Troubleshooting of site-specific GGG-K(N3)-S7Abu conjugation to XCL1
| Troubleshooting | Outcome | Conclusion |
| Lower equivalents of peptide in reaction | Selective aggregation of sortagged product | Insolubility 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 product | 10% (v/v) glycerol did not improve solubility of XCL1(CC3)-K(N3)-S7Abu |
| Addition of 10 mM l-arginine·HCl37 51 | Selective aggregation of sortagged product | l-arginine·HCl did not prevent aggregation of insoluble XCL1(CC3)-K(N3)-S7Abu |
| Immobilization of sortase on NiNTA resin | Selective aggregation of sortagged product | Sortase immobilization did not prevent aggregation of product |
| SUMO solubility domain removal after XCL1 modification | Selective aggregation of sortagged product | Presence 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 buffer | Purification of XCL1(CC3)-K(PEG5k)-S7Abu possible on large scale |