1. A Bacterial Expression Platform for Production of Therapeutic Proteins Containing Human-like O-Linked Glycans.
- Author
-
Du T, Buenbrazo N, Kell L, Rahmani S, Sim L, Withers SG, DeFrees S, and Wakarchuk W
- Subjects
- Bacterial Proteins genetics, Bacterial Proteins metabolism, Campylobacter jejuni enzymology, Escherichia coli metabolism, Galactosyltransferases genetics, Galactosyltransferases metabolism, Glycosylation, Growth Hormone genetics, Humans, Interferon alpha-2 genetics, N-Acetylgalactosaminyltransferases genetics, N-Acetylgalactosaminyltransferases metabolism, Protein Disulfide-Isomerases genetics, Protein Disulfide-Isomerases metabolism, Recombinant Proteins biosynthesis, Recombinant Proteins isolation & purification, UDPglucose 4-Epimerase genetics, UDPglucose 4-Epimerase metabolism, Polypeptide N-acetylgalactosaminyltransferase, Growth Hormone metabolism, Interferon alpha-2 metabolism, Polysaccharides metabolism
- Abstract
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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF