Table I.
Properties of HSA-DKK2C2 fusion proteins
| Construct | Substitutions | Heparin-Sepharose elution mM NaCl (mS/cm)a | IC50 (µg/ml) biotin–heparin bindingb | IC50 (nM) LRP6 bindingc | IC50 (nM) Wnt inhibitiond |
|---|---|---|---|---|---|
| ACE464 | – | 650 (44) | 0.26 | 72 | 51 |
| ACE486 | – | NA | NA | 59 | 92 |
| ACE502 | R185N | 590 (42) | 0.13 | 120 | 96 |
| BKM231 | K202E | 580 (42) | 0.41 | 400 | 300 |
| BKM230 | R197E | 560 (40) | 0.50 | 900 | NA |
| BKM227 | H223E | 560 (40) | 0.60 | 21 | 34 |
| ACE507 | S248N/K250S | 560 (40) | 0.76 | 42 | 66 |
| ACE506 | K250E | 540 (38) | 0.11 | 300 | 170 |
| BKM232 | K216S/H223T | 490 (34) | >50 | 61 | 110 |
| BKM225 | K220N | 490 (34) | >50 | 67 | 130 |
| BKM226 | K220E | 490 (33) | >50 | 51 | 130 |
| BKM233 | K216S/K220S | 450 (31) | >50 | 65 | 82 |
| ACE504 | K240E/K243E | 440 (29) | >50 | 140 | 57 |
| ACE505 | K216E/K250E | 370 (22) | >50 | 1000 | 190 |
| BKM228 | K216E/H223E | 370 (22) | >50 | 300 | 130 |
| BKM229 | K216E/K220E | 300 (16) | >50 | 800 | 150 |
| ACE503 | K202E/K220E | <150 (13) | >50 | 100 000 | 410 |
| BKM195 | H198A/K205A | NA | 0.21 | 35 000 | NA |
| BKM199 | R230A | NA | 0.64 | 75 000 | NA |
aNaCl concentration (conductivity) at elution during Heparin-Sepharose chromatography.
bIC50 values for monomeric biotin–heparin binding measured by ELISA.
cIC50 values for LRP6 binding measured by FACS assay.
dIC50 values for Wnt3a-stimulated canonical Wnt signaling inhibition measured by STF.
Mutants are organized in the table based on binding characteristics to Heparin-Sepharose from tightest to weakest binders.
NA - not tested.