Expression Systems

There are a variety of different expression hosts used to produce glycoproteins. Each has advantages and disadvantages, of which the user should be aware.

Mammalian cell lines such as Chinese Hamster Ovary cells (CHO), NS0 murine myeloma cells and Human Embryonic Kidney cells (HEK)
Advantages Disadvantages
  • Produces mammalian-like glycosylation
  • Some cell lines have been modified to produce humanized glycoproteins
  • Glycosylation pattern may not be fully human
  • Can have immunogenic nonhuman epitopes such as Gal ⍺1-3 Gal and N-glycolylneuraminic acid residues (1) (Fig. 1)
Insect cell lines using a Baculovirus expression system
Advantages Disadvantages
  • Heterologous genes are well expressed and the protein produced is often correctly folded
  • Post-translational modification and oligomerization are often identical to that which occurs in mammalian cells
  • Some cell lines have been modified to reduce or eliminate nonmammalian glycosylation (1)
  • Glycoproteins can have N-linked glycans with simpler side chains called paucimannose N-glycans (2) (Fig. 1)
  • N-glycans are often modified at the GlcNAc core by an ⍺1-3 fucose (Fig. 1) known to be allergenic in humans
  • These modified glycans are resistant to PNGase F cleavage but not PNGase A
Yeast strains, such as Saccharomyces cerevisiae, Pichia pastoris and Kluyveromyces lactis
Advantages Disadvantages
  • Relatively easy to grow in shake flasks or fermentors
  • Their biosynthetic pathways resemble higher eukaryotic cells
  • Work has been done to alter Pichia to produce nearly human N-glycosylation (3)
  • Generally unable to produce complex N-linked glycans (Fig. 1)
  • Both S.cerevisiae and K.lactis produce hypermannosylated N-linked glycans (Fig. 1)
Several plant-based expression systems
Advantages Disadvantages
  • Can produce high yields of proteins
  • A variety of different proteins have been successfully expressed including membrane proteins, multiple sub-unit domains as well as monoclonal antibodies
  • Work has been done to modify glycosylation pathways to avoid nonhuman modifications (4)
  • Plants typically modify their N-linked glycans with the same ⍺1-3 fucose as seen on some insect-produced proteins (Fig. 1)
  • These modified glycans are resistant to PNGase F cleavage but not PNGase A
  • N-glycans can also be modified with a xylose residue on the b-mannose of the glycan core (Fig. 1)

 

Figure 1: Summary of N-glycans commonly found in glycoproteins in various host systems. Typical glycoforms for each system are shown, some of these modifications can occur in the same glycan molecule. Mammalian cells have a more diverse range of modifications than any of the other organisms. Certain motifs extending the N-glycan arms (lactosamine chains, terminal GalNAc, terminal sialylation, fucosylation) are found in mammalian O-glycans as well.
 
  1. Galili U., et al (1988) J Biol Chem. 25;263(33):17755-62. PMID: 2460463
  2. Shi, X., Jarvis, D.L. (2007) Current Drug Targets. 8(10):1116-25. PMID: 17979671
  3. Hamilton, S.R., Gerngross, T.U. (2007) Curr Opin Biotechnol. 18(5):387-92. PMID: 17951046
  4. Gomord, V., et al (2010) Plant Biotech J. 8(5):564-87. PMID: 20233335

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