This invention relates to the stable and high-yield production of a high-potency toxin protein called DT390-EGF. This toxin was developed for the treatment of EGF-receptor-positive cancers, including bladder cancer. Initial methods for synthesizing DT390-EGF relied on the use of E. coli. However, the production in E. coli was difficult to prepare and had limited stability. Repeated efforts to standardize the process in E. coli gave poor yields, purity, and high variation.

To overcome the problems with production using E. coli, this invention employs the Pichia pastoris strain of yeast. To produce DT390-EGF in P. pastoris, the DNA encoding the DT390-EGF protein was modified to (a) introduce an N-terminal alanine, (b) optimize codon usage for efficient translation in P. pastoris, (c) abolish N-linked glycosylation sites, and (d) add a (G4S)3 linker between the DT390 and EGF moieties. With these changes, DT390-EFG can be reproducibly produced in P. pastoris with more stability and potency compared to that produced in E. coli.