Extracellular Self-Assembly of Functional and Tunable Protein Conjugates from Bacillus subtilis

Abstract

The ability to stably and specifically conjugate recombinant proteins to one another is a powerful approach for engineering multifunctional enzymes, protein therapeutics, and novel biological materials. While many of these applications have been illustrated through in vitro and in vivo intracellular protein conjugation methods, extracellular self-assembly of protein conjugates offers unique advantages: simplifying purification, reducing toxicity and burden, and enabling tunability. Exploiting the recently described SpyTag-SpyCatcher system, we describe here how enzymes and structural proteins can be genetically encoded to covalently conjugate in culture media following programmable secretion from Bacillus subtilis. Using this approach, we demonstrate how self-conjugation of a secreted industrial enzyme, XynA, dramatically increases its resilience to boiling, and we show that cellular consortia can be engineered to self-assemble functional protein-protein conjugates with tunable composition. This novel genetically encoded modular system provides a flexible strategy for protein conjugation harnessing the substantial advantages of extracellular self-assembly.

Engineered Living Materials: Prospects and Challenges for Using Biological Systems to Direct the Assembly of Smart Materials

“…Nevertheless, some progress has been made in reimagining extracellular assembly modes for secreted proteins. In one example, researchers employed the genetically encoded SpyTag–SpyCatcher conjugation scheme to create modular extracellular polymers . First, genes were constructed encoding for proteins with form SpyPart–protein–SpyPart, in which arbitrary protein sequences were flanked by different combinations of SpyTag and SpyCatcher.…”

From Synthesis to Characterization of Site-Selective PEGylated Proteins

“…The SpyTag/SpyCatcher system allows the specific and covalent conjugation of proteins through two short polypeptide tags (Zakeri et al, 2012). The larger partner, the SpyCatcher, adopts an immunoglobulin-like conformation that specifically binds the SpyTag (γ-carbon of Asp-117), leading the formation of an extremely resistant intermolecular bond between two amino acid side chains (Gilbert et al, 2017). In this extremely fast method, no exogenous enzymes need to be added or removed (Fisher et al, 2017) and despite its recent description, this system has already been used in the production of synthetic vaccines (Brune et al, 2016), thermo-stable enzymes (Schoene et al, 2016;Wang et al, 2016), and other applications (Fierer et al, 2014;Dovala et al, 2016;Lakshmanan et al, 2016).…”

Post‐translational Assembly of Protein Parts into Complex Devices by Using SpyTag/SpyCatcher Protein Ligase

“…The concept of enzyme tunability can be exploited by using the SpyTag/SpyCatcher system as exemplified by Gilbert, et al . when they extracellularly self‐assembled secreted enzymes of industrial value.…”

“…The concept of enzyme tunability can be exploitedb yu sing the SpyTag/SpyCatcher system as exemplified by Gilbert, et al [37] when they extracellularly self-assembleds ecretede nzymeso fi ndustrial value. They sought first to create thermotolerant enzymes for biotechnology throughS pyTag/SpyCatchermediated cyclization for export extracellularly.S econdly, they engineered self-organizing protein assemblies by fine-tuning relative inoculation ratios in bacterial cultures, without the need for further genetice ngineering methods or alteration of promoters, thus demonstrating the tunability of protein architectures outside of the cell ( Figure 2B).…”