We have been developing bacterial expression systems for human mucin-type O-glycosylation on therapeutic proteins, which is initiated by the addition of α-linked GalNAc to serine or threonine residues by enzymes in the GT-27 family of glycosyltransferases. Substrate preference across different isoforms of this enzyme is influenced by isoform-specific amino acid sequences at the site of glycosylation, which we have exploited to engineer production of Core 1 glycan structures in bacteria on human therapeutic proteins. Using RP-HPLC with a novel phenyl bonded phase to resolve intact protein glycoforms, the effect of sequon mutation on O-glycosylation initiation was examined through in vitro modification of the naturally O-glycosylated human interferon α-2b, and a sequon engineered human growth hormone. As part of the development of our glycan engineering in the bacterial expression system we are surveying various orthologues of critical enzymes to ensure complete glycosylation. Here we present an in vitro enzyme kinetic profile of three related GT-27 orthologues on natural and engineered sequons in recombinant human interferon α2b and human growth hormone where we show a significant change in kinetic properties with the amino acid changes. It was found that optimizing the protein substrate amino acid sequence using Isoform Specific O-Glycosylation Prediction (ISOGlyP, http://isoglyp.utep.edu/index.php) resulted in a measurable increase in kcat/Km, thus improving glycosylation efficiency.  We showed that the Drosophila orthologue showed superior activity with our human growth hormone designed sequons compared to the human enzyme

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