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First production of a human protein with complex glycosylation in genetically modified yeast

ting safer, more effective therapeutics."

Humanizing the Yeast Glycosylation Pathway

The GlycoFi team began their work with the yeast P. pastoris, a robust organism commonly used in fermentation processes, which can be grown to high cell density in a chemically defined growth medium. This yeast normally produces non-human N-glycans of the high mannose type, which have no therapeutic value for humans. The scientists modified the yeast by first eliminating endogenous yeast glycosylation pathways, while sequentially engineering into the organism five active eukaryotic proteins, including mannosidases I & II, N-acetylglucosaminyl transferases I & II and UDP-N-acetylglucosamine transporter. The targeted localization of these enzymes enabled the generation of a synthetic in vivo glycosylation pathway that enabled the yeast to produce a complex human N-glycan, GlcNAc2Man3GlcNAc2, in vivo. However, unlike the glycosylation pathway in mammalian cell lines, which typically produces an array of glycoforms, the genetically modified yeast yielded essentially homogeneous glycoforms.

"We have essentially been able to humanize the yeast, where it is able to make a single glycoform of exceptional uniformity," Dr. Gerngross said. "Unfortunately, using mammalian cell culture, it is difficult to isolate individual protein glycoforms and even more difficult to produce specific structures at a commercial scale. The ability to express a single protein in a library of genetically engineered yeasts each producing a defined and uniform glycoform will enable the generation of glycoprotein libraries that can be used both to elucidate specific structure-function relationships and to identify the most efficacious molecule for a particular therapeutic use. Moreover, once identified, a particular protein glycoform can be readily produced at industrial scale using the relevant yeast, due to the well-established rapidity with which yeast fermentations can be scaled u
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28-Aug-2003


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