression system, utilizing a recently characterized calmodulin methyltransferase (CaM KMT), to trimethylate a combinatorial library of the calmodulin central linker region. We show that this system is robust, with the successful overexpression and post- translational modification performed in E. coli. Furthermore we show that trimethylation differentially affected the conformational dynamics of the protein upon the binding of calcium, and the thermal stability of the apoprotein. Collectively, these data support that when applied to an appropriately designed protein library scaffold, CaM KMT is able to produce a post-translationally modified library of protein sequences, thus providing a powerful tool for future protein library designs and constructions. Thus, could prove essential in Kentucky's emerging the biotechnology industry.