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 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.

High-throughput recombinant protein expression in Escherichia coli : current status and future perspectives

“…In E. coli, recombinant proteins can be ubiquitinated by co-overexpressing the target protein, ubiquitin and ubiquitin ligases [130]. Additionally, methylation, myristoylation and acetylation have been successfully performed in E. coli by coexpressing a methyltransferase, myristoyltransferase and acetylase, respectively [131][132][133]. Therefore, target proteins can be post-translationally modified in E. coli expression systems by coexpressing genes related to the modifications of interest.…”

TypiCal but DeliCate Ca++re: Dissecting the Essence of Calcium Signaling Network as a Robust Response Coordinator of Versatile Abiotic and Biotic Stimuli in Plants

“…In addition to the crosstalk between the NRT1.1 sensor and Ca 2+/ CaM signaling components, Calmodulin-lysine N-methyltransferase (CaM KMT) has been identified as an important modulator of several signal transductions (Banerjee et al, 2013). CaM KMT trimethylates CaM at a specific lysyl residue and the methylation status of CaM plays an important role in the determination of CaM-binding partners in presence of Ca 2+ (Magnani et al, 2010(Magnani et al, , 2012. The earlier report depicted that undermethylation of CaM in CaM KMT knockout mutant plants made it relatively more tolerant to salt, drought, and thermal stresses compared to CaM KMT overexpressed and control Arabidopsis plants through possible changes in CaM-binding partners (Banerjee et al, 2013).…”

Escherichia coli Extract-Based Cell-Free Expression System as an Alternative for Difficult-to-Obtain Protein Biosynthesis

“…Although protein glycosylation in the E. coli CF system is the only successful example of PTM and considerable improvement is still required, there is hope for the expansion of controllable PTM. Other types of PTM were recently applied for recombinant protein modification in an E. coli background, including phosphorylation achieved due to the activity of expressed human Jun N-terminal kinase 1, JNK1 [167], sumoylation-in the presence of SUMO (small ubiquitin-like modifier) and SUMO E3 ligase [168], and methylation catalyzed by calmodulin lysine methyltransferase, CaM KMT [169].…”