EXPRESSION OF PROTEINS BY EUKARYOTIC CELLS
Although bacterial cells have successfully expressed many eukaryotic proteins, there are cases where it is best to express eukaryotic proteins using eukaryotic cells. Some eukaryotic proteins are unstable or inactive after being made by bacterial cells. This is especially true of proteins that require posttranslational modification.
A variety of eukaryotic modifications may occur after the polypeptide chain has been made (Fig. 10.8). These include:
(a) Chemical modifications that form novel amino acids in the polypeptide chain.
(b) Formation of disulfide bonds between correct cysteine partners (e.g., the assembly of insulin).
(c) Glycosylation, that is, the addition of sugar residues at specific locations on the protein. Many cell surface proteins are glycosylated and will not assemble correctly into membranes or function properly if lacking their glycosyl components.
(d) Addition of a variety of extra groups, such as fatty acid chains, acetyl groups, phosphate groups, sulfate groups.
(e) Cleavage of precursor proteins. This may occur in several stages, as illustrated by insulin. Cleavage may be involved with secretion, correct folding, and/or activation of proteins.
The enzymes required for modification and processing are normally absent from bacterial cells, making it necessary to express eukaryotic proteins in eukaryotic cells. Related processing enzymes are often present in a range of higher organisms; thus it is rarely absolutely necessary to express a protein in its original organism. Here we are concerned with protein production in cultured cells. However, as discussed, it is now possible to engineer whole transgenic animals or plants to produce recombinant proteins. A further advantage of expressing eukaryotic proteins in eukaryotic cells is that contamination with bacterial components is avoided. Despite purification, bacterial components that are toxic or promote immune reactions or cause fever may be present in traces if bacteria are used for production.
As discussed in Previews Pages, shuttle vectors are designed to move genes between different groups of organisms. Because genetic engineering is more difficult for eukaryotes, most expression vectors for eukaryotic cells are in fact shuttle vectors. Such vectors allow genetic engineering to be carried out in bacteria, usually E. coli, and allow transfer to other organisms for gene expression. We will consider the use of yeasts, insect cells, and mammalian cells for expression of recombinant proteins.
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