Exploring a combined Escherichia coli-based glycosylation and in vitro transglycosylation approach for expression of glycosylated interferon alpha. (1st March 2021)

• Record Type: Journal Article
• Authors: Prabhu, Sunaina Kiran; Yang, Qiang; Tong, Xin; Wang, Lai-Xi
• Is Part Of: Bioorganic & medicinal chemistry. Volume 33(2021)
• Publication Date: 2021-03-01
• Subjects: E. coli glycosylation -- N-glycosyltransferase -- Endoglycosidase -- Transglycosylation -- Sugar oxazoline -- Eukaryotic protein; Bioorganic chemistry -- Periodicals; Pharmaceutical chemistry -- Periodicals; Biochemistry -- Periodicals; Chemistry, Clinical -- Periodicals; Chemistry, Organic -- Periodicals; Chimie bio-organique -- Périodiques; Chimie pharmaceutique -- Périodiques; 615.19
• DOI: 10.1016/j.bmc.2021.116037
• Languages: English
• ISSNs: 0968-0896

Abstract

Graphical abstract: Two-step glycosylation of proteins using E. coli . Abstract: The conventional use of E. coli system for protein expression is limited to non-glycosylated proteins. While yeast, insect and mammalian systems are available to produce heterologous glycoproteins, developing an engineered E. coli -based glycosylation platform will provide a faster, more economical, and more convenient alternative. In this work, we present a two-step approach for production of a homogeneously glycosylated eukaryotic protein using the E. coli expression system. Human interferon α-2b (IFNα) is used as a model protein to illustrate this glycosylation scheme. In the first step, the N -glycosyltransferase from Actinobacillus pleuropneumoniae (ApNGT) is co-expressed for in vivo transfer of a glucose residue to IFNα at an NX(S/T) N -glycosylation sequon. Several E. coli systems were examined to evaluate the efficiency of IFNα N -glucosylation. In the second step, the N -glucosylated protein is efficiently elaborated with biantennary sialylated complex-type N -glycan using an in vitro chemoenzymatic method. The N -glycosylated IFNα product was found to be biologically active and displayed significantly improved proteolytic stability. This work presents a feasible E. coli -based glycosylation machinery for producing therapeutic eukaryotic glycoproteins.