Table 2

Troubleshooting of site-specific GGG-K(N3)-S7Abu conjugation to XCL1

TroubleshootingOutcomeConclusion
Lower equivalents of peptide in reactionSelective aggregation of sortagged productInsolubility of XCL1(CC3)-K(N3)-S7Abu in sortase buffer
Adding 10% final (v/v) glycerol to reaction36 (interacts with large hydrophobic patches and improves solubility)Selective aggregation of sortagged product10% (v/v) glycerol did not improve solubility of XCL1(CC3)-K(N3)-S7Abu
Addition of 10 mM l-arginine·HCl37 51Selective aggregation of sortagged productl-arginine·HCl did not prevent aggregation of insoluble XCL1(CC3)-K(N3)-S7Abu
Immobilization of sortase on NiNTA resinSelective aggregation of sortagged productSortase immobilization did not prevent aggregation of product
SUMO solubility domain removal after XCL1 modificationSelective aggregation of sortagged productPresence of the solubility domain did not enable isolation of SUMO-XCL1(CC3)-K(N3)-S7Abu
PEGylation of peptide38 39 52 at K(N3)Solubility of peptide and product in sortase bufferPurification of XCL1(CC3)-K(PEG5k)-S7Abu possible on large scale