Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy

Abstract

NMR spectroscopy has distinct advantages for providing insight into protein structures, but faces significant resolution challenges as protein size increases. To alleviate such resonance overlap issues, the ability to produce segmentally labeled proteins is beneficial. Here we show that the S. aureus transpeptidase sortase A can be used to catalyze the ligation of two separately expressed domains of the same protein, MecA (B. subtilis). The yield of purified, segmentally labeled MecA protein conjugate is ∼40%. The resultant HSQC spectrum obtained from this domain-labeled conjugate demonstrates successful application of sortase A for segmental labeling of multi-domain proteins for solution NMR study.

Enzymatic activity of circular sortase A under denaturing conditions: An advanced tool for protein ligation

“…Sortase practical applications require large amounts of the protein because of the low turnover of the catalyzed transpeptidation, requiring stoichiometric amounts of the enzyme for protein coupling [14,36]. However, cyclization allows a significant increase in the turnover of the reaction in the presence of 2-2.5 M urea.…”

Site‐Specific Protein Labeling via Sortase‐Mediated Transpeptidation

“…In terms of reaction reversibility, a handful of strategies have been introduced that drive reaction equilibrium through selective removal or deactivation of transpeptidation products. These strategies may be as simple as running the reaction under dialysis conditions to separate out low molecular weight by-products (Pritz et al, 2007; Kobashigawa et al, 2009; Refaei et al, 2011), but also include the use of β-hairpins (Yamamura et al, 2011), depsipeptides (Williamson et al, 2012; Liu et al, 2014), or masked metal binding peptides (Row, et al 2015) that prevent certain transpeptidation products from re-entering the catalytic cycle. Finally, advances have been made in expanding the substrate scope of sortase-catalyzed transpeptidations beyond the LPXTG motif.…”

Bilden und Brechen von Peptidbindungen: Protein‐Engineering mithilfe von Sortase

“…Auch über die teilweise Markierung des MecA-Proteins von Bacillus subtilis durch Verwendung von Sortase in einer NMR-spektroskopischen Studie wurde berichtet. [66] Mit Sortase wurden auch zahlreiche Immundetektionsreagentien erzeugt. Weiterhin gelangen Protein-Protein-Ligationen zwischen dem Fc-Bindungsmodul (ZZ-Domäne) und mehreren Detektionsenzymen (alkalische Phosphatase, Luciferase, Glucoseoxidase) mit Sortase.…”