N-Linked Glycosylation in
            <i>Campylobacter jejuni</i>
            and Its Functional Transfer into
            <i>E. coli</i>

Wacker, Michael; Linton, Dennis; Hitchen, Paul G.; Niță-Lazăr, Mihai; Haslam, Stuart M.; North, Simon J.; Panico, Maria; Morris, Howard R.; Dell, Anne; Wren, Brendan W.; Aebi, Markus

doi:10.1126/science.298.5599.1790

Cited by 723 publications

(753 citation statements)

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“…In another neoglycoprotein production experiment, ssPelB and ssTorA were successfully used for the transfer of the N-glycosylation target protein AcrA of C. jejuni to the periplasm of E. coli, demonstrating that bacterial N-glycosylation can occur independently of the protein translocation machinery. [21,29] Concerning periplasmic targeting of S-layer proteins in general, MBP has been reported to export the S-layer protein SbsA of G. stearothermophilus PV/72 to the periplasm of E. coli. [32] The different S-layer fusion proteins were expressed in E. coli BL21 Star (DE3) carrying the respective expression plasmids.…”

Section: Periplasmic Targeting Of Sgsementioning

confidence: 99%

“…For this purpose, the plasmid pACYCpgl harboring the complete pgl gene cluster of C. jejuni that is responsible for heptasaccharide biosynthesis and its transfer to the protein was transformed into E. coli BL21 Star (DE3). [21] As a positive control, to confirm that the glycosylation machinery of C. jejuni functions under the chosen conditions, the plasmid pEC(AcrA-per), expressing periplasmic soluble AcrA of C. jejuni with the PelB signal peptide and a Cterminal hexahistidine tag, was transferred to electrocompetent E. coli BL21 Star (DE3) cells containing pACYCpgl. [29] Analysis of the expression product by Western immunoblotting, using a heptasaccharide specific antibody (anti-pgl antibody), confirmed glycosylation of AcrA ( Figure 3, lane 8).…”

Section: Design Of An Sgse Neoglycoprotein Carrying a C Jejuni Heptamentioning

confidence: 99%

“…[11] Recent demonstrations of the functional transfer of protein glycosylation pathways into the experimental model organism Escherichia coli at the genetic level opens new avenues for carbohydrate engineering of S-layer proteins. [21,22] Based on the availability of molecular tools, the Pgl protein N-glycosylation system from Campylobacter jejuni [23] and the E. coli O7 antigen biosynthesis system [24] were used for the design of SgsE-neoglycoproteins. The C. jejuni Pgl enzymes synthesize a heptasaccharide with the structure D-GalNAc-α1,4-D-GalNAc-α1,4-(D-Glc-β1,3)-D-GalNAc-α1,4-D-GalNAc-α1,4-D-GalNAc-α1,3-D-Bac, where Bac is 2,4-diacetamido-2,4,6-trideoxy-D-Glc, [25] involving the lipid carrier undecaprenylpyrophosphate.…”

Section: Introductionmentioning

confidence: 99%

“…In Gram-negative bacteria, the oligosaccharyl:protein transferase is proposed to act in the periplasm [21] in a mechanism, which is, in contrast to eukaryotes, independent of the protein translocation machinery, and is able to glycosylate folded and unfolded proteins. [29] The aim of this study is the transfer of the C. jejuni heptasaccharide and the E. coli O7 polysaccharide onto the SgsE S-layer protein of G. stearothermophilus NRS 2004/3a, and the successful expression of the S-layer neoglycoproteins in E. coli.…”

Section: Introductionmentioning

confidence: 99%

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Recombinant Glycans on an S‐Layer Self‐Assembly Protein: A New Dimension for Nanopatterned Biomaterials

Steiner

Hanreich

Kainz

et al. 2008

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Crucial biological phenomena are mediated through carbohydrates that are displayed in a defined manner and interact with molecular scale precision. We lay the groundwork for the integration of recombinant carbohydrates into a "biomolecular construction kit" for the design of new biomaterials, by utilizing the self-assembly system of the crystalline cell surface (S)-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a. SgsE is a naturally O-glycosylated protein, with intrinsic properties that allow it to function as a nanopatterned matrix for the periodic display of glycans. By using a combined carbohydrate/protein engineering approach, two types of S-layer neoglycoproteins are produced in Escherichia coli. Based on the identification of a suitable periplasmic targeting system for the SgsE self-assembly protein as a cellular prerequisite for Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts protein glycosylation, and on engineering of one of the natural protein O-glycosylation sites into a target for N-glycosylation, the heptasaccharide from the AcrA protein of Campylobacter jejuni and the O7 polysaccharide of E. coli are co-or post-translationally transferred to the S-layer protein by the action of the oligosaccharyltransferase PglB. The degree of glycosylation of the Slayer neoglycoproteins after purification from the periplasmic fraction reaches completeness. Electron microscopy reveals that recombinant glycosylation is fully compatible with the S-layer protein self-assembly system. Tailor-made ("functional") nanopatterned, self-assembling neoglycoproteins may open up new strategies for influencing and controlling complex biological systems with potential applications in the areas of biomimetics, drug targeting, vaccine design, or diagnostics.

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Section: Periplasmic Targeting Of Sgsementioning

confidence: 99%

Section: Design Of An Sgse Neoglycoprotein Carrying a C Jejuni Heptamentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Recombinant Glycans on an S‐Layer Self‐Assembly Protein: A New Dimension for Nanopatterned Biomaterials

Steiner

Hanreich

Kainz

et al. 2008

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“…14 Intriguingly, it has been shown that the glycosylation machinery can be transferred to E. coli , resulting in E. coli cells capable of N-glycosylation of proteins. 41–43 Introduction of this machinery into E. coli SHuffle cells may enable the production of N-glycosylated antibodies. Such an improvement would afford a mAb more similar to its mammalian-expressed therapeutic counterpart.…”

Section: Discussionmentioning

confidence: 99%

Platform development for expression and purification of stable isotope labeled monoclonal antibodies inEscherichia coli

Reddy

Brinson

Hoopes

et al. 2018

mAbs

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The widespread use of monoclonal antibodies (mAbs) as a platform for therapeutic drug development in the pharmaceutical industry has led to an increased interest in robust experimental approaches for assessment of mAb structure, stability and dynamics. The ability to enrich proteins with stable isotopes is a prerequisite for the in-depth application of many structural and biophysical methods, including nuclear magnetic resonance (NMR), small angle neutron scattering, neutron reflectometry, and quantitative mass spectrometry. While mAbs can typically be produced with very high yields using mammalian cell expression, stable isotope labeling using cell culture is expensive and often impractical. The most common and cost-efficient approach to label proteins is to express proteins in Escherichia coli grown in minimal media; however, such methods for mAbs have not been reported to date. Here we present, for the first time, the expression and purification of a stable isotope labeled mAb from a genetically engineered E. coli strain capable of forming disulfide bonds in its cytoplasm. It is shown using two-dimensional NMR spectral fingerprinting that the unlabeled mAb and the mAb singly or triply labeled with 13C, 15N, 2H are well folded, with only minor structural differences relative to the mammalian cell-produced mAb that are attributed to the lack of glycosylation in the Fc domain. This advancement of an E. coli-based mAb expression platform will facilitate the production of mAbs for in-depth structural characterization, including the high resolution investigation of mechanisms of action.

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Glycosylation and Disease

Dwek

Markiv

2018

Encyclopedia of Life Sciences

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Glycosylation is the process of attachment of carbohydrates (glycans) to proteins and lipids to form the glycoproteins and glycolipids found in eukaryotic organisms. Glycosylation reactions are amongst the most common posttranslational modifications that have been reported on proteins, and they function to alter protein size, stability, charge and antigenicity. Glycosylation plays an important role in cancer, inherited diseases, pathogen–host interactions and immune recognition. Key Concepts The analysis of glycosylation is more complex than DNA and RNA or protein analysis as glycosylation is a non‐template‐driven event. N‐ linked glycosylation commences in the endoplasmic reticulum and is completed in the Golgi apparatus. O‐ linked glycosylation commences and is completed in the Golgi apparatus. N‐ linked glycosylation occurs on asparagine residues in the consensus sequon Asn‐X‐Ser/Thr. O‐ linked glycosylation occurs on serine and threonine residues. Many enzymes are involved in both N‐ and O‐ linked glycosylation. Glycans play a role in sperm–egg interactions, the implantation process and embryonic development. Congenital disorders in glycosylation result from autosomally dominant mutations in enzymes involved in glycosylation pathways. Glycosylation of proteins influences their half‐life in the serum. N‐ and O‐ linked glycosylation have been reported to be altered in cancer and implicated in all of the steps in the metastatic cascade.

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N-Linked Glycosylation in Campylobacter jejuni and Its Functional Transfer into E. coli

Cited by 723 publications

References 20 publications

Recombinant Glycans on an S‐Layer Self‐Assembly Protein: A New Dimension for Nanopatterned Biomaterials

Recombinant Glycans on an S‐Layer Self‐Assembly Protein: A New Dimension for Nanopatterned Biomaterials

Platform development for expression and purification of stable isotope labeled monoclonal antibodies inEscherichia coli

Glycosylation and Disease

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