High level in vivo mucin-type glycosylation in Escherichia coli

Journal Article · Fri Oct 26 00:00:00 EDT 2018 · Microbial Cell Factories

DOI:https://doi.org/10.1186/s12934-018-1013-9· OSTI ID:1580336

Mueller, Phillipp ^[1]; Gauttam, Rahul ^[2]; Raab, Nadja ^[1]; Handrick, René ^[1]; Wahl, Claudia ^[3]; Leptihn, Sebastian ^[4]; Zorn, Michael ^[5]; Kussmaul, Michaela ^[5]; Scheffold, Marianne ^[5]; Eikmanns, Bernhard ^[2]; Elling, Lothar ^[3]; Gaisser, Sabine ^[1]

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1. Biberach Univ. of Applied Sciences (Germany)
2. Univ. of Ulm (Germany)
3. RWTH Aachen Univ. (Germany)
4. Zhejiang Univ. (China)
5. Boehringer Ingelheim Pharma GmbH and Co.KG, Biberach (Germany)

Background::Increasing 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. Results:The 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. Conclusion:In 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.

• Research Organization: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
• Sponsoring Organization: USDOE Office of Science (SC)
• Grant/Contract Number: AC02-05CH11231
• OSTI ID: 1580336
• Journal Information: Microbial Cell Factories, Journal Name: Microbial Cell Factories Journal Issue: 1 Vol. 17; ISSN 1475-2859
• Publisher: BioMed Central
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Chaperone co-expression
Escherichia coli
Glycosyltransferase GalNAcT2
In vivo glycosylation
Mucin-type O-glycosylation
Protein expression
UDP-GlcNAc 4-epimerase WbgU