Fig. 4.

a SDS-PAGE analysis of the extracellular G-CSF produced by transformants containing single deletion in the pro-secretory sequence fused to the WT-GCSF gene. Equal volume (10 μl) of the culture filtrate was loaded after 120 h of cultivation in a 500 ml Erlenmeyer flask. Lane 1: Molecular weight ladder; Lane 2; empty vector; Lane 3: Matα:WT fused to the WT-GCSF gene; Lanes 4 & 5: Matα:Δ57–70, Cl #s 6 & 16; Lanes 6 & 7: Matα:Δ30–43, Cl #s 10 &16; Lanes 8 & 9: Matα:Δ47–49, Cl #s 24, 36; Lane 10: Filgrastim as a standard (1.5 μg). b SDS-PAGE analysis of G-CSF produced by transformants containing double deletions in the pro-secretory sequence fused to the WT-GCSF gene. Lane 11: Molecular weight ladder; Lane 12: MATα:WT fused to the WT-GCSF gene; Lanes 13 &14: Matα:Δ57–70, Cl #s 6, 16; Lanes 15 &16: Matα:Δ57–70;47–49, Cl #s 22, 25; Lanes 17 & 18: Matα:Δ57–70;30–43, Cl #s 21, 34; Lane 19: Filgrastim (1.5 μg). c Cell O.D, total extracellular protein and G-CSF titre at 120 h post-methanol induction. The units for cell O.D. are the same as shown on the Y-axis