In order to develop reagents for the systematic structure function analysis of IL-7, as well as the genetic construction of the IL-7R targeted fusion toxin DAB389 IL-7, we have designed and synthesized a gene encoding IL-7 using Escherichia coli condon usage bias. The synthetic IL-7 gene has been cloned in an expression vector and biologically active IL-7 has been expressed. The long term goals of this segment of the NCDDG application are to use an iterative process to develop molecular models of the tertiary structure of Il-7. We have used computational analysis of the secondary structure of Il-7, and then have used this information with the X-ray crystallographic data on two related cytokines (IL-4 and G-CSF) in order to construct molecular models of Il-7. Since these models differ considerably, we have been able to make structural predictions for each model. The determination of the disulfide bond assignment in recombinant Il-7 will allow us to pick one of these models for further structural refinement. We shall conduct site-directed mutagenesis studies in order to resolve those regions of Il-7 that re involved with receptor binding and signal transduction. We anticipate the isolation nd characterization of mutant forms of Il-7 that will be receptor antagonists, and further anticipate that these mutants will serve as candidate compounds for the treatment of many of the acute leukemias. In addition to the structure function analysis of native IL-7, we have also designed, constructed, expressed and purified, a novel fusion toxin (DAB389IL-7) that is targeted toward the IL-7 receptor. This fusion toxin is highly receptor specific and potent and will serve as the prototype of an IL-7R targeted cytotoxic agent.
The specific aims of this component of the NCDDG are to produce these and other mutant forms of Il-7 and DAB389IL-7 for experimental use in experiments proposed in the subsequent sections of this application by Drs. Foss, Rich, and Waters.
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