title: Expression of the functional recombinant human glycosyltransferase GalNAcT2 in Escherichia coli.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Applied Biotechnology, Biberach University of Applied Sciences, Biberach, Germany. Jennifer.Lauber@hochschule-bc.de.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lauber
authors.affiliation: Institute of Applied Biotechnology, Biberach University of Applied Sciences, Biberach, Germany. rene.handrick@hochschule-bc.de.
authors.forename: René
authors.initials: R
authors.lastname: Handrick
authors.affiliation: Institute of Microbiology and Molecular Biology, University of Hohenheim, Stuttgart, Germany. sebastian.leptihn@uni-hohenheim.de.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Leptihn
authors.affiliation: Institute of Microbiology and Biotechnology, University of Ulm, Ulm, Germany. peter.duerre@uni-ulm.de.
authors.forename: Peter
authors.initials: P
authors.lastname: Dürre
authors.affiliation: Institute of Applied Biotechnology, Biberach University of Applied Sciences, Biberach, Germany. gaisser@hochschule-bc.de.
authors.forename: Sabine
authors.initials: S
authors.lastname: Gaisser
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003124:Colorimetry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000097763:Polypeptide N-acetylgalactosaminyltransferase
chemical_list: D016179:Granulocyte Colony-Stimulating Factor; D006639:Histidine; D017350:N-Acetylgalactosaminyltransferases; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C471213:His-His-His-His-His-His