Figure 6.

(A) Representative image of western blot analyses of extracellular (a, b) and intracellular (c, d) samples from Saccharomyces cerevisiae cultures that express recombinant human granulocyte colony‐stimulating factor (rhG‐CSF) using the propeptides designed in the third stage. Western blot results of Endo H‐treated extracellular and intracellular samples are also shown in b and d, respectively (Standard, the commercial standard of human granulocyte colony‐stimulating factor [hG‐CSF]; Control, samples from the culture of S. cerevisiae Y280; B, original propeptide; BN3‐N7,15, best propeptide sequence in the first stage; BN3‐N7,15‐K23D, best propeptide sequence in the second stage; BN3‐N7,15‐K23D‐GS to BN3‐N7,15‐K23D‐(GSG)₂; BN3‐N7,15‐K23D‐derived propeptides designed in the third stage). (B) The chemiluminescent bands (extracellular mature rhG‐CSF) were quantified through extrapolation based on the band intensity of the standard. Each bar represents the average with the error bars indicating standard deviations (n = 3). The p‐values were analyzed by Student's t‐test (**p < 0.001).