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Cited by 30Glycosylation and Glycoproteins ResearchViral Infectious Diseases and Gene Expression in InsectsProtein purification and stability

Expression of the functional recombinant human glycosyltransferase GalNAcT2 in Escherichia coli

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Lauber, Jennifer, Handrick, René, Leptihn, Sebastian, Dürre, Peter, Gaisser, Sabine·2015·Microbial Cell Factories
DOI 10.1186/s12934-014-0186-0PMC PMC4299809
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Abstract

BACKGROUND: Recombinant protein-based therapeutics have become indispensable for the treatment of many diseases. They are produced using well-established expression systems based on bacteria, yeast, insect and mammalian cells. The majority of therapeutic proteins are glycoproteins and therefore the post-translational attachment of sugar residues is required. The development of an engineered Escherichia coli-based expression system for production of human glycoproteins could potentially lead to increased yields, as well as significant decreases in processing time and costs. RESULTS: This work describes the expression of functional human-derived glycosyltransferase UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 2 (GalNAcT2) in a recombinant E. coli strain. For expression, a codon-optimised gene encoding amino acids 52-571 of GalNAcT2 lacking the transmembrane N-terminal domain was inserted into a pET-23 derived vector encoding a polyhistidine-tag which was translationally fused to the N-terminus of the glycosyltransferase (HisDapGalNAcT2). The glycosyltransferase was produced in E. coli using a recently published expression system. Soluble HisDapGalNAcT2 produced in SHuffle® T7 host cells was purified using nickel affinity chromatography and was subsequently analysed by size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) and circular dichroism spectroscopy to determine molecular mass, folding state and thermal transitions of the protein. The activity of purified HisDapGalNAcT2 was monitored using a colorimetric assay based on the release of phosphate during transfer of glycosyl residues to a model acceptor peptide or, alternatively, to the granulocyte-colony stimulating growth factor (G-CSF). Modifications were assessed by Matrix Assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry analysis (MALDI-TOF-MS) and Electrospray Mass Spectrometry analysis (ESI-MS). The results clearly indicate the glycosylation of the acceptor peptide and of G-CSF. CONCLUSION: In the present work, we isolated a human-derived glycosyltransferase by expressing soluble HisDapGalNAcT2 in E. coli. The functional activity of the enzyme was shown in vitro. Further investigations are needed to assess the potential of in vivo glycosylation in E. coli.

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  • Significantly higher activities were detected for rhGalNAc-T2 compared to HisDapGalNAc-T2.

    “In general, significantly higher activities were detected for rhGalNAc-T2 as compared to HisDapGalNAc-T2 (Fig. 3b), thus confirming previously published results [6].”

    From High level in vivo mucin-type glycosylation in Escherichia coli (2018) · cites this paper as [6]

    The Results (Analysis of HisDapGalNAcT2 enzymatic activity; page unavailable) report higher specific activity for rhGalNAcT2 with both tested substrates, at approximately twice that of HisDapGalNAcT2. The paper does not report a statistical significance test for these differences, so the word “significantly” is not established if intended in its statistical sense. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

  • Glycosylation of the synthetic peptide EA2 derived from Rattus norvegicus submandibular apomucin 10 (MUC10) has been demonstrated in various in vitro assays in the presence of soluble GalNAc-T2.

    “As an example, glycosylation of the synthetic peptide EA2 (PTTDSTTPAPTTK)—derived from Rattus norvegicus submandibular apomucin 10 (MUC10)—has been demonstrated in various in vitro assays in the presence of soluble GalNAc-T2 [6, 14, 15].”

    From High level in vivo mucin-type glycosylation in Escherichia coli (2018) · cites this paper as [6]

  • O-glycosylation of eukaryotic target sites using a truncated form of human GalNAc-T2 has been shown without altering the target peptide sequence.

    “In contrast, 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].”

    From High level in vivo mucin-type glycosylation in Escherichia coli (2018) · cites this paper as [6]

  • An active human GalNAcT2 derivative comprising amino acids 52-571 with a translationally fused N-terminal HisDap tag has been isolated from a SHuffle® T7 E. coli strain pre-/co-expressing chaperones.

    “As a first step to establish an E. coli-based expression platform enabling the glycosylation of full-length biopharmaceuticals in vivo, an active human GalNAcT2 derivative comprising amino acids 52–571 with a translationally fused N-terminal HisDap (diamino peptidase) tag has been isolated from a SHuffle® T7 E. coli strain pre-/co-expressing chaperones [6].”

    From High level in vivo mucin-type glycosylation in Escherichia coli (2018) · cites this paper as [6]

  • The activity of the purified glycosyltransferase has been demonstrated using the mucin peptide derivative EA2 and filgrastim as acceptor substrates in vitro.

    “The activity of the purified glycosyltransferase has been demonstrated using the mucin peptide derivative EA2 and filgrastim (Granulocyte-colony stimulating factor, G-CSF) as acceptor substrates in vitro [6].”

    From High level in vivo mucin-type glycosylation in Escherichia coli (2018) · cites this paper as [6]

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Research works printed but not linked: 36

  1. [1]Skretas G Carroll S DeFrees S Schwartz MF Johnson KF Georgiou G Expression of active human sialyltransferase ST6GalNAcI in Escherichia coli Microb Cell Fact 2009 8 50 10.1186/1475-2859-8-50 19788761 PMC2762462 · DOI
  2. [2]Walsh G Jefferis R Post-translational modifications in the context of therapeutic proteins Nat Biotechnol 2006 24 1241 52 10.1038/nbt1252 17033665 · DOI
  3. [3]Lobstein J Emrich CA Jeans C Faulkner M Riggs P Berkmen M SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm Microb Cell Fact 2012 11 56 10.1186/1475-2859-11-56 22569138 PMC3526497 · DOI
  4. [4]Huang CJ Lin H Yang X Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements J Ind Microbiol Biotechnol 2012 39 383 99 10.1007/s10295-011-1082-9 22252444 · DOI
  5. [5]Demain AL Vaishnav P Production of recombinant proteins by microbes and higher organisms Biotechnol Adv 2009 27 297 306 10.1016/j.biotechadv.2009.01.008 19500547 · DOI
  6. [6]Merritt JH Ollis AA Fisher AC DeLisa MP Glycans-by-design: engineering bacteria for the biosynthesis of complex glycans and glycoconjugates Biotechnol Bioeng 2013 110 1550 64 10.1002/bit.24885 23456823 · DOI · Paper page
  7. [7]Nothaft H Szymanski CM Protein glycosylation in bacteria: sweeter than ever Nat Rev Microbiol 2010 8 765 78 10.1038/nrmicro2383 20948550 · DOI
  8. [8]White T Bennett EP Takio K Sorensen T Bonding N Clausen H Purification and cDNA cloning of a human UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase J Biol Chem 1995 270 24156 65 10.1074/jbc.270.41.24156 7592619 · DOI
  9. [9]Wildt S Gerngross TU The humanization of N-glycosylation pathways in yeast Nat Rev Microbiol 2005 3 119 28 10.1038/nrmicro1087 15685223 · DOI
  10. [11]Schwarz F Huang W Li C Schulz BL Lizak C Palumbo A A combined method for producing homogeneous glycoproteins with eukaryotic N-glycosylation Nat Chem Biol 2010 6 264 6 10.1038/nchembio.314 20190762 PMC2842479 · DOI
  11. [12]Walsh G Post-translational modifications of protein biopharmaceuticals Drug Discov Today 2010 15 773 80 10.1016/j.drudis.2010.06.009 20599624 · DOI
  12. [13]Valderrama-Rincon JD Fisher AC Merritt JH Fan YY Reading CA Chhiba K An engineered eukaryotic protein glycosylation pathway in Escherichia coli Nat Chem Biol 2012 8 434 6 10.1038/nchembio.921 22446837 PMC3449280 · DOI
  13. [14]Iwashkiw JA Seper A Weber BS Scott NE Vinogradov E Stratilo C Identification of a general O-linked protein glycosylation system in Acinetobacter baumannii and its role in virulence and biofilm formation PLoS Pathog 2012 8 e1002758 10.1371/journal.ppat.1002758 22685409 PMC3369928 · DOI
  14. [15]Faridmoayer A Fentabil MA Haurat MF Yi W Woodward R Wang PG Extreme substrate promiscuity of the Neisseria oligosaccharyl transferase involved in protein O-glycosylation J Biol Chem 2008 283 34596 604 10.1074/jbc.M807113200 18930921 PMC3259870 · DOI
  15. [17]Hatahet F Ruddock LW Protein disulfide isomerase: a critical evaluation of its function in disulfide bond formation Antioxid Redox Signal 2009 11 2807 50 10.1089/ars.2009.2466 19476414 · DOI
  16. [18]Taus C Lucini C Sato T Furukawa K Grabherr R Staudacher E Expression and characterization of the first snail-derived UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase Glycoconj J 2013 30 825 33 10.1007/s10719-013-9486-6 23877648 PMC3825155 · DOI
  17. [19]Ten Hagen KG Fritz TA Tabak LA All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases Glycobiology 2003 13 1R 16 10.1093/glycob/cwg007 12634319 · DOI
  18. [20]Sorensen T White T Wandall HH Kristensen AK Roepstorff P Clausen H UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. Identification and separation of two distinct transferase activities J Biol Chem 1995 270 24166 73 10.1074/jbc.270.41.24166 7592620 · DOI
  19. [21]Wandall HH Hassan H Mirgorodskaya E Kristensen AK Roepstorff P Bennett EP Substrate specificities of three members of the human UDP-N-acetyl-α-D-galactosamine:Polypeptide N-acetylgalactosaminyltransferase family, GalNAc-T1, −T2, and -T3 J Biol Chem 1997 272 23503 14 10.1074/jbc.272.38.23503 9295285 · DOI · Paper page
  20. [22]DeFrees S Wang ZG Xing R Scott AE Wang J Zopf D GlycoPEGylation of recombinant therapeutic proteins produced in Escherichia coli Glycobiology 2006 16 833 43 10.1093/glycob/cwl004 16717104 · DOI
  21. [23]Johnson WC Jr Protein secondary structure and circular dichroism: a practical guide Proteins 1990 7 205 14 10.1002/prot.340070302 2194218 · DOI
  22. [24]Kelly SM Price NC The application of circular dichroism to studies of protein folding and unfolding Biochim Biophys Acta 1997 1338 161 85 10.1016/S0167-4838(96)00190-2 9128135 · DOI
  23. [25]Wu ZL Phosphatase-coupled universal kinase assay and kinetics for first-order-rate coupling reaction PLoS One 2011 6 e23172 10.1371/journal.pone.0023172 21853082 PMC3154929 · DOI · Paper page
  24. [26]Albone EF Hagen FK VanWuyckhuyse BC Tabak LA Molecular cloning of a rat submandibular gland apomucin J Biol Chem 1994 269 16845 52 8207007
  25. [27]Pratt MR Hang HC Ten Hagen KG Rarick J Gerken TA Tabak LA Deconvoluting the functions of polypeptide N-α-acetylgalactosaminyltransferase family members by glycopeptide substrate profiling Chem Biol 2004 11 1009 16 10.1016/j.chembiol.2004.05.009 15271359 · DOI · Paper page
  26. [28]Faulkner MJ Veeravalli K Gon S Georgiou G Beckwith J Functional plasticity of a peroxidase allows evolution of diverse disulfide-reducing pathways Proc Natl Acad Sci U S A 2008 105 6735 40 10.1073/pnas.0801986105 18456836 PMC2373332 · DOI
  27. [29]Krause M Ukkonen K Haataja T Ruottinen M Glumoff T Neubauer A A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures Microb Cell Fact 2010 9 11 10.1186/1475-2859-9-11 20167131 PMC2841585 · DOI
  28. [30]Panula-Perala J Siurkus J Vasala A Wilmanowski R Casteleijn MG Neubauer P Enzyme controlled glucose auto-delivery for high cell density cultivations in microplates and shake flasks Microb Cell Fact 2008 7 31 10.1186/1475-2859-7-31 19017379 PMC2588551 · DOI
  29. [31]Ukkonen K Vasala A Ojamo H Neubauer P High-yield production of biologically active recombinant protein in shake flask culture by combination of enzyme-based glucose delivery and increased oxygen transfer Microb Cell Fact 2011 10 107 10.1186/1475-2859-10-107 22152005 PMC3258199 · DOI
  30. [32]Horynova M Takahashi K Hall S Renfrow MB Novak J Raska M Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells Protein Expr Purif 2012 81 175 80 10.1016/j.pep.2011.10.006 22033505 PMC3637031 · DOI
  31. [33]Gomez H Rojas R Patel D Tabak LA Lluch JM Masgrau L A computational and experimental study of O-glycosylation. Catalysis by human UDP-GalNAc polypeptide:GalNAc transferase-T2 Org Biomol Chem 2014 12 2645 55 10.1039/c3ob42569j 24643241 PMC4744471 · DOI · Paper page
  32. [34]Hoglund M Glycosylated and non-glycosylated recombinant human granulocyte colony-stimulating factor (rhG-CSF)–what is the difference? Med Oncol 1998 15 229 33 10.1007/BF02787205 9951685 · DOI
  33. [35]Gascon P Presently available biosimilars in hematology-oncology: G-CSF Target Oncol 2012 7 Suppl 1 S29 34 10.1007/s11523-011-0190-9 22258705 PMC3291849 · DOI
  34. [36]Oh-eda M Hasegawa M Hattori K Kuboniwa H Kojima T Orita T O-linked sugar chain of human granulocyte colony-stimulating factor protects it against polymerization and denaturation allowing it to retain its biological activity J Biol Chem 1990 265 11432 5 1694845
  35. [37]Sambrock J Fritsch EF Maniatis T Molecular Cloning: A Laboratory Manual 1989 2 New-York Cold Spring Harbor Laboratory Press
  36. [38]Krishna Rao DV Rao JV Narasu ML Bhujanga Rao AK Optimization of the AT-content of codons immediately downstream of the initiation codon and evaluation of culture conditions for high-level expression of recombinant human G-CSF in Escherichia coli Mol Biotechnol 2008 38 221 32 10.1007/s12033-007-9018-3 18040898 · DOI

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Authors
Lauber, J., Handrick, R., Leptihn, S., Dürre, P., & Gaisser, S.
Affiliations
  • University of Applied Sciences Biberach (DE)
  • Universität Ulm (DE)
Corresponding author
Gaisser, Sabine
ORCID
Venue
Microbial Cell Factories
Publisher
Springer Science and Business Media LLC
Published
2015-01-13
Volume
14
Issue
1
Pages
3-3
ISSN
1475-2859 (ISSN-L)
DOI
10.1186/s12934-014-0186-0
PMC
PMC4299809
OpenAlex
W2164257156
Cited by
30
References
2
Citations by year
2026: 2 · 2025: 1 · 2024: 2 · 2023: 1 · 2022: 2 · 2021: 5 · 2020: 5 · 2019: 2 · 2018: 4 · 2017: 1 · 2016: 4 · 2015: 1
FWCI
1.45
Type
journal article
Language
en
Open access
Licence
Locations
  • Microbial Cell Factories (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
Keywords
N-acetylgalactosaminyltransferase, glycosyltransferase expression, recombinant therapeutic proteins, glycoprotein synthesis, soluble protein expression, His-tag fusion protein, codon optimization
MeSH terms
Polypeptide N-acetylgalactosaminyltransferase, Amino Acid Sequence, Chromatography, Affinity, Chromatography, Gel, Circular Dichroism, Colorimetry, Electrophoresis, Polyacrylamide Gel, Escherichia coli, Genetic Vectors, Glycosylation, Histidine, Humans, Molecular Sequence Data, Molecular Weight, Oligopeptides, Recombinant Fusion Proteins, Immunoblotting, Granulocyte Colony-Stimulating Factor, N-Acetylgalactosaminyltransferases, Protein Folding, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

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