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Fro. 3.29. A summary of the steps in in vitro genetic recombination. Both plasmid vector and foreign DNA are cut by the restriction endonuclease, EcoRI, producing linear double-stranded DNA fragments with single-stranded cohesive projections. EcoRI recognizes the oligonucleotide sequence and will cut any double-stranded DNA molecule to yield fragments with the same cohesive ends c , if-rAAG. On mixing vector and foreign DNA, hybrids form into circular molecules which can be covalently joined using DNA ligase. Transformation of E. coli results in the low-frequency uptake of hybrid molecules whose presence can be detected by the ability of the plasmid to confer drug resistance on the host (Atherton et al., 1979).

Fro. 3.29. A summary of the steps in in vitro genetic recombination. Both plasmid vector and foreign DNA are cut by the restriction endonuclease, EcoRI, producing linear double-stranded DNA fragments with single-stranded cohesive projections. EcoRI recognizes the oligonucleotide sequence and will cut any double-stranded DNA molecule to yield fragments with the same cohesive ends c , if-rAAG. On mixing vector and foreign DNA, hybrids form into circular molecules which can be covalently joined using DNA ligase. Transformation of E. coli results in the low-frequency uptake of hybrid molecules whose presence can be detected by the ability of the plasmid to confer drug resistance on the host (Atherton et al., 1979).

prokaryotic cell. Nagata et al. (1980) used the reverse transcriptase method to produce the genes coding for human interferon. Complementary single-stranded DNA was prepared from a mixture of messenger RNA extracted from virus-induced human lymphocytes. The DNA was then introduced into E. coli at random using a plasmid vector and the recipient cells screened for the presence of the interferon gene. Those colonies that contained the gene were then examined for interferon production. Using this method, Nagata's group succeeded in demonstrating the expression of the interferon gene in E. coli. Regrettably, a detailed explanation of the techniques involved in gene cloning is outside the scope of this book, but excellent accounts of the various methods are given by Gingold (1993) Slater (1993) and Curran and Bugeja (1993), all in the same text (Walker and Gingold, 1993).

The most publicized application of recombinant DNA technology in the context of fermentation is the construction of strains capable of synthesizing foreign proteins. Although this chapter is concerned with strain improvement it is the obvious place to consider such chimeric strains. The use of the technique for the improvement of microbial product synthesis has also been very successful and this will be discussed in a later section.

(i) The production of heterologous proteins

The rationale for the commercial production of foreign proteins in micro-organisms depends on the protein under consideration. The first commercial heterologous protein to be produced was human growth hormone (hGH) which is used to treat hypopituitary dwarfism and, prior to its manufacture by fermentation, was extracted from the brains of human cadavers! Naturally, this source was not readily available and carried the additional disadvantage of the risk of contamination with human pathogens. The successful production of recombinant hGH from E. coli both satisfied the demand for the compound and eliminated the risks associated with the human source (Dykes, 1993). Factor VIII is a blood clotting agent used in the treatment of haemophilia. Prior to its production as a recombinant protein it was extracted from human blood with the associated risk of contamination with HIV, so that the logic behind its production by fermentation is very similar to that of hGH — availability and safety.

The logic for the development of recombinant human insulin is not quite as clear because diabetes had been treated successfully for many years with animal insulin. However, it was assumed that the recombinant human product would cause fewer immunological difficulties and it would be pure and not contaminated with such pancreatic peptides as proinsulin, glucagon, somatostatin, pancreatic polypeptides and vaso-active intestinal peptides. Furthermore, the incidence of diabetes was also expected to increase due to changes in diet, the improved care of pregnant diabetic women (resulting in an increase of diabetes in the gene pool) and the increased life expectancy of diabetics (Dykes, 1993). Recombinant human insulin was first marketed in the U.K. in 1982 and by 1989 had become the most common form in use (Dykes, 1993).

Many other human proteins are synthesized at very low levels and the only practical way to produce them in sufficient quantities for use as therapeutic agents is as recombinant proteins. Examples of such products include the interferons and erythropoietin. Table 3.8 lists the recombinant proteins which have been licensed for therapeutic use. According to Dykes (1993) although insulin and hGH have been two of the most successful products (in terms of sales) some newer products have greater potential. For example, erythropoietin stimulates the production of erythrocytes from immature erythroid progenitor cells and has been used for the successful treatment of renal failure-induced anaemia. Predicted annual sales of the protein in the mid-1990s are in the region of $1200 million. The potential markets of some other recombinant human proteins are considered in Chapter 1.

Hepatitis B virus causes an infection of the liver giving rise to chronic viral hepatitis which may lead to progressive liver disease. There is no effective cure of the disease and, thus, vaccination is critical. The first hepatitis B vaccine became available in 1982, prepared from hepatitis B surface antigen (HBsAg) purified from the plasma of human carriers of the disease. Although the vaccine was successful its origin obviously presented the same difficulties of availability and contamination as discussed for hGH and Factor VIII. These difficulties provided the impetus to develop a recombinant vaccine which was achieved by expressing the HBsAg gene in Saccharomyces cereuisiae.

The vast majority of the early work on recombinant DNA technology concerned the transfer of genetic material into E. coli and this led to the adoption of the organism as the host for the production of several heterologous proteins. The bacterium had several advantages in its favour, primarily:

The availability of genetic knowledge.

The very wide range of vectors available.

A simple fermentation process using cheap media.

Promising protein yields in the range of 2-5 g dm^3.

Despite these advantages E. coli also presents several problems for the production of heterologous proteins:

Proteins are formed as insoluble aggregates.

The proteins are not secreted.

Lack of post-translational modification.

By the mid-1980s the use of E. coli had declined considerably and yeasts, filamentous fungi and animal cells in culture were being investigated as alternative hosts. However, by 1992 E. coli was once again in favour. The protein yields obtained from animal cells were disappointing and the understanding of secretion and protein folding had progressed such that soluble proteins could be secreted by engineered E. coli (Hockney, 1994). However, mammalian cells are still the preferred host when the protein activity depends upon post-translational modification.

Whichever organism is used for the synthesis of an heterologous protein it is important that the strain is constructed such that expression of the gene may be controlled during the fermentation process. This may be achieved by inserting the gene into an inducible system, such as /3-galactosidase, so that expression of the gene may be initiated by the addition of the inducer. This aspect is discussed in Chapter 4.

As an example of the production of an heterologous protein the development of the Genencor process for the production of recombinant chymosin will be con-

Table 3.8. Recombinant proteins licensed for therapeutic use (Dykes, 1993)

Protein

Clinical use

Insulin

Growth hormone

Tissue plasminogen activator

Erythropoietin

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