STRATEGIES FOR OPTIMAL SYNTHESIS AND SECRETION OF HETEROLOGOUS PROTEINS IN THE METHYLOTROPHIC YEAST PICHIA-PASTORIS

Numerous heterologous proteins have been produced at greater than gram per liter levels in the methylotrophic yeast, Pichia pastoris, using the methanol oxidase promoter. The factors that drastically influence protein production in this system include: copy number of the expressi on cassette, site and mode of chromosomal integration of the expressio n cassette, mRNA 5'- and 3'-untranslated regions (UTR), translational start codon (AUG) context, A+T composition of cDNA, transcriptional an d translational blocks, nature of secretion signal, endogenous proteas e activity, host strain physiology, media and growth conditions, and f ermentation parameters. All these factors should be considered in desi gning an optimal production system. The inherent ability of P. pastori s to convert the zymogen (pro-enzyme) form of matrix metalloproteinase s (MMP) into active mature farms (which tend to self-degrade, and in s ome instances also cause damage to cells), largely limits the use of t his system for the production of MMP. However, this problem can be par tly alleviated by co-expression of tissue inhibitor of MMP (TIMP-1).