Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells
Highlights
► Recombinant proteins expressed in HEK and CHO cells have different glycosylation. ► Different isoform patterns for recombinant proteins expressed in HEK and CHO cells. ► Different sialic acid contents in HEK and CHO derived recombinant proteins. ► The N-linked glycostructures released are different.
Introduction
The properties of a protein are largely determined by its amino acid sequence. However, protein characteristics are also modified and regulated by a large number of post-translational modifications, taking place during or after synthesis of the polypeptide chain (Walsh et al., 2005). One of the most common post-translational modifications is glycosylation and approximately half of all human proteins are estimated to be glycoproteins (Wong, 2005). Protein glycosylation is a conserved mechanism that occurs in yeast, plants and animals (Lommel and Strahl, 2009). Simple forms of glycosylation have also been identified in bacteria (Nothaft and Szymanski, 2010). Glycosylation are enzymatic processes where glycans are added to specific amino acids in the polypeptide chain. Two types of glycosylation, N-linked and O-linked, occur in proteins. N-linked glycosylation starts as a co-translational process in the endoplasmic reticulum (ER), where a branched presynthesized oligomeric glycan structure is attached to the nitrogen of an aspargine in the protein chain (Yan and Lennarz, 2005). The glycan structure is then trimmed before the protein is transferred to the Golgi apparatus (GA) where the glycan structure is further modified. O-linked glycosylation is a post-translational process occurring in the GA (Peter-Katalinic, 2005). Single monosaccharides are attached to the hydroxyl group of serine or threonine residues. The glycan structures are subsequently built up by addition of individual monosaccharides. In addition to the two major glycosylation pathways, glycans can also be attached to arginine, tyrosine, hydroxylysine, hydroxyproline and tryptophan residues (Spiro, 2002). These modifications are less common and restricted to specific proteins. Protein glycosylation can be very heterogeneous, resulting in a large number of isoforms of the protein (Hua et al., 2011). Potential glycosylation sites can be either occupied or unmodified, and each site can be occupied by a different glycan structure in different protein molecules.
Although, glycosylation was long considered as an unimportant protein decoration, it is now clear that it has important functions. Glycosylation affects protein folding, stability, solubility, protein–protein interactions and in vivo bioavailability, biodistribution, pharmacokinetics and immunogenecity (Kaushik et al., 2011, Kayser et al., 2011, Li and d’Anjou, 2009, Oberg et al., 2011, Opanasopit et al., 2001). In general the activity of a protein is determined by its primary amino acid sequence however, there are several examples where glycosylation affects and regulates the activity (Rajagopalan et al., 2010, Straumann et al., 2006, Su et al., 2010). Impaired or changed protein glycosylation is associated with a number of human pathologies, including, rheumatoid arthritis, Leroy disease, and leukocyte-adhesion deficiency type II (Gornik and Lauc, 2008, Koscielak, 1995). Alterations in protein glycosylation are frequently associated with different cancers, although the function or implication of those changes mostly remains unclear (Dennis et al., 1999, Orntoft and Vestergaard, 1999).
Most proteins used for in vitro and in vivo studies today are produced as recombinant proteins in various cell culture based expression systems. Widely used expression systems are, in addition to bacteria, where little post-translational modifications occur, yeast, insect cells and different mammalian cell lines. It is well known that the glycosylation structures are different between mammalian, yeast and insect cells (Brooks, 2006). However, it is much less known what differences exist between mammalian cell lines. Two frequently used mammalian cell lines are HEK and CHO. HEK is a cell line originally derived from Human Embryonic Kidney tissue. HEK cells are easy to grow and transfect, and transient transfection frequently gives good expression levels, which has made this cell line widely used in research. CHO is derived from Chinese Hamster Ovary. This cell line is frequently used for expression after stable transfections, it has a good long-term stable gene expression with high expression levels. For transient expression CHO is more difficult to transfect and the expression levels are frequently lower compared to transiently transfected HEK cells.
For this study 12 proteins containing different numbers of potential N- and O-linked glycosylation sites were selected and each one was expressed and purified from HEK and CHO cells. The purified proteins were analyzed by SDS-PAGE, gel IEF and/or capillary IEF. To verify the contribution of glycosylation to the differences in protein patterns detected the proteins were treated with deglycosylating enzymes and reanalyzed after the treatment. For the more in depth analyses subsets of the proteins were analyzed by mass spectrometry and released N-linked glycans were analyzed by capillary gel electrophoresis.
Section snippets
Reagents
The Poros 20 MC resin was from Applied Biosystems. Superdex 75 and Sephadex G-25 resins were from GE Healthcare. The NuPAGE 10% Bis-Tris gels, IEF 3–10 gels, IEF 3–7 gels, MES-SDS running buffer, MOPS-SDS running buffer and BenchMark protein ladder were from Invitrogen. InstantBlue staining solution was purchased from Expedeon. Sialidase, O-glycosidase, β-galactosidase, glucosaminidase, corresponding reaction buffers and denaturation solutions were from QA-Bio. PNGase F was either form QA-Bio
Results
The aim of this study was to examine whether the glycosylation pattern of proteins expressed in the HEK and CHO cell lines is significantly different. A set of 12 proteins of different sizes, between 9.5 kDa and 52 kDa, containing various numbers of potential N- and/or O- glycosylation sites were selected and expressed in HEK293 EBNA and CHO-S cells (Table 1). The two cell lines were transiently transfected and grown in standard conditions usually applied for production of recombinant protein as
Discussion
Virtually all proteins used in biological and biomedical research today are recombinant proteins expressed and purified from different expression systems. In addition, the number of proteins approved for therapeutic use is increasing rapidly (Durocher and Butler, 2009). All new therapeutic proteins are produced through recombinant procedures, mostly in mammalian expression systems in order to maintain mammalian posttranslational modifications. Recombinant proteins produced in mammalian
Conclusion
Although some differences in glycosylation of HEK derived proteins were detected when the proteins were expressed in HEK EBNA cells or in a variant HEK 6E, and when the culture media and conditions were changed, the major differences in the glycosylation was detected between the CHO and HEK cell lines. The results clearly show that proteins expressed in two frequently used mammalian cell lines have very significant differences in their glycosylation pattern. The size and number of the
Contributions
Amelie Croset and Laurence Delafosse performed most of the experiments and participated in the planning of the experiments.
Ana Krstanovic and Flavie Robert planned and performed the capillary gel electrophoresis glycoanalysis.
Francis Vilbois and Damien Begue performed the LC–MS analysis.
Christophe Losberger performed the capillary IEF.
Loic Glez supervised the bioprocessing work.
Jean-Philippe Gaudry and Christian Arod supervised the protein purification.
Laurent Chevalet and Bruno Antonsson
Acknowledgements
A. Krstanovic and F. Robert thank Erdmann Rapp and René Hennig from Glyxera®, and Francesca Cutillo and Horst Bierau from Merck Serono SpA Italy.
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2023, Advanced Drug Delivery ReviewsCitation Excerpt :First, CMP-Neu5Ac hydroxylase is inactive in human cells but its activity in CHO cells allows for the glycosylation of proteins with Neu5Gc sialic acids, which can provoke antibody responses[105]. Interestingly, the ability to synthesize Neu5Gc enhances the sialylation of CHO-driven proteins compared to those from human HEK293 cells[106,107]. Second, CHO cells may also possess α1,3-galactosyltransferase activity, which is absent in humans, and generate protein products with Galα1,3-Gal residues (α-Gal) that are known to elicit adverse anaphylaxis reactions[108].
- 1
These two authors contributed equally to the work.
- 2
Present address: Institut Curie, Centre Universitaire, Orsay, France.
- 3
Present address: National Research Council – Biotechnology Research Institute, Montréal, Quebec, Canada.
- 4
Present address: Novartis Pharma AG, Basel, Switzerland.