Protein Glycosylation: Implications for In Vivo Functions and Therapeutic Applications - Advances in Biochemical Engineering Biotechnology - page 177

5.1.1
Yeasts

A wide range of yeast species, including Saccharomyces cerevisiae, Pichia pasto-
ris, Hansenula polymorpha, Kluyveromyces lactis, Schizosaccharomyces pombe
and Yarrowia lipolytica have been tested for the production of heterologous
eukaryotic glycoproteins [168,169].Major advantages in yeasts include the rela-
tively low culture costs, high expression levels and presence of a secretory
pathway similar to that in higher eukaryotes.Yeasts may successfully use secre-
tion signals from other organisms, and secretion of heterologous proteins into
the culture medium simplifies product purification due to low levels of native
secretory proteins.All the steps involved in higher eukaryotic protein trafficking
and post-translational modification may not be equivalent in yeast. Thus,
although a number of recombinant glycoproteins with pharmaceutical or in-
dustrial value has been obtained using the yeast expression system, they often
have altered biological properties and functions with respect to solubility,sensi-
tivity to proteases or serum half-life, mostly due to differences in protein glyco-
sylation.In S.cerevisiae,N -linked glycans are processed to highly mannosylated
structures and not to complex-type structures containing Fuc, Gal and SA as
found in higher eukaryotes [170]. The Man 8 Gn 2 oligosaccharide structure is
elongated in the Golgi by the action of a series of mannosyltransferases to form
the large mannan oligosaccharides, usually containing 50-150 mannose residue
and some of them have Man-P-Man sequences [171]. Such glycoproteins are
recognized by Man receptors and removed from circulation. In addition,
nonhuman glycosylation in expressed proteins are potentially immunoreactive.
A mannosylation defective mutant strain has been used to avoid hyper manno-
sylation. These mutants however, do not grow well like other yeast strains [172].
Species like P. pastoris and H. polymorpha express glycoproteins of average Man
chain length less than 30 monomers, and furthermore the expressed proteins
are devoid of highly antigenic terminal 1,3-Man linkage, which is obtained in

S.cerevisiae system [168]. Besides glycosylation variants, proteolytic instability
of the secretory proteins is another significant problem in yeast expression
systems. There is a need to develop protease-deficient mutants to improve the
quality and yields of the expressed proteins.

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5.1.2
Mammalian Cells

The major advantage of using mammalian cells to produce heterologous
eukaryotic proteins are that the expressed proteins are correctly folded and
glycosylated to near native structures [173].Because of these unique post-trans-
lational modifications, various mammalian expression systems have been
reported time to time for the production of therapeutic proteins [173-176].
However, the high cost of cell culture, low productivity (5-50 pg cell -1 day -1 ),
and difficulty in growing in large scale imposes serious restrictions on their