title: Utilization of a calmodulin lysine methyltransferase co-expression system for the generation of a combinatorial library of post-translationally modified proteins.
abstract: 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. As an initial step of incorporating post-translational modifications into combinatorial libraries, 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 over-expression and post-translational modification performed in Escherichia 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.
authors.affiliation: Department of Horticulture, University of Kentucky, Lexington, KY 40546, USA.
authors.forename: Roberta
authors.initials: R
authors.lastname: Magnani
authors.forename: Brian
authors.initials: B
authors.lastname: Chaffin
authors.forename: Emerson
authors.initials: E
authors.lastname: Dick
authors.forename: Michael L
authors.initials: ML
authors.lastname: Bricken
authors.forename: Robert L
authors.initials: RL
authors.lastname: Houtz
authors.forename: Luke H
authors.initials: LH
authors.lastname: Bradley
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008322:Mammals; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D058615:Synthetic Biology / Q000379:methods
chemical_list: D011994:Recombinant Proteins; D008780:Methyltransferases; C038999:calmodulin-lysine methyltransferase