EXPRESSION
OF MULTIPLE SUBUNITS IN MAMMALIAN CELLS
To further complicate
recombinant protein expression, many mammalian proteins have multiple subunits
that must assemble inside a mammalian cell. Thus, manufacturing the separate
subunits in separate cultures and mixing them later fail to yield active
protein.
In this case it may be
necessary to synthesize more than one polypeptide in the same cell (Fig.
10.14). This may be done in three main ways:
(a)
Two separate vectors are used, each carrying the gene for one of
the subunits.
(b)
A single vector is used that carries two separate genes for the two
subunits, each under control of its own promoter.
(c)
A single vector is used that carries an artificial “operon” in
which the two genes are expressed using the same promoter. Transcription gives
a single polycistronic mRNA carrying both genes. As
discussed in Previews Pages, eukaryotic cells normally only translate the first open
reading frame in an mRNA. However, certain animal viruses have sequences known
as internal ribosomal entry sites
(IRESs) that allow the translation of multiple coding sequences on the same
message. Note that the mRNA is read twice by two different ribosomes that bind
in two different places (the normal 5’-end and the IRES). This is different
from the translation of polycistronic mRNA in bacteria where a single
ribosome proceeds along the mRNA and translates all the open reading frames in
the operon.
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