Sortase A as a cross-linking enzyme in tissue engineering

Abstract

Enzymatic crosslinking has immense appeal for tissue engineers as one of the most biocompatible methods of hydrogel crosslinking. Sortase A has a number of unique advantages over previous systems. We show an impressive and tunable range of crosslinking kinetics, from almost instantaneous gelation to several minutes. We also demonstrate that Sortase A crosslinked hydrogels have good cytocompatibility and cause no immune reaction when implanted in vivo. With its additional benefits of excellent stability in solution and easy large-scale synthesis available to any lab, we believe this novel crosslinking modality will find multiple applications in high throughput screening, tissue engineering, and biofabrication.

Enzyme‐catalysed polymer cross‐linking: Biocatalytic tools for chemical biology, materials science and beyond

“…These proteins are stable, and do not exhibit a loss in enzyme activity following incubation at room temperature for >24 hours. [38,39] The ability of sortases to proficiently fuse proteins or peptides to poly(glycine) containing substrates has encouraged researchers to explore the use of these enzymes across a range of application areas. [40][41][42] It should be noted that the enzyme in most of these applications is linking linear segments to create a longer chain rather than catalysing cross-linking between chains through non-terminal locations.…”

“…More recently, these results have been reproduced in a hyaluronic acidbased polymer system. [39] Notably, and with respect to the wider potential applications of this technology, the purity of the recombinant SrtA employed in these studies enabled the formation of cross-linked F I G U R E 1 A, Catalytic mechanism of transglutaminase. [189] The enzyme catalyses intermolecular covalent bond formation between substrate glutamyl and lysyl side chains.…”

“…also been shown to be comparable to those produced by Matrigel, or chemically cross-linked PEG-based hydrogels. [38,39] One additional intriguing observation is that SrtA may also be employed as an agent of hydrogel dissolution ( Figure 4). [47] Valdez et al…”

“…The reversibility of sortase cross-linking does risk the reaction not proceeding to completion, even in the presence of significant quantities of enzyme, resulting in unfavourable material properties (altering hardness, and gel fraction). [39,49] To mitigate this problem, it is necessary to conduct SrtA catalysed cross-linking reac- (Figure 4). [49] A further application of sortase cross-linking can be seen in the development of the sortase-mediated transpeptidation or 'Sortagging'…”

“…this enzyme is only moderately stable at room temperature and the kinetics of factor XIII gelation rapidly reach a plateau where further addition of enzyme does not increase gelation rate. [39] Encouragingly, the past decade has witnessed significant improvements in recombinant protein production technologies that go some way towards circumventing the issues of scaling up produc- SrtA enzymes have been shown to retain activity for >48 hours at room temperature, and up to 140-fold improvement in coupling activity, and exhibiting linear reaction kinetics with respect to enzyme concentration. [74] SrtA can cross-link polymers via an analogous mechanism to factor XIII, through functionalisation with vinylsulfones followed by conjugation to thiol containing peptides via Michael addition, so the suitability of this enzyme for manufacture at scale along with its favourable kinetics and stability should encourage the wider adoption of this biocatalyst for cross-linking applications.…”