Engineering of Microbial Expression Systems for the In Vitro Production of Anticancer Peptides ^†

Introduction: Cancer causes nearly 10 million deaths worldwide each year, with approximately 704,000 new cases in Brazil. Conventional therapies face toxicity and resistance, emphasizing safer alternatives. Anticancer peptides offer selective cytotoxicity by targeting tumor membranes and microenvironments. Among them, CrotAMP14 shows antitumor activity, and heterologous microbial expression offers biocompatible and sustainable large-scale production. Methodology: pPICZαA was used as the expression vector to carry the CrotAMP14 gene with α-factor, 6xHis, and FLAG tags. E. coli DH5α and Pichia pastoris X33 were transformed via electroporation, with positive clones confirmed by PCR and sequencing. Colonies were grown in BMGY and induced in BMMY with 0.5% methanol every 12 h for 72 h. Samples were purified via nickel IMAC, refined by HPLC, and analyzed by MALDI. Results: A total of 100 μg of vector (3693 bp) was synthesized and verified by gel electrophoresis. Initial PmeI linearization presented multimers (>10,000 bp). Colony PCR and sequencing confirmed correct integration in most clones. Small-scale expression (100 mL BMMY) produced a faint ≈3 kDa band, indicating low expression. Cloning was repeated, gel-purified, transformed into E. coli DH5α to remove multimers, and re-linearized, yielding a single band after PmeI cleavage. The re-transformation into P. pastoris yielded a stronger ≈3 kDa band, confirmed by MALDI-MS/MS; though partially degraded. Conclusions: The heterologous system is functional but affected by proteolysis, likely from endogenous proteases or non-optimized purification. Optimizing expression and purification is essential for reliable production of CrotAMP14.