High level in vivo mucin-type glycosylation in Escherichia coli

Phillipp Mueller,Sabine Gaisser,Claudia Wahl,René Handrick,Michael Zorn,Rahul Gauttam,Marianne Scheffold,Michaela Kussmaul,Bernhard Eikmanns,Lothar Elling,Nadja Raab,Sebastian Leptihn

doi:10.1186/s12934-018-1013-9

Abstract

BackgroundIncreasing efforts have been made to assess the potential of Escherichia coli strains for the production of complex recombinant proteins. Since a considerable part of therapeutic proteins are glycoproteins, the lack of the post-translational attachment of sugar moieties in standard E. coli expression strains represents a major caveat, thus limiting the use of E. coli based cell factories. The establishment of an E. coli expression system capable of protein glycosylation could potentially facilitate the production of therapeutics with a putative concomitant reduction of production costs.ResultsThe previously established E. coli strain expressing the soluble form of the functional human-derived glycosyltransferase polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2) was further modified by co-expressing the UDP-GlcNAc 4-epimerase WbgU derived from Plesiomonas shigelloides. This enables the conversion of uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) to the sugar donor uridine 5′-diphospho-N-acetylgalactosamine (UDP-GalNAc) in the bacterial cytoplasm. Initially, the codon-optimised gene wbgU was inserted into a pET-derived vector and a Tobacco Etch Virus (TEV) protease cleavable polyhistidine-tag was translationally fused to the C- terminus of the amino acid sequence. The 4-epimerase was subsequently expressed and purified. Following the removal of the polyhistidine-tag, WbgU was analysed by circular dichroism spectroscopy to determine folding state and thermal transitions of the protein. The in vitro activity of WbgU was validated by employing a modified glycosyltransferase assay. The conversion of UDP-GlcNAc to UDP-GalNAc was shown by capillary electrophoresis analysis. Using a previously established chaperone pre-/co- expression platform, the in vivo activity of both glycosyltransferase GalNAc-T2 and 4-epimerase WbgU was assessed in E. coli, in combination with a mucin 10-derived target protein. Monitoring glycosylation by liquid chromatography electrospray ionization mass spectrometry (LC–ESI–MS), the results clearly indicated the in vivo glycosylation of the mucin-derived acceptor peptide.ConclusionIn the present work, the previously established E. coli- based expression system was further optimized and the potential for in vivo O-glycosylation was shown by demonstrating the transfer of sugar moieties to a mucin-derived acceptor protein. The results offer the possibility to assess the practical use of the described expression platform for in vivo glycosylations of important biopharmaceutical compounds in E. coli.

Highlights

Increasing efforts have been made to assess the potential of Escherichia coli strains for the production of complex recombinant proteins
We successfully show in vivo glycosylation by co-expressing the 4-epimerase WbgU, the truncated form of the glycosyltransferase GalNAc-T2, and the MUC10 target protein in the established E. coli system pre-/coexpressing the chaperones sulfhydryl oxidase Erv1p and the human protein disulfide isomerase PDI, as previously described [6, 8]
Soluble expression of WbgUTEV6H (41 kDa) was found to be best in Terrific Broth (TB) medium without Isopropylβ-d-thiogalactopyranosid (IPTG) induction and with the pre-/co-expression of the redox folding helper proteins Erv1p and PDI encoded by pMJS9 under the control of an arabinose promotor

Summary

Introduction

Increasing efforts have been made to assess the potential of Escherichia coli strains for the production of complex recombinant proteins. The lack or insufficient capability of E. coli strains to perform post-translational modifications, including disulfide bond formation and glycosylations [2, 3], has contributed to the rise of mammalian rather than nonmammalian expression systems for the commercial production of biopharmaceutical products [4]. To enable recombinant expression of complex proteins in E. coli, research has focused on improving protein folding, disulfide bond formation, as well as N- and O-linked glycosylation [5,6,7,8,9,10,11]. As a result, engineering of the polypeptide sequence of the target protein or altering the substrate specificity of PglB is required to promote correct N-glycosylation of recombinant proteins expressed in the periplasm of E. coli [13]. O-glycosylation of eukaryotic target sites using a truncated form of the human glycosyltransferase GalNAc-T2 has been shown without altering the target peptide sequence [6, 9, 14, 15]

Methods

Results

Discussion

Conclusion