Rapid addition of unlabeled silent solubility tags to proteins using a new substrate-fused sortase reagent

Abstract

Many proteins can’t be studied using solution NMR methods because they have limited solubility. To overcome this problem, recalcitrant proteins can be fused to a more soluble protein that functions as a solubility tag. However, signals arising from the solubility tag hinder data analysis because they increase spectral complexity. We report a new method to rapidly and efficiently add a non-isotopically labeled Small Ubiquitin-like Modifier protein (SUMO) solubility tag to an isotopically labeled protein. The method makes use of a newly developed SUMO-Sortase tagging reagent in which SUMO and the Sortase A (SrtA) enzyme are present within the same polypeptide. The SUMO-Sortase reagent rapidly attaches SUMO to any protein that contains the sequence LPXTG at its C-terminus. It modifies proteins at least 15-times faster than previously described approaches, and does not require active dialysis or centrifugation during the reaction to increase product yields. In addition, silently tagged proteins are readily purified using the well-established SUMO expression and purification system. The utility of the SUMO-Sortase tagging reagent is demonstrated using PhoP and green fluorescent proteins, which are ~90% modified with SUMO at room temperature within four hours. SrtA is widely used as a tool to construct bioconjugates. Significant rate enhancements in these procedures may also be achieved by fusing the sortase enzyme to its nucleophile substrate.

Sortase Transpeptidases: Structural Biology and Catalytic Mechanism

“…Moreover, the activity of SaSrtA has been improved using directed evolution approaches, resulting in tetramutant enzyme that is ~140-fold more active than the native SaSrtA (Chen et al, 2011). Additional rate enhancements have been achieved by altering the reaction conditions and by fusing the nucleophile substrate to SaSrtA (Amer, Macdonald, Jacobitz, Liauw, & Clubb, 2016). The reader is referred to a number of excellent reviews describing the use of sortase as a bioconjugation reagent (Proft, 2010; Tsukiji & Nagamune, 2009; Wu & Guo, 2012).…”

Recent advances in sortase-catalyzed ligation methodology

“…Both N- and C-terminal fusions have been described, typically in the context of new approaches for recombinant protein expression and purification [3, 24–30]. Of these, fusions at the N-terminus are particularly intriguing as it has been suggested that the N-termini of these constructs can access the enzyme active site in an intramolecular fashion, thereby driving transpeptidation due to the increase in local reactant concentration [3, 27, 30]. As an example, Amer et al demonstrated an increase in ligation rates when a construct consisting of wild-type SrtA staph fused at its N-terminus to an aminoglycine-containing SUMO module was reacted with a separate LPXTG substrate [3].…”

“…Of these, fusions at the N-terminus are particularly intriguing as it has been suggested that the N-termini of these constructs can access the enzyme active site in an intramolecular fashion, thereby driving transpeptidation due to the increase in local reactant concentration [3, 27, 30]. As an example, Amer et al demonstrated an increase in ligation rates when a construct consisting of wild-type SrtA staph fused at its N-terminus to an aminoglycine-containing SUMO module was reacted with a separate LPXTG substrate [3]. In this case, reaction rates involving the SUMO-SrtA staph fusion were significantly enhanced relative to a control reaction involving separate SrtA staph , aminoglycine SUMO, and LPXTG substrate.…”

“…It is well established that the removal of reaction by-products or residual sortase enzyme can be achieved through the strategic inclusion of His 6 affinity handles, or the use of the elastin-like polypeptide as a controlled solubility switch [3, 26, 27, 31]. SrtA staph has also been covalently immobilized on sepharose or PEGA resins to facilitate enzyme removal and enzyme recycling [14, 32].…”

“…With regard to NMR, sortagging has found use in segmental isotope labeling, wherein an isotopically-enriched protein fragment is ligated to an NMR “silent” fragment. Examples include the attachment of unlabeled solubility enhancing tags (GB1, SUMO), as well as the construction of multidomain proteins (MecA, TIA-1, Hsp90, BRD4) where only certain domains are isotopically labeled to simplify the complexity of NMR spectra [3, 36–38, 54]. Interestingly, the particular challenges of segmental labeling have provided excellent opportunities for refining sortagging methods.…”