Recombinant DNA Approaches

Development of Proteinaceous Drug Targeting Constructs Using Chemical and Recombinant DNA Approaches... 

Figure 12.2 Recombinant DNA approaches for gene multimerization (a) and cloning steps involved in rPBPs expression (b). RE represents the restriction enzyme site for compatible sticky-ends. (Adapted with permission from [76], Copyright 2011, American Chemical Society.)...

Whole cells are grown for a variety of reasons. The cells may perform a desired transformation of the substrate, e.g., wastewater treatment the cells themselves may be the desired produce, e.g., yeast production or the cells may produce a desired product, e.g., penicillin. In the later case, the desired product may be excreted, as for the penicillin example, and recovered in relatively simple fashion. If the desired product is retained within the cell walls, it is necessary to lyse (rupture) the cells and recover the product from a complex mixture of cellular proteins. This approach is often needed for therapeutic proteins that are created by recombinant DNA technology. The resulting separation problem is one of the more challenging aspects of biochemical engineering. However, culture of the cells can be quite difficult experimentally and is even more demanding theoretically. 

Experimental approaches that have afforded major insights to the processes described in this chapter include (1) use of yeast mutants (2) application of recombinant DNA techniques (eg, mutating or eliminating particular sequences in proteins, or fusing new sequences onto them and (3) development of in vitro... 

In approaching the study of the molecular mechanisms of heredity, this chapter first discusses the structural and functional roles of the genetic material, DNA. This includes an analysis of its replication and susceptibility to mutation. The health-related aspects of the use of recombinant DNA techniques are considered, and examples of then-use in the analysis of several human genetic diseases are used to illustrate the biochemical side of genetics. 

Before the era of artificially recombinant DNA, affinity chromatography emerged as a potentially highly selective approach to protein purification [13]. It revived a laborious art based on selective precipitations and a limited range of chromatographic media. 

Another potential class of antivirals is those that interfere with the ability of virus to enter cells. If the virus entry process is inhibited, then spread of infection within an individual might be inhibited. As discussed earlier, HIV virus particles initially attach to cells by way of the cellular receptor for CD4 protein, which is embedded in the surface of normal T lymphocytes and macrophages. Recently, recombinant DNA techniques have been used to make large amounts of a part of the pure CD4 protein. Test-tube experiments have shown that if this CD4 protein fragment is incubated with T lymphocytes or macrophages, it can saturate all the CD4 receptors and prevent subsequent infection with HIV. It is possible that this approach might be effective in people, as well. 

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