Cc c g

'Rluflr Ends'

*|=Cleavage points.

*|=Cleavage points.

Figure 12.1 Insertion of foreign DNA into a bacterial plasmid.

Resistant cells can then be isolated, propagated and tested to determine whether the required protein is being synthesized by the cells. This is achieved by exposing dishes on which cultures are growing to cellulose nitrate filters. If the cultures are producing the required protein this protein will be adsorbed onto the surface of the filter. Subsequent exposure of the filter to protein-specific antibodies carrying a radioactive label produces radioactive patches corresponding to the location of cultures producing the required protein.

A major problem that can occur is the proteolytic degradation of the synthesised polypeptide. This can be prevented by fusing the synthetic gene to the gene of a larger protein associated with the plasmid, e.g. beta-galactosidase or beta-lactamase. The fusion protein is resistant to proteolysis and the required polypeptide can then be cleaved off and isolated. A number of proteins undergo post-translational modification such as modifications of signal or leader amino acid sequences and glycosylation of the protein. An example of how this is achieved for insulin is discussed in Section 12.1.3.2. Choice of the host cell is important since different host organisms have differing capacities to perform post translational changes and may differ in their efficiency of recombinant protein production. Currently the most popular hosts are Escherichia coli, Bacillus subtilis, yeast, and cultured cells of higher eukaryotes such as insect and mammalian cells. For proteins such as insulin which require post-translational modifications and require formation of disulphide bonds to achieve the active product, E. coli is not the vector of choice. In that case the yeast Saccharomyces cerevisiae has been successfully used as the host. Also in contrast to E. coli, coproduction of pyrogens and endotoxins is not a problem with S. cerevisiae.

12.1.3 Production of polypeptides using recombinant DNA technology

12.1.3.1 Somatostatin

Somatostatin is a small polypeptide (14 amino acids long) and the gene is relatively easily synthesised. The synthetic gene is illustrated in Figure 12.3. It should be noted that the initiation amino acid, methionine, preceded the NH2 terminal amino acid of somatostatin and that the COOH-terminal amino acid is followed by two stop codons. An Eco RI

Figure 12.2 Diagrammatic representation of a vector plasmid.

WcJ Att_ t>, Crl im Avt fllijf fill iri J_hr pl,r fly Stf Sm>a SiOO

5'AATTC ATG SCf GST TOT AAG TTC TTT IfiG AAG ACT TIC ACT TCÜ TCI TGA TAG

G TAC CGA C&A ACA TTC nc AAft AAA ACC TTC TÜA AAG T(LA AÜC ACA ACT ATCCTAG i

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