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Chapter: Biotechnology Applying the Genetic Revolution: Recombinant Proteins

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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.

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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