Fundamentals of Biochemical Engineering Part 1 - Bailey J., Ollis D. 1989

Molecular Genetics and Regulatory Systems
Modification of Cellular DNA Structure
Cell Fusion

Profound genetic modifications, including the crossover of genetic material between different species, can be achieved through the fusion of Cells of various types. As shown in the diagram of microbial Cell fusion (Fig. 6.17), the initial stage of this complex process is the preparation of protoplasts—cells stripped of their outer walls and enclosed solely by Plasma Membranes. Various Enzymes (Cell wall Hydrolases) are employed for this purpose, and the Hydrolysis is carried out in a strictly hypotonic medium to minimize protoplast lysis caused by osmotic pressure. The incubation of protoplasts leads to the regeneration of cell walls and the restoration of normal cellular Morphology. This factor is critical for the entire process because, ultimately, the fusion of protoplasts from different strains is intended to yield an Organism that can be subsequently cultivated under standard conditions, including large-scale industrial production. Protoplast fusion is usually performed in a polyethylene glycol (PEG) solution, which induces the process, while Selection Methods similar to those used in traditional microbial genetics are applied to isolate the fusion products (Table 6.3).

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FIG. 6.16. Genetic Map of E. coli K12, showing the relative positions of various genes on the circular chromosome. [Reprinted from: Taylor A. L., Trotter С. D., Revised Linkage Map of E. coli, Bacteriol. Rev., 31, 332 (1967).]

FIG. 6.17. Main stages in obtaining and isolating new strains via the fusion of protoplasts from two different parent strains. [Reprinted from: Peberdy J. F., Protoplast Fusion — A Tool for Genetic Manipulation and Breeding in Industrial Microorganisms, Enzyme Microb. Technol., 2, 25 (1980).]

Hybrid cells of various Penicillium, Aspergillus, Streptomyces, and Cephalosporium strains, as well as Yeasts and Bacteria, have been successfully generated using this approach. Interspecific hybrids have been obtained, notably, between various Penicillium and Aspergillus species, between Saccharomyces cerevisiae and Saccharomyces diastaticus, and between Candida tropicalis and Saccharomyces fibuligera. Hybrids of significantly divergent species tend to be insufficiently stable and often revert to one of the parental species after a few generations; nevertheless, overall, protoplast fusion represents a highly compelling method for the substantial restructuring of cellular genetic material in a single step. Currently, researchers are exploring the possibility of fusing protoplasts from fast-growing Streptomyces strains with mutant, highly productive, yet slow-growing strains.

The Cell fusion technique can also be applied to produce hybrids of plant or animal cells. A prime example is hybridomas, which are already of great practical significance and immense potential; they are formed by fusing antibody-producing leukocytes with mouse or other animal myeloma (Skin Cancer) cells. Each hybridoma cell synthesizes only a single type of antibody; therefore, a cell culture derived from a single hybridoma produces Monoclonal Antibodies. Conversely, when antibodies are obtained by introducing a protein or other antigen into an organism, such as a rabbit, that organism simultaneously synthesizes a multitude of antibodies specific to various Regions of the antigen molecule. Monoclonal antibodies already serve as an essential research tool and form The basis of numerous established and developing disease diagnostic methods. In an immobilized form, monoclonal antibodies function as highly specific affinity adsorbents suitable for the Isolation and Purification of Antigens on both laboratory and industrial scales. Looking forward, monoclonal antibodies may prove to be invaluable agents for tumor imaging and targeted drug delivery to specific cell types within the body.

Small quantities of monoclonal antibodies are conveniently obtained by inoculating hybridoma cells into the peritoneal cavity of an immunologically compatible mouse. The developing tumor releases monoclonal antibodies into the surrounding Body Fluids, where antibody concentrations reach 1–20 mg/mL. Hybridomas can also be cultivated in vitro. Currently, considerable attention is being devoted to the design and Structure/175.html">Implementation of novel bioreactor configurations that enhance cell density and antibody productivity, as well as to finding ways to reduce the cost of large-scale monoclonal antibody production.