Insulin glargine production: Codon engineering in Pichia pastoris X-33 for enhanced therapeutic yield

Aims This study aims to investigate the optimization and expression of insulin glargine, a long-acting insulin analogue, in Pichia pastoris X-33. The focus is on enhancing the production efficiency of recombinant insulin glargine by utilizing codon optimization to increase protein yield. Materials and methods The insulin glargine gene was codon-optimized to enhance translational efficiency. Both the codon-optimized and non-optimized versions of the gene were introduced into Pichia pastoris X-33 via transformation with the pPICZalpha -G plasmid. Gene expression was induced using methanol-driven fermentation in ½ BSM. Protein production was evaluated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to identify protein bands characteristic of insulin glargine. Subsequent purification was achieved through High-Performance Liquid Chromatography (HPLC) to confirm the successful production of insulin glargine. Results The expression of both the codon-optimized and non-optimized insulin glargine genes in P. pastoris X-33 was successful, with the optimized gene yielding significantly higher quantities of insulin glargine protein. Characteristic proteins corresponding to insulin glargine were observed, validating gene expression. Conclusion This study demonstrates the codon optimization significantly boosting protein yields of insulin glargine. The findings pave the way for further optimization of fermentation techniques and progress towards clinical-scale production of therapeutic insulin glargine for diabetes treatment.