RESEARCH n er e b tion systems for recombinant proteins include bacteria, bonds are preferentially expressed in eukaryotic CHO,
Lauber et al. Microbial Cell Factories (2015) 14:3
DOI 10.1186/s12934-014-0186-0produce compounds with high yield and quality [4,5].
So far, E. coli-based systems have been employed to
Full list of author information is available at the end of the articleyeast, insect and mammalian cells. Bacterial expression systems for recombinant human-derived proteins are widely used, but limited as most bacteria lack certain posttranslational modification (PTM) mechanisms, including those for glycosylation . In general, glycoproteins are yeast and insect cells, a production process that is often time consuming and cost-intensive .
About a third of the currently approved recombinant protein therapeutics are produced in E. coli strains .
The use of bacteria as expression hosts provides many advantages such as rapid growth, low-cost media, a versatility of cloning tools combined with the potential to* Correspondence: email@example.com 1Institute of Applied Biotechnology, Biberach University of Applied Sciences,part of approved biotechnology-based medicines. Produc-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.
Keywords: Protein expression, Escherichia coli, Glycosyltransferase GalNAcT2, Erv1p/PDI co-expression, in vitro glycosylation,
Filgrastim, Recombinant glycosyltransferase, Enzymatic activity glycosyltransferase, Secondary structure glycosyltransferase
Recombinant protein therapeutics comprise a significant produced in eukaryotic cell lines such as Chinese Hamster
Ovary (CHO), murine myeloma (NS0) or Baby Hamster
Kidney (BHK) . Proteins stabilised by multiple disulfideExpression of the functio glycosyltransferase GalNA
Jennifer Lauber1, René Handrick1, Sebastian Leptihn2, Pet
Background: Recombinant protein-based therapeutics hav© 2015 Lauber et al.; licensee BioMed Central.
Commons Attribution License (http://creativec reproduction in any medium, provided the or
Dedication waiver (http://creativecommons.or unless otherwise stated.Open Access al recombinant human cT2 in Escherichia coli
Dürre3 and Sabine Gaisser1* ecome indispensable for the treatment of manyThis is an Open Access article distributed under the terms of the Creative ommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and iginal work is properly credited. The Creative Commons Public Domain g/publicdomain/zero/1.0/) applies to the data made available in this article,
Lauber et al. Microbial Cell Factories (2015) 14:3 Page 2 of 12express non-glycosylated proteins, however, the development of engineered strains allowing the expression of complex therapeutics including correct folding, disulfide bond formation, and glycosylation modifications are highly desirable and may potentially lead to a significant decrease in process time and costs [2,6].
Glycosylation represents one of the most common posttranslational modifications, with N- and O-linked glycosylation requiring the activity of specific glycosyltransferases [2,7]. N-glycosylations take place on an amide nitrogen of the amino acid asparagine and O-glycosylations on mucin are initiated by the addition of the monosaccharide N-acetylgalactosamine to the hydroxyl group of the amino acids serine or threonine [8-11]. The presence, composition and pattern of glycosylation potentially influence various parameters such as glycoprotein functional activity, folding, stability and immunogenicity [6,12]. Various strategies have been pursued to isolate glycoengineered
E. coli strains including the functional transfer of glycosylation pathways . As an example, the production of eukaryotic N-glycoproteins has been demonstrated in E. coli engineered to express the N-glycosylation machinery of Campylobacter jejuni [11,13]. Similarly, the construction of an engineered E. coli strain has been described to successfully transfer glycans to target proteins by the expression of several heterologous glycosyltransferases from