Development and Characterization of a Model Post-Translationally Modified Protein Library

Emily MajorFollow

Grade Level at Time of Presentation

Junior

Major

Agricultural and Medical Biotechnology

Minor

Neuroscience and Psychology

Institution

University of Kentucky

KY House District #

71

KY Senate District #

22

Department

Department of Neuroscience; Department of Horticulture; Department of Molecular and Cellular Biology

Abstract

Protein engineering is a platform biotechnology that alters a protein sequence for a variety of applications in research, medicine, and agriculture. For example, the biopharmaceutical industry relies on protein engineering to produce over a quarter of all emerging drugs (a $60 billion a year industry). However, as the applications for engineered proteins becomes more widespread, new innovations are needed to increase their stringency. While numerous approaches, including the use of combinatorial libraries, have been utilized for engineering protein binding specificity through altering protein sequence, the incorporation of post-translational modifications, which nature uses to alter protein activity, have been overlooked. To incorporate these regulatory elements into protein combinatorial libraries, we developed a powerful bacterial post-translational co-expression system utilizing calmodulin methyltransferase (CaM KMT) to completely trimethylate a diverse protein library of the calmodulin (CaM) central linker region. Characterization of 17 randomly selected library members show that all library sequences were over-expressed and post-translationally modified [1]. In addition, we show that trimethylation differentially altered the conformational changes of CaM associated with the binding of calcium, CaM’s thermal stability, and binding specificity towards CaM-peptide target sequences. To guide future library designs and applications, the specificity of the CaM KMT needs to be defined. Forty mutations were designed to alter the residues around the Lysine-115, the trimethlyation site on CaM. The characterization of individual mutants show that certain positions and residues govern the recognition of CaM KMT, with those positions closest to Lysine-115 having the largest effect. These data further enhance the ability of our post-translationally modified library to unbiasedly target novel sequences, providing a more advanced technology for designing and generating protein with stringent protein-target specificities for biomedicine.

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Development and Characterization of a Model Post-Translationally Modified Protein Library

Protein engineering is a platform biotechnology that alters a protein sequence for a variety of applications in research, medicine, and agriculture. For example, the biopharmaceutical industry relies on protein engineering to produce over a quarter of all emerging drugs (a $60 billion a year industry). However, as the applications for engineered proteins becomes more widespread, new innovations are needed to increase their stringency. While numerous approaches, including the use of combinatorial libraries, have been utilized for engineering protein binding specificity through altering protein sequence, the incorporation of post-translational modifications, which nature uses to alter protein activity, have been overlooked. To incorporate these regulatory elements into protein combinatorial libraries, we developed a powerful bacterial post-translational co-expression system utilizing calmodulin methyltransferase (CaM KMT) to completely trimethylate a diverse protein library of the calmodulin (CaM) central linker region. Characterization of 17 randomly selected library members show that all library sequences were over-expressed and post-translationally modified [1]. In addition, we show that trimethylation differentially altered the conformational changes of CaM associated with the binding of calcium, CaM’s thermal stability, and binding specificity towards CaM-peptide target sequences. To guide future library designs and applications, the specificity of the CaM KMT needs to be defined. Forty mutations were designed to alter the residues around the Lysine-115, the trimethlyation site on CaM. The characterization of individual mutants show that certain positions and residues govern the recognition of CaM KMT, with those positions closest to Lysine-115 having the largest effect. These data further enhance the ability of our post-translationally modified library to unbiasedly target novel sequences, providing a more advanced technology for designing and generating protein with stringent protein-target specificities for biomedicine.