SpyTag/SpyCatcher Cyclization Confers Resilience to Boiling on a Mesophilic Enzyme

Abstract

SpyTag is a peptide which spontaneously forms an amide bond to its protein partner SpyCatcher. Here we fused SpyTag at the N-terminus of β-lactamase and SpyCatcher at the C-terminus, so the partners could cyclize to lock together the termini of the enzyme. Wild-type enzyme aggregates above 37 °C, with irreversible loss of activity. Cyclized β-lactamase was soluble even after heating at 100 °C; after cooling, the catalytic activity was restored. SpyTag/SpyCatcher-cyclization achieved > 60 °C increase in stability, much larger than from point mutation or alternative approaches to cyclization. Cyclized dihydrofolate reductase was similarly resilient. Analyzing unfolding calorimetrically and via mutants, cyclization did not increase the unfolding temperature of β-lactamase, but facilitated refolding after thermal stress. SpyTag and SpyCatcher sandwiching represents a simple and efficient route for enzyme cyclization, with potential to greatly enhance the robustness of biocatalysts.

Kinetic Controlled Tag-Catcher Interactions for Directed Covalent Protein Assembly

“…Although, our current assembly was accomplished with EGFP fusions, we do not foresee any difficulty fusing other proteins onto the Tag-Catchers as shown from previous studies [11, 12, 15]. To further enhance yields, solid phase addition, as demonstrated by SnoopTag [16], or step-wise purifications could also be utilized.…”

“…Although, circular proteins have been constructed previously using SpyTag-SpyCatcher [11, 12], development of the SdyTag presents us with an opportunity to generate specific internally-circularized proteins with specific free Tags to be used for further protein assembly possibilities.…”

“…SpyTag-SpyCatcher is an important addition to the protein engineering toolbox as it is robust, rapid and flexible in its placement within the protein. Applications for this protein ligation system have been numerous; these include controlled macromolecular assembly [11], protein cyclisation [12], cell labelling [13], antibody engineering [14] and hydrogel synthesis [15]. To realize the goal of assembling higher order macroassemblies from modular protein parts, multiple orthogonal interaction domains are needed.…”

B 12 -dependent photoresponsive protein hydrogels for controlled stem cell/protein release

“…Because of its high efficiency and modularity, this chemistry has led to a number of applications, including control of biomacromolecular topology, synthesis of bioactive and "living" materials, and biomolecular imaging (16,18,(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). It has proven to be a powerful method for constructing complex biomolecular architectures both in vitro and in vivo.…”

Editorial: Protein stabilization – crossroad for protein‐based processes and products

“…In parallel, an increasing number of bioinformatics and computational biology approaches [8] are starting to provide a rational physical basis for the design of biocatalysts with improved performance and stability [9][10][11]. See the article by Suplatov et al in this Special Issue [12].…”