O Manipulation Of Dna Sequence Information

Perhaps the greatest impact of rDNA technology lies in its ability to alter a DNA sequence and create entirely new molecules that, if reintroduced into the genome, can be inherited and propagated in perpetuity. The ability to alter a DNA sequence, literally in a test tube, at the discretion of an individual, corporation, or nation, brings with it important questions about ownership, ethics, and social responsibility. There is no question, however, that potential benefits to the treatment of human disease are great.

There are three principal reasons for using rDNA technology to alter DNA sequences. The first is simply to clone the DNA to facilitate subsequent manipulation. The second is to intentionally introduce mutations so that the site-specific effect on protein structure and function can be studied.93,94 The third reason is to add or remove sequences to obtain some desired attribute in the recombinant protein. For example, recent studies with factor VIII show that the protein contains a small region of amino acids that are the major determinant for the generation of anti-factor VIII antibodies in a human immune system. This autoimmune response of the patient inhibits the activity of factor VIII, which is obviously a serious therapeutic complication for patients who are using factor VIII for the treatment of hemophilia. By altering the DNA sequence encoding, this determinant, however, the amino acid sequence can be changed both to reduce the antigenicity of the factor VIII molecule and to make it transparent to any existing anti-factor VIII antibodies (i.e., changing the epitope eliminates the existing antibody recognition sites).

It is possible to combine elements of two proteins into one new recombinant protein. The resulting protein, referred to as a chimeric or fusion protein, may then have some of the functional properties of both of the original proteins. This is illustrated in Figure 4.8 for two receptors labeled A and B. Each receptor has functional domains that are responsible for ligand binding, integration into the plasma membrane, and activation of intracellular signaling pathways. Using rDNA techniques, one can exchange these functional domains to create chimeric receptors that, for example, contain the ligand-binding domain of receptor B but the transmembrane and intracellular signaling domains of receptor A. The application of the fusion protein strategy is discussed further in connection with the hGH receptor (under "Novel Drug-Screening Strategies") and with denileukin diftitox.

Another reason for combining elements of two proteins into one recombinant protein is to facilitate its expression and purification. For example, recombinant glutathione S-transferase (GST), cloned from the parasitic worm Schistosoma japonicum, is strongly expressed in e. coli and has a binding site for glutathione. Heterologous sequences encoding the functional domains from other proteins can be fused, in frame, to the carboxy terminus of GST, and the resulting fusion protein is often expressed at the same levels as GST itself. In addition, the resulting fusion protein retains the ability to bind glutathione, which means that affinity chromatography, using glutathione that has been covalently bonded to agarose, can be used for a single-step purification of the fusion protein. The functional activity of the heterol-ogous domains that have been fused to GST can then be

Receptor A Receptor B

^Chimeric Receptors Figure 4.8 • Chimeric receptors.

studied either as part of the fusion protein or separately following treatment of the fusion protein with specific proteases that cleave at the junction between GST and the het-erologous domain. Purified fusion proteins can also be used to generate antibodies to the heterologous domains and for other biochemical studies. Sometimes, fusion proteins are made to provide a recombinant protein that can be easily identified. An example of this is a technique called epitope tagging, in which well-characterized antibody recognition sites are fused with recombinant proteins. The resulting recombinant protein can then be identified by immunofluores-cence or can be purified with antibodies that recognize the epitope.

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