The Role Of Genetic Manipulation In Bioseparations

The ability to manipulate genetic material has revolutionized biotechnology by giving unprecedented access to the activities of biological systems and their control. DNA can be transferred between species so that foreign proteins are expressed in new host cells. The properties of industrial species can therefore be changed in a specific and targeted way which is no longer limited by the constraints of the cells' natural metabolism and the vagaries of natural recombination. Unique combinations of biological processes can thus be brought together. DNA can be manipulated to create products with new properties, to optimize the synthesis of desired metabolites, or to tailor selected cell functions. The ability to engineer biological systems is not limited to the control of expression and catalytic function of proteins, but can also be used to facilitate protein purification. This makes it possible to consider product recovery as an integrated part of the cloning and expression of recombinant proteins.

There are a number of ways in which recombinant products can be more amenable to purification than native, naturally-expressed proteins. For example, the level of expression of the cloned gene can be increased by using efficient promoters and multi-copy vectors. Recombinant products expressed intracellularly in Escherichia coli characteristically accumulate to levels up to 30% of total cell protein, although expression levels of 50% of total cell protein are occasionally reported (Marston, 1986; Kane & Hartley, 1988). If the product is in a high concentration in the crude feedstock it is easier and cheaper to purify than a compound which is a minor component of a complex starting material. As many protein products no longer have to be produced by the native source organism, a wide range of host organisms is available. These may be selected to overcome production problems associated with the use of biomass which is difficult to obtain or is only available in small amounts. Examples relevant to the food industry include the microbial production of animal growth hormones (Schoner et al, 1985; Chen et al, 1992), recombinant chymosin (rennin) for use in cheese manufacture (Emtage et al, 1983; Cullen et al, 1987) and viral proteins for use as animal vaccines (Kleid et al, 1981). The recovery of these products from overproducing bacteria in large-scale culture is much less problematic and more economic than their recovery from animal tissues. A few well-characterized cell systems can be used for the synthesis of proteins from a wide variety of sources. This minimizes problems associated with fermentation and biomass handling as familiar processing plant and techniques may be used for different products. In addition, proteins produced in nature by pathogenic microorganisms can now be cloned into organisms which are more suitable for industrial use.

The recombinant product can take various forms in the host organism and this can influence the downstream purification process. Many are synthesised in a soluble form in the cell cytoplasm. The crude cell extract which contains these products will also contain all the normal cell components in a complex mixture. As stated above, the purification problems can be minimised if the desired product is the major component in the mixture. In some cases, particularly when proteins are cloned into foreign hosts, or are expressed in high levels, the recombinant product forms an intracellular insoluble aggregate (inclusion bodies) (Kane & Hartley, 1988). This is due to the specific association of partially folded recombinant peptide chains produced during the folding process (Mitraki & King 1989). Inclusion bodies differ from other insoluble cell components in size and density and can therefore be isolated relatively easily by differential centrifugation (Taylor et al 1986). However, the insoluble protein is not biologically active and must be refolded into the correct 3-dimensional conformation. This is achieved by first denaturing the protein in the presence high concentrations of agents such as guanidine HC1 or urea. On removal of the denaturant by, for example, dialysis or size exclusion chromatography, the protein may refold into the active confirmation.

Some genetic manipulation procedures and expression systems, which are currently used on a commercial scale for the production of recombinant proteins, result in the extracellular liberation of the product. The recovery of the product from the growth medium is easier than from cell extracts as it is a less complex mixture which is likely to contain few other proteins in high concentrations. Certain microorganisms have well-developed systems for the transport of proteins across the cell membrane into the external medium. In particular, those species which are capable of hydrolysing extracellular substrates are often capable of secreting large amounts of enzymes such amylases, lipases, cellulases or proteases. Cullen et al (1987) reported a strain of the filamentous fungus Aspergillus niger which could liberate glucoamylase to a concentration of 5 g/1.

Secreted proteins are synthesised as precursors which include an additional series of amino acids on the N-terminal end. This "signal sequence" identifies the protein as a product for export and directs its passage through the cell membrane (Randall et al, 1987). The incorporation of signal sequence code into recombinant protein genes often results in the liberation of the desired product into the growth medium. This strategy has been widely investigated for the production of bovine chymosin by microorganisms. Cullen et al (1987) obtained high levels of chymosin secretion from Aspergillus niger by coupling the structural gene of the enzyme to the transcriptional, translational and secretory control regions of the fungal glucoamylase gene. High secretion levels have also been achieved in the bacterium Proteus mirabilis using the secretion and expression control regions of streptococcal exotoxin (Klessen et al, 1989). Another example of a food-related target for this type of technology is the plant protein thaumatin which is 100 000 times sweeter than sucrose and has potential as a natural sweetener. Illingworth et al (1988) expressed thaumatin in Bacillus subtilis and obtained extracellular liberation using the a-amylase signal peptide. Hahm & Batt (1990) demonstrated that recombinant thaumatin is secreted by Aspergillus oryzae using the native plant signal.

A variation on the theme of extracellular liberation of recombinant proteins to facilitate production and downstream recovery is the expression of the desired product in the milk of transgenic animals. This is achieved by fusing a structural gene to the regulatory regions of a milk protein and inserting the construct into a transgenic host. This approach was developed using the expression of human tissue plasminogen activator in the milk of lactating mice by means of the promoter and regulatory sequences of murine whey acid protein (Gordon et al, 1987). Similar methods have since been investigated for the expression of foreign proteins in the milk of various other animals, including rabbits, sheep and goats. Some examples of this technology are described in Table 2. The majority of applications of this new technology which are currently under investigation concern the production of high-value therapeutic proteins. This may yet be extended to the production of enzymes and proteins for food use when the technology is more mature. Combining dairy animal husbandry with the production of biotechnological proteins for use by the food industry is certainly an attractive idea.

The advances in genetic technology described above demonstrate that expression systems for recombinant proteins can be selected which give the greatest advantage for subsequent product isolation. Genetic manipulation also enables the actual physio-chemical character of the product to be changed in order to facilitate its purification. Just as signal sequences can be incorporated into the product to control secretion from the cell, other sequences of amino acids can be added which confer particular properties which can be used as a "handle" during purification. Sassenfeld & Brewer (1984) and Smith et al (1984) introduced the code for polyarginine into the gene for urogastrone and cloned the construct in E. coli. The expressed protein was therefore urogastrone with a C-terminal polyarginine fusion. The great majority of cell proteins are acidic and are therefore negatively charged at physiological pH. The incorporation of the polyarginine tail made the resulting fusion unusually basic and thus very amenable to purification by ion-exchange chromatography. The tail was then removed by cleavage using a carboxypeptidase. By this method, Sassenfeld & Brewer obtained an urogastrone preparation of purity greater than 95% using a 2-step process. Similar techniques have since been applied to a variety of recombinant proteins. For example, polyaspartic acid fusions have been used to make p-galactosidase (Parker et al, 1990) and glucoamylase (Suominen et al, 1992) more amenable to purification by polyethyleneimine precipitation. Persson et al (1988) added code for 4 cysteine residues to the galactokinase gene and code for 11 phenylalanine residues to the P-galactosidase gene. The recombinant proteins were then expressed in E. coli. In the former case, the fusion sequence increased the binding of galactokinase to thiol groups during covalent chromatography, the protein being recovered after elution with reducing agents. Polyphenylalanine increased the hydrophobicity of P-galactosidase to improve purification of the enzyme by hydrophobic interaction chromatography.

Table 2

The expression of human proteins in the milk of transgenic animals

Table 2

The expression of human proteins in the milk of transgenic animals

Protein

Host organism

Concentration (ilg ml"1)

Reference

t-PA1

Mouse

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