THE USE OF CMY GENE (BOS TAURUS CHYMOSIN) OPTIMIZED WITH A SYNTHETIC SEQUENCE FOR THE PRODUCTION OF RECOMBINANT CHYMOSIN, FOR ITS EXPRESSION IN PICHIA PASTORIS.

摘要

The present invention relates to the study in the bovine prochymosin gene codon optimization, performed for its expression in Pichia Pastoris. Specifically, related to an artificial gene coding designed from a wild chymosin gene for the same amino-acid sequence (aa), thereby replacing atypical or low-frequency codons use in Pichia Pastoris (15%), by those using preferably high-frequency yeast. The modified nucleotides of up to 332, therefore, optimizing up to 286 codons and the designed gene cloned into the expression vector pPICZa and transformed into P. pastoris. The strains transformed with the gene cultured in artificial media enriched with 1% yeast extract, 2% peptone and 1% glycerol, thereby increasing the biomass to subsequently being cultured in a similar way, thus replacing glycerol with methanol of up to 0.5% as a carbon source to initiate gene induction. Furthermore, the chymosin activation of prochymosin synthesized with the designed gene by acidification-neutralization, thus showing coagulant activity directly from the supernatant of up to 146.11 U/mL, representing an approximate increase of up to 12 times, comparable to the wild gene corresponding to 12.2 U/ml. Said chymosin activation, showed a stability range of up 25°C to 50°C with an optimum of up 35°C to 40°C and a range of up 5pH to 6.9pH with an optimum of up to 5.0. These results show that codon optimization in the bovine prochymosin gene is a viable alternative to improve expression levels in P. pastoris.