Table 2.
Summary of Ligation Conditions and Yields of each Oxidation and Ligation Approach
| strategy | KIIIA precursor oxidation yielda | PaurTx3 precursor oxidation yielda | conditionc | reaction time | ligation yieldb |
|---|---|---|---|---|---|
| SrtA5° | 40% | 50% | 100 μM PaurTx3[LPATGG]:300 μM [G8]KIIIA:30 μM enzyme 50 mM Tris:150 mM NaCl:10 mM CaCl2, pH 7.4, 37 °C |
15 min | 60% |
| wt-OaAEP 1 | 38% | 28% | 250 μM PaurTx3[NGL]:750 μM [G8]KIIIA:3 μM enzyme 0.1 M Na2HPO4:0.1 M NaCl, pH 6.5 |
4 h | 12% |
| C247A-OaAEP 1 | 40% | 18% | 20 μM PaurTx3[NGL]:60 μM [GVG6]KIIIA:75 nM enzyme 0.1 M Na2HPO4:0.1 M NaCl, pH 6.5 |
6 h | 35% |
| butelase 1 | 40% | 29% | 20 μM PaurTx3[NHV]:60 μM [GVG6]KIIIA:100 nM enzyme 0.1 M Na2HPO4:0.1 M NaCl, pH 6.5 |
6 h | 38% |
| KAHA | 25% | 28% | 22 mM PaurTx3-ka:15 mM [OprG8]-KIIIA | 6 h | 36% |
| peptiligased | 40% | n/d | 90% DMSO:10% H2O, 0.1 M oxalic acid, 60 °C 10 mM PaurTx3[linker]-OCamL:15 mM [linker]-KIIIA 10% DMSO in 1 M potassium phosphate, pH 8–8.5 (with/without 2 |
n/a | n/d |
a
Oxidation yield was calculated based on the precursor weight and isolated oxidized peptide weight.
b
Ligation yield was calculated by dividing the observed weight by the theoretical weight calculated from PaurTx3 analogs.
c
Reactions carried out at room temperature unless specified.
d
Ligation was not carried due to insolubility of peptide fragments.