Activity of rHuEPO Variants
| Location . | Mutation . | Expression* (U/mL) . | Receptor Binding† (% of total) . | In Vitro Activity‡ (% of total) . | RIA-9G8Aρ (% of total) . |
|---|---|---|---|---|---|
| Human EPO | 67 | 130 | 187 | 100 | |
| Helix A | Val11/Ser | 45 | 25 | 6.3 | 380 |
| Arg14/Ala | 44 | 40 | 11 | 590 | |
| Arg14/Gln | 21 | 28 | 1.7 | 82 | |
| Tyr15/lle | 11 | 28 | <0.43 | 150 | |
| A/B Loop | Pro42/Asn | 0.8 | <1 | <0.01 | 1,600 |
| Thr44/lle | 7 | 52 | 8.7 | 640 | |
| Lys45/Asp | 53 | 0.2 | <0.03 | 180 | |
| Val46/Ala | 9 | 20 | 7.8 | 774 | |
| Typ51/Phe | 97 | 40 | 34 | 340 | |
| Helix C | Ser100/Glu | 79 | 8 | <0.01 | 150 |
| Ser100/Thr | 14 | 11 | <0.2 | 58 | |
| Arg103/Ala | 7.7 | 20 | <0.12 | 86 | |
| Ser104/lle | 30 | 7.3 | <0.2 | 100-240 | |
| Ser104/Ala | 12 | 36 | 12 | 150 | |
| Leu108/Lys | 35 | 10 | <0.02 | 330 | |
| Helix D | Asn147/Lys | 5 | 1.2 | 0.3 | 260 |
| Arg150/Ala | 47 | 19 | 8 | 97 | |
| Gly151/Ala | 29 | <0.7 | <0.03 | 760 | |
| Leu155/Ala | 42 | 48 | 16 | 439 |
| Location . | Mutation . | Expression* (U/mL) . | Receptor Binding† (% of total) . | In Vitro Activity‡ (% of total) . | RIA-9G8Aρ (% of total) . |
|---|---|---|---|---|---|
| Human EPO | 67 | 130 | 187 | 100 | |
| Helix A | Val11/Ser | 45 | 25 | 6.3 | 380 |
| Arg14/Ala | 44 | 40 | 11 | 590 | |
| Arg14/Gln | 21 | 28 | 1.7 | 82 | |
| Tyr15/lle | 11 | 28 | <0.43 | 150 | |
| A/B Loop | Pro42/Asn | 0.8 | <1 | <0.01 | 1,600 |
| Thr44/lle | 7 | 52 | 8.7 | 640 | |
| Lys45/Asp | 53 | 0.2 | <0.03 | 180 | |
| Val46/Ala | 9 | 20 | 7.8 | 774 | |
| Typ51/Phe | 97 | 40 | 34 | 340 | |
| Helix C | Ser100/Glu | 79 | 8 | <0.01 | 150 |
| Ser100/Thr | 14 | 11 | <0.2 | 58 | |
| Arg103/Ala | 7.7 | 20 | <0.12 | 86 | |
| Ser104/lle | 30 | 7.3 | <0.2 | 100-240 | |
| Ser104/Ala | 12 | 36 | 12 | 150 | |
| Leu108/Lys | 35 | 10 | <0.02 | 330 | |
| Helix D | Asn147/Lys | 5 | 1.2 | 0.3 | 260 |
| Arg150/Ala | 47 | 19 | 8 | 97 | |
| Gly151/Ala | 29 | <0.7 | <0.03 | 760 | |
| Leu155/Ala | 42 | 48 | 16 | 439 |
The rHuEPO constructs were transfected into Cos cells. After 3 to 5 days, the concentration (U/mL) of rHuEPO was immunoassay (see text).
Receptor binding assays were performed using OCIM-1 cells (human erythroleukemia cells) and cell conditioned media from COS cells transfected with the wild-type or mutant EPO constructs. The assay measures displacement of 125I rHuEPO. Specific activity was calculated by dividing by the concentration determined by immunoassay. Results are expressed as a percentage of total.
In vitro activity was the ability of cell conditioned media containing wild-type or mutant EPO to stimulate 3H thymidine uptake in 32D+EPOR (32D cells transfected with a murine EPO receptor). Specific activity was calculated by dividing by the concentration determined by immunoassay. Results are expressed as a percentage of total.
ρ 9G8A assays were radioimmunoassays using the anti-EPO monoclonal antibody 9G8A. Specific activity was calculated by dividing by concentration determined by immunoassay. Results are expressed as a percentage of total. Increased immunoreactivity is indicative of altered folding.