Glycoengineering of human IgG1-Fc through combined yeast expression and in vitro chemoenzymatic glycosylation

Yadong Wei, Cishan Li, Wei Huang, Bing Li, Scott Strome, Lai Xi Wang

Research output: Contribution to journalArticlepeer-review

Abstract

The presence and precise structures of the glycans attached at the Fc domain of monoclonal antibodies play an important role in determining antibodies' effector functions such as antibody-dependent cell cytotoxicity (ADCC), complement activation, and anti-inflammatory activity. This paper describes a novel approach for glycoengineering of human IgG1-Fc that combines high-yield expression of human IgG1-Fc in yeast and subsequent in vitro enzymatic glycosylation, using the endoglycosidase-catalyzed transglycosylation as the key reaction. Human IgG1-Fc was first overproduced in Pichia pastoris. Then the heterogeneous yeast glycans were removed by Endo-H treatment to give the GlcNAc-containing IgG1-Fc as a homodimer. Finally, selected homogeneous glycans were attached to the GlcNAc-primer in the IgG1-Fc through an endoglycosidase-catalyzed transglycosylation, using sugar oxazolines as the donor substrates. It was found that the enzymatic transglycosylation was efficient with native GlcNAc-containing IgG1-Fc homodimer without the need to denature the protein, and the reaction could proceed to completion to give homogeneous glycoforms of IgG1-Fc when an excess of oligosaccharide oxazolines was used as the donor substrates. The binding of the synthetic IgG1-Fc glycoforms to the FcγIIIa receptor was also investigated. This novel glycoengineering approach should be useful for providing various homogeneous, natural or synthetic glycoforms of IgG1-Fc for structure-function relationship studies, and for future clinical applications.

Original languageEnglish (US)
Pages (from-to)10294-10304
Number of pages11
JournalBiochemistry
Volume47
Issue number39
DOIs
StatePublished - Sep 30 2008

ASJC Scopus subject areas

  • Biochemistry

Fingerprint Dive into the research topics of 'Glycoengineering of human IgG1-Fc through combined yeast expression and in vitro chemoenzymatic glycosylation'. Together they form a unique fingerprint.

Cite this