Table 4. Solubility test of point mutants of DCase.
Hydrophilicity of amino acids is from weak to strong: L, A, G, T, Y, H, E, D, N, K, and R. Solubility of proteins was analysed after cell lysis by ultrasonication, and then supernatant fractions were resolved by SDS/PAGE. The intensity of the target protein band of muteins was compared with that of wild-type DCase. −, no increase in solubility. Total expression of the target protein (DT) was calculated by adding the integrated density of the soluble (DS) and insoluble fractions (DI), and the soluble fraction was defined as SF=DS/DT [11]. ΔSF=(SF of mutein−SF of WT)/SF of WT.
| Ala18 | Tyr30 | Lys34 | |||
|---|---|---|---|---|---|
| Mutation | ΔSF (%) | Mutation | ΔSF (%) | Mutation | ΔSF (%) |
| A18L | − | Y30L | − | K34L | − |
| A18T | ∼34 | Y30A | − | K34A | − |
| A18Y | ∼40 | Y30G | − | K34G | − |
| A18E | ∼47 | Y30E | ∼81 | K34H | − |
| A18D | ∼44 | Y30D | ∼122 | K34E | ∼81 |
| A18N | ∼42 | Y30N | ∼55 | K34D | ∼37 |
| A18K | − | Y30K | − | K34R | − |
| A18R | − | Y30R | − | ||