Insertion of a Protease-binging Loop into Interleukin-1B

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One of the central problems in protein chemistry is to discover what determines three-dimensional structure. Proteolytic enzymes are involved in regulation of a great number of physiological processes. In some cases, proteases cleave a fragment from an inactive precursor in order to convert it to an active protein; in other cases, proteolysis ends a cycle of enzymatic activity. Proteases may also act to remove damaged tissue, as part of the repair process. It is of interest to design new inhibitors for proteases, both with an eye to possible therapeutic uses of these inhibitors, and also with the aim of studying the interaction between protease and inhibitor. Many small organic molecules have been shown to be effective inhibitors of serine proteases in vitro, but they do not mimic the fit between the natural inhibitor and its target protein. Rather, they mimic the fit between substrate and protease. This project will entail inserting a sequence from antitrypsin into human interleukin so that it will inhibit elastase from human leukocytes. Unique restriction sites in the synthetic gene being used allow the gene to be cut in the region to be mutated, and the appropriate DNA to be ligated. Expression of the mutated gene will be followed by purification of the desired protein. The new protein will be tested for binding to elastase and for elastase-inhibitory activity and will be examined by X-ray crystallography. This project should help to answer the question of whether the three- dimensional structure is determined purely by the sequence of amino acids, or whether the rest of the protein plays a role in stabilizing, or even determining the characteristics of the loop.