Cell Metabolism - Bioprocess Engineering: Kinetics, Biosystems, Sustainability and Reactor Design - page 490

preform is what is made from the m-RNA, but the actual active form is the mature form. The
preform is the signal sequence plus the mature form.
In prokaryotes, secretion of proteins occurs through the cytoplasmic membrane. In E. coli
and most gram-negative bacteria, the outer membrane blocks release of the secreted protein
into the extracellular compartment. In gram-positive cells, secreted proteins readily pass the
cell wall into the extracellular compartment. Whether a protein product is retained in a cell or
released has a major impact on bioprocess design.
In eukaryotic cells, proteins are released by two pathways. Both involve exocytosis, where
transport vesicles fuse with the plasma membrane and release their contents. Transport vesi-
cles mediate the transport of proteins and other chemicals from the endoplasmic reticulum
(ER) to the Golgi apparatus and from the Golgi apparatus to other membrane-enclosed
compartments. Such vesicles bud from a membrane and enclose an aqueous solution with
specific proteins, lipids, or other compounds. In the secretory pathway vesicles, carrying
proteins bud from the ER, enter the cis face of the Golgi apparatus, exit the Golgi trans
face, and then fuse with the plasma membrane. Only proteins with a signal sequence are pro-
cessed in the ER to enter the secretory pathway. Two pathways exist. One is the constitutive
exocytosis pathway, which operates at all times and delivers lipids and proteins to the plasma
membrane. The second is the regulated exocytosis pathway, which typically is in specialized
secretory cells. These cells secrete proteins or other chemicals only in response to specific
chemical signals.
Other modifications to proteins can take place, particularly in higher eukaryotic cells.
These modifications involve the addition of nonamino acid components (for example, sugars
and lipids) and phosphorylation. Glycosylation refers to the addition of sugars. These modifica-
tions can be quite complex and are important considerations in the choice of host organisms
for the production of proteins. A bioprocess engineer must be aware that many proteins are
subject to extensive processing after the initial polypeptide chain is made.
A particularly important aspect of post translational processing is N-linked glycosylation.
The glycosylation pattern can serve to target the protein to a particular compartment or to
control its degradation and removal from the organism. For therapeutic proteins injected
into the human body, these issues are critical ones. A protein product may be ineffective if
the N-linked glycosylation pattern is not humanlike, as the protein may not reach the target
tissue or may be cleared (i.e. removed) from the body before it exerts the desired action.
Furthermore, undesirable immunogenic responses can occur if a protein has a nonhuman-
like pattern. Thus, the glycoform of a protein product is a key issue in bioprocesses to
make therapeutic proteins.
The process of N-linked glycosylation occurs only in eukaryotic cells and involves both the
ER and Golgi. Thus, the use of prokaryotic cells, such as E. coli, to serve as hosts for expres-
sion of human therapeutic proteins is limited to those proteins where N-linked glycosylation
is not present or unimportant. However, not all eukaryotic cells produce proteins with
humanlike, N-linked glycosylation. For example, yeasts, lower fungi, and insect cells often
produce partially processed products. Even mammalian cells (including human cells) will
show altered patterns of glycosylation when cultured in bioreactors, and these patterns
can shift upon scale-up in bioreactor size.
The process of N-linked glycosylation is depicted in Fig. 10.11 . The pattern shown is
“typical,” and many variants are possible. The natural proteins in the human body usually