skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: High level in vivo mucin-type glycosylation in Escherichia coli

Abstract

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 inmore » 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.« less

Authors:
ORCiD logo [1];  [2];  [1];  [1];  [3];  [4];  [5];  [5];  [5];  [2];  [3];  [1]
  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)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1580336
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
Microbial Cell Factories
Additional Journal Information:
Journal Volume: 17; Journal Issue: 1; Journal ID: ISSN 1475-2859
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Protein expression; Escherichia coli; Glycosyltransferase GalNAcT2; Chaperone co-expression; In vivo glycosylation; UDP-GlcNAc 4-epimerase WbgU; Mucin-type O-glycosylation

Citation Formats

Mueller, Phillipp, Gauttam, Rahul, Raab, Nadja, Handrick, René, Wahl, Claudia, Leptihn, Sebastian, Zorn, Michael, Kussmaul, Michaela, Scheffold, Marianne, Eikmanns, Bernhard, Elling, Lothar, and Gaisser, Sabine. High level in vivo mucin-type glycosylation in Escherichia coli. United States: N. p., 2018. Web. doi:10.1186/s12934-018-1013-9.
Mueller, Phillipp, Gauttam, Rahul, Raab, Nadja, Handrick, René, Wahl, Claudia, Leptihn, Sebastian, Zorn, Michael, Kussmaul, Michaela, Scheffold, Marianne, Eikmanns, Bernhard, Elling, Lothar, & Gaisser, Sabine. High level in vivo mucin-type glycosylation in Escherichia coli. United States. doi:10.1186/s12934-018-1013-9.
Mueller, Phillipp, Gauttam, Rahul, Raab, Nadja, Handrick, René, Wahl, Claudia, Leptihn, Sebastian, Zorn, Michael, Kussmaul, Michaela, Scheffold, Marianne, Eikmanns, Bernhard, Elling, Lothar, and Gaisser, Sabine. Fri . "High level in vivo mucin-type glycosylation in Escherichia coli". United States. doi:10.1186/s12934-018-1013-9. https://www.osti.gov/servlets/purl/1580336.
@article{osti_1580336,
title = {High level in vivo mucin-type glycosylation in Escherichia coli},
author = {Mueller, Phillipp and Gauttam, Rahul and Raab, Nadja and Handrick, René and Wahl, Claudia and Leptihn, Sebastian and Zorn, Michael and Kussmaul, Michaela and Scheffold, Marianne and Eikmanns, Bernhard and Elling, Lothar and Gaisser, Sabine},
abstractNote = {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.},
doi = {10.1186/s12934-018-1013-9},
journal = {Microbial Cell Factories},
number = 1,
volume = 17,
place = {United States},
year = {2018},
month = {10}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 3 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Mucin dynamics and enteric pathogens
journal, March 2011

  • McGuckin, Michael A.; Lindén, Sara K.; Sutton, Philip
  • Nature Reviews Microbiology, Vol. 9, Issue 4
  • DOI: 10.1038/nrmicro2538

An engineered eukaryotic protein glycosylation pathway in Escherichia coli
journal, March 2012

  • Valderrama-Rincon, Juan D.; Fisher, Adam C.; Merritt, Judith H.
  • Nature Chemical Biology, Vol. 8, Issue 5
  • DOI: 10.1038/nchembio.921

Post-translational modifications in the context of therapeutic proteins
journal, October 2006

  • Walsh, Gary; Jefferis, Roy
  • Nature Biotechnology, Vol. 24, Issue 10
  • DOI: 10.1038/nbt1252

Glycopeptide N-acetylgalactosaminyltransferase specificities for O-glycosylated sites on MUC5AC mucin motif peptides
journal, June 2001

  • Tetaert, Daniel; Ten Hagen, Kelly G.; Richet, Colette
  • Biochemical Journal, Vol. 357, Issue 1
  • DOI: 10.1042/bj3570313

Production of Secretory and Extracellular N-Linked Glycoproteins in Escherichia coli
journal, December 2010

  • Fisher, Adam C.; Haitjema, Charles H.; Guarino, Cassandra
  • Applied and Environmental Microbiology, Vol. 77, Issue 3
  • DOI: 10.1128/AEM.01901-10

Increased Understanding of the Biochemistry and Biosynthesis of MUC2 and Other Gel-Forming Mucins Through the Recombinant Expression of Their Protein Domains
journal, January 2013

  • Bäckström, Malin; Ambort, Daniel; Thomsson, Elisabeth
  • Molecular Biotechnology, Vol. 54, Issue 2
  • DOI: 10.1007/s12033-012-9562-3

Definition of the bacterial N-glycosylation site consensus sequence
journal, April 2006


SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm
journal, May 2012

  • Lobstein, Julie; Emrich, Charlie A.; Jeans, Chris
  • Microbial Cell Factories, Vol. 11, Issue 1
  • DOI: 10.1186/1475-2859-11-56

Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria
journal, August 2015

  • Robinson, Michael-Paul; Ke, Na; Lobstein, Julie
  • Nature Communications, Vol. 6, Issue 1
  • DOI: 10.1038/ncomms9072

A computational and experimental study of O-glycosylation. Catalysis by human UDP-GalNAc polypeptide:GalNAc transferase-T2
journal, January 2014

  • Gómez, Hansel; Rojas, Raúl; Patel, Divya
  • Org. Biomol. Chem., Vol. 12, Issue 17
  • DOI: 10.1039/C3OB42569J

Deconvoluting the Functions of Polypeptide N-α-Acetylgalactosaminyltransferase Family Members by Glycopeptide Substrate Profiling
journal, July 2004


Porcine Submaxillary Mucin Forms Disulfide-linked Multimers through Its Amino-terminal D-domains
journal, June 1998

  • Perez-Vilar, Juan; Eckhardt, Allen E.; DeLuca, Alexander
  • Journal of Biological Chemistry, Vol. 273, Issue 23
  • DOI: 10.1074/jbc.273.23.14442

Expression of the functional recombinant human glycosyltransferase GalNAcT2 in Escherichia coli
journal, January 2015

  • Lauber, Jennifer; Handrick, René; Leptihn, Sebastian
  • Microbial Cell Factories, Vol. 14, Issue 1
  • DOI: 10.1186/s12934-014-0186-0

Protein Disulfide Isomerase: A Critical Evaluation of Its Function in Disulfide Bond Formation
journal, November 2009

  • Hatahet, Feras; Ruddock, Lloyd W.
  • Antioxidants & Redox Signaling, Vol. 11, Issue 11
  • DOI: 10.1089/ars.2009.2466

Substrate Specificities of Three Members of the Human UDP- N -Acetyl-α-d-galactosamine:Polypeptide N -Acetylgalactosaminyltransferase Family, GalNAc-T1, -T2, and -T3
journal, September 1997

  • Wandall, Hans H.; Hassan, Helle; Mirgorodskaya, Ekaterina
  • Journal of Biological Chemistry, Vol. 272, Issue 38
  • DOI: 10.1074/jbc.272.38.23503

The metastability of human UDP-galactose 4′-epimerase (GALE) is increased by variants associated with type III galactosemia but decreased by substrate and cofactor binding
journal, November 2014

  • Pey, Angel L.; Padín-Gonzalez, Esperanza; Mesa-Torres, Noel
  • Archives of Biochemistry and Biophysics, Vol. 562
  • DOI: 10.1016/j.abb.2014.07.030

Biopharmaceutical benchmarks 2014
journal, October 2014


Function of the CysD domain of the gel-forming MUC2 mucin
journal, April 2011

  • Ambort, Daniel; van der Post, Sjoerd; Johansson, Malin E. V.
  • Biochemical Journal, Vol. 436, Issue 1
  • DOI: 10.1042/BJ20102066

The Carboxyl-terminal 90 Residues of Porcine Submaxillary Mucin Are Sufficient for Forming Disulfide-bonded Dimers
journal, March 1998

  • Perez-Vilar, Juan; Hill, Robert L.
  • Journal of Biological Chemistry, Vol. 273, Issue 12
  • DOI: 10.1074/jbc.273.12.6982

Site-Specific Modification of Recombinant Proteins: A Novel Platform for Modifying Glycoproteins Expressed in E. coli
journal, May 2011

  • Henderson, Grant E.; Isett, Kevin D.; Gerngross, Tillman U.
  • Bioconjugate Chemistry, Vol. 22, Issue 5
  • DOI: 10.1021/bc100510g

Dynamic Association between the Catalytic and Lectin Domains of Human UDP-GalNAc:Polypeptide α- N -Acetylgalactosaminyltransferase-2
journal, January 2006

  • Fritz, Timothy A.; Raman, Jayalakshmi; Tabak, Lawrence A.
  • Journal of Biological Chemistry, Vol. 281, Issue 13
  • DOI: 10.1074/jbc.M513590200

Cellular engineering for therapeutic protein production: product quality, host modification, and process improvement
journal, December 2016


Therapeutic insulins and their large-scale manufacture
journal, December 2004


Glycans-by-design: Engineering bacteria for the biosynthesis of complex glycans and glycoconjugates
journal, March 2013

  • Merritt, Judith H.; Ollis, Anne A.; Fisher, Adam C.
  • Biotechnology and Bioengineering, Vol. 110, Issue 6
  • DOI: 10.1002/bit.24885

    Works referencing / citing this record:

    New tools for recombinant protein production in Escherichia coli : A 5‐year update
    journal, May 2019

    • Rosano, Germán L.; Morales, Enrique S.; Ceccarelli, Eduardo A.
    • Protein Science, Vol. 28, Issue 8
    • DOI: 10.1002/pro.3668

    High level in vivo mucin-type glycosylation in Escherichia coli
    text, January 2018