The techniques provided by modern molecular biology have made it possible to rapidly clone and sequence new proteins at a rate that has rapidly outpaced our ability to perform structure-activity analysis using conventional methods. Some of these proteins have significant therapeutic potential. Interleukin-2 has been a prototypical example. Although it has been only five years since the CDNA sequence of IL-2 was reported, recombinant IL-2 is already being used clinically in cancer and AIDS trials and is under investigition as an adjuvant and linked to diptheria toxin, as an immunosuppressant. Although structure-function analysis of IL-2 has been attempted with a multitude of modern techniques, the specific receptor binding region remain unidentified. To achieve the long term goals of the production of IL-2 agonists and antagonists and the elucidation of the novel IL-2 receptor system, we have pursued a rational protein design strategy. Based on a computer generated model and working in a semi-synthetic system, we have begun to utilize design principles successfully employed by Kaiser and others in small peptide systems for the analysis and design of a real protein.
The specific aims of this investigation are: 1) to explore the critical biophysical parameters governing stability of secondary and tertiary conformation of IL-2; 2) to alter the binding conformation of IL-2 by manipulating these interactions thus generating agonists and antogonists an 3) to utilize these mutants to elucidate the novel IL-2-multi component receptor interaction. To accomplish these goals, cassette, multiple cassette and site directed mutagenesis will be employed on a custom synthetic IL-2 gene. The mutants will be isolated, refolded and purified. They will be characterized biochemically to ascertain purity and confirm the presence of the desired mutations biochemically to monitor conformation, stability and folding characteristics and biologically to quantify bioactivity and binding to each of the receptor proteins on cell lines and normal T cells. This study will not only generate agonists and antagonists for IL-2 based on protein design principles, but should also contribute to the understanding of folding for this class of proteins and provide fundamental knowledge concerning ligandreceptor interactions within the immune system.
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