A Bacterial Expression Platform for Production of Therapeutic Proteins Containing Human-like O-Linked Glycans

• Published in: Cell chemical biology Vol. 26; no. 2; pp. 203 - 212.e5
• Main Authors: Du, Ting, Buenbrazo, Nakita, Kell, Laura, Rahmani, Sadia, Sim, Lyann, Withers, Stephen G, DeFrees, Shawn, Wakarchuk, Warren
• Format: Journal Article
• Language: English
• Published: United States 21.02.2019
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Campylobacter jejuni - enzymology; Escherichia coli - metabolism; Galactosyltransferases - genetics; Galactosyltransferases - metabolism; Glycosylation; Growth Hormone - genetics; Growth Hormone - metabolism; Humans; Interferon alpha-2 - genetics; Interferon alpha-2 - metabolism; N-Acetylgalactosaminyltransferases - genetics; N-Acetylgalactosaminyltransferases - metabolism; Polypeptide N-acetylgalactosaminyltransferase; Polysaccharides - metabolism; Protein Disulfide-Isomerases - genetics; Protein Disulfide-Isomerases - metabolism; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; UDPglucose 4-Epimerase - genetics; UDPglucose 4-Epimerase - metabolism; glycosylation; interferon-α2b; T antigen; human growth hormone; operon
• ISSN: 2451-9456; 2451-9448; 2451-9456
• DOI: 10.1016/j.chembiol.2018.10.017

We have developed an Escherichia coli strain for the in vivo production of O-glycosylated proteins. This was achieved using a dual plasmid approach: one encoding a therapeutic protein target, and a second encoding the enzymatic machinery required for O-glycosylation. The latter plasmid encodes human polypeptide N-acetylgalactosaminyl transferase as well as a β1,3-galactosyl transferase and UDP-Glc(NAc)-4-epimerase, both from Campylobacter jejuni, and a disulfide bond isomerase of bacterial or human origin. The effectiveness of this two-plasmid synthetic operon system has been tested on three proteins with therapeutic potential: the native and an engineered version of the naturally O-glycosylated human interferon α-2b, as well as human growth hormone with one engineered site of glycosylation. Having established proof of principle for the addition of the core-1 glycan onto proteins, we are now developing this system as a platform for producing and modifying human protein therapeutics with more complex O-glycan structures in E. coli.