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Technology

This technology is an improved vector system to drive very high expression of heterologous proteins in methylotrophic yeast. These inventors systematically identified promoter sequences, translation initiation (Kozak) sequences, secretion signal sequences, and mRNA codons that resulted in drastic increases in heterologous protein expression. The promoter sequences chosen drive very high levels of protein expression regardless of integration location, and integrations at the endogenous locus, alternate loci, or even as an episomal vector result in efficient protein production. Analysis of optimal Kozak translation initiation sequence was previously limited in methylotrophic yeast. These inventors determined the optimal Kozak sequence that increases protein production by increasing translation initiation. Biologic protein products frequently include a signal sequence that codes for extracellular secretion to facilitate isolation of the product. These inventors determined the native signal sequences in methylotrophic yeast and identified those that resulted in optimal secretion of a heterologous protein product. Finally, the inventors analyzed the effects of three-dimensional mRNA folding on protein translation, and demonstrated that codon optimization can further increase expression of heterologous protein. Taken together, these vector improvements drastically increase the per-cell yield of heterologous protein product in methylotrophic yeast.