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.

Keywords: Protein ligation; SUMO; Silent solubility tag; Sortase.

Figure 1

Schematic of a typical 6xHis-SUMO-Protein purification and of the Silent Tagging reaction by SUMO-SrtA (A) A schematic of a typical SUMO fusion purification (B) A schematic of the Silent Tagging reaction by SUMO-SrtA. The target protein is expressed in isotopically enriched media producing a labeled fusion protein. The target protein is expressed as a SUMO fusion protein with a C-terminal sortase, LPETG, recognition motif, aiding in overall expression and solubility. The unlabeled one-reagent SUMO-SrtA fusion tagging reagent will rapidly recognize the LPETG motif and cleave between the carbonyl of the threonine and glycine residues present in the motif. This reaction is resolved when the sortase Gly₅ secondary substrate nucleophile, fused to the N-terminus of the unlabeled SUMO, enters the active site and the enzyme performs a transpeptidation reaction yielding the amino terminus of the penta-glycine motif appended to the LPET motif present in the target protein. This ligation mixture is then incubated with Ulp1, the SUMO protease, releasing both the labelled SUMO solubility tag, and the sortase enzyme from the ligation product. This allows for a rapid purification of the ligation product by cobalt (or nickel) affinity purification.

Figure 2

A. ImageJ analysis (top) of the SDS-PAGE results (bottom) of conversion of ¹⁵N-SUMO-PhoPC-LPETG into ¹⁵N-SUMO-PhoPC-LPETG-¹⁴N-SUMO. This reaction was carried out as a 5:1 mixture of the SUMO-SrtA single tagging reagent to ¹⁵N-SUMO-PhoPC-LPETG resulting in ~90% conversion to product in 4 hours at room temperature (25°C). P, ligation product, R, SUMO-SrtA reagent, and S, SUMO-PhoPC-LPETG substrates. Lane 1, molecular weight ladder, lane 2, SUMO-SrtA tagging reagent, lane 3, ¹⁵N-SUMO-PhoPC-LPETG control, lane 4, 5:1 reagent: ¹⁵N-SUMO-PhoPC-LPETG ligation reaction 0 hour, lane 5, 1 hour, lane 6, 2 hours, lane 7, 3 hours, lane 8, 4 hours, lane 9, 6 hours. B. ImageJ analysis (top) of the representative SDS-PAGE results (bottom) indicate that the 5:1 incubation of the SUMO-SrtA ligation reagent with GFP-LPETG results in the conversion of approximately 90% of the initial substrate into ligated product in approximately 5 hours at room temperature (25°C). Lane 1, SUMO-SrtA tagging reagent, lane 2, GFP-LPETG, lane 3, 0 hour reaction, lane 4, 1 hour, lane 5, 2.5 hour, lane 6, 4 hours, lane 7, 6 hours. C. ImageJ analysis (top) of the SDS-PAGE results (bottom) indicate that the 5:1 incubation of the SUMO-SrtA ligation reagent with GFP-LPETG results in the conversion of approximately 90% of the initial substrate into ligated product in approximately 18 hours at low temp (4°C). Reactions done in the cold prevent degradation of the ligated product, tagging reagent, and target protein. Lane 1, GFP-LPETG control, lane 2, 5:1 reagent:GFP-LPETG ligation reaction 0 hour, lane 3, ligation reaction 1 hour, lane 4, 2 hours, lane 5, 3 hours, lane 6, 4 hours, lane 7, 5 hours, lane 8, 6 hours, lane 9, 7 hours, lane 10, 18 hours. D. 3 component reaction does not have high yield or efficiency. ImageJ analysis (top) of the SDS-PAGE results (bottom) of conversion of GFP-LPETG into ligation product. This reaction was carried out as a 5:5:1 mixture of the Gly₅-SUMO and SrtA-6xHis to GFP-LPETG resulting in ~55% conversion to product in 6 hours at room temperature (25°C). Lane 1, reaction 0 hour, lane 2, reaction 1 hour, lane 3, reaction 2 hours, lane 4, 3 hour, lane 5, 4 hours, lane 6, 5 hours, lane 7, 6 hours.

Figure 3

HSQCs of SUMO tagged ¹⁵N-PhoPC. ¹⁵N-SUMO-PhoPC-LPETG prior to the SUMO-SrtA silent ligation reaction (top), and after SUMO-SrtA silent tagging reaction (bottom). The boxed regions show regions where signals from the 15N-SUMO tag are removed by replacing it with the silent SUMO tag.