Flow‐Based Enzymatic Ligation by Sortase A

Abstract

Sortase-mediated ligation (sortagging) is a versatile, powerful strategy for protein modification. Because the sortase reaction reaches equilibrium, a large excess of polyglycine nucleophile is often employed to drive the reaction forward and suppress sortase-mediated side reactions. A flow-based sortagging platform employing immobilized sortase A within a microreactor was developed that permits efficient sortagging at low nucleophile concentrations. The platform was tested with several reaction partners and used to generate a protein bioconjugate inaccessible by solution-phase batch sortagging.

Methods for the directed evolution of proteins

“…Owing to the unfavourable kinetics of wild-type SrtA, efficient bioconjugation typically requires equimolar concentration of substrate and enzyme. Iterated rounds of FACS screening with increasing stringency produced evolved variants of SrtA (eSrtA) with 140-fold higher k cat /K m values, enabling new applications [78][79][80][81][82][83] .…”

Recent advances in sortase-catalyzed ligation methodology

“…SrtA staph has also been covalently immobilized on sepharose or PEGA resins to facilitate enzyme removal and enzyme recycling [14, 32]. A useful extension of sortase-immobilization has been the construction of flow-based systems in which reactants are passed over immobilized sortase columns [14, 33]. The flow-based reactor described by Pentelute and coworkers is particularly noteworthy in that the authors demonstrated that flow-based sortagging increased isolated product yields, and reduced contamination by hydrolytic, cyclic, or oligomeric by-products relative to analogous reactions performed using a standard solution-phase protocol [33].…”

“…Indeed, sortagging reactions conducted under dialysis conditions or in centrifugal filtration units can significantly enhance reaction conversion through selective removal of low molecular weight aminoglycine by-products [35–38]. Similarly, affinity immobilization strategies combined with sortase-substrate fusions or the aforementioned flow-based sortagging platform have been shown to minimize the need for excess reagents through the selective removal of various reaction components [27, 33]. All of these approaches are straightforward, and typically do not require changes in the sortase substrate recognition site.…”

Recent Advances in Chemoenzymatic Bioconjugation Methods

“…Pentelute and coworkers designed a flow-based process that allows the use of lower concentrations of nucleophile without degradation of product yield. 49 Liu and coworkers chose to evolve SrtA using a yeast display technology, which lead to a mutant with 140-fold greater activity over wild type. 50 A subsequent evolution project resulted in two new SrtA mutants with altered sequence specificity (LPXSG and LAXTG).…”