SAR OF INTERLEUKIN 2 AND IL-2 RELATED PEPTIDES

现代分子生物学提供的技术使之成为可能 以快速克隆和测序新蛋白质的速度， 超越了我们的能力进行结构活性分析， 常规方法。其中一些蛋白质具有显著的治疗作用， 潜力白细胞介素-2就是一个典型的例子。虽然 自IL-2的cDNA序列被报道以来只有五年， 重组IL-2已经在临床上用于癌症和艾滋病试验 作为一种佐剂，与白喉毒素有关， 免疫抑制剂 尽管已经尝试了IL-2的结构-功能分析， 许多现代技术，特异性受体结合区仍然存在， 身份不明为了实现生产IL-2的长期目标， 激动剂和拮抗剂以及新IL-2受体的阐明 系统，我们追求了合理的蛋白质设计策略。基于 计算机生成的模型和工作在一个半合成系统，我们有 开始利用凯撒成功采用的设计原则， 其他人在小肽系统的分析和设计一个真实的 蛋白 本研究的具体目的是：（1）探索关键的 控制第二和第三系稳定性的生物物理参数 IL-2的结合构象; 2）通过改变IL-2的结合构象， 操纵这些相互作用，从而产生激动剂和拮抗剂， 3)利用这些突变体来阐明新的IL-2多组分 受体相互作用 为了实现这些目标，卡匣、多个卡匣和位点定向 诱变将被用于定制的合成 IL-2基因。 的 突变体将被分离、重折叠和纯化。 他们将 进行生物化学表征，以确定纯度并确认 所需突变的生物化学监测构象，稳定性 和折叠特性，并在生物学上量化生物活性， 与细胞系和正常T细胞上的每种受体蛋白结合， 细胞 本研究不仅将产生基于IL-2的激动剂和拮抗剂， 蛋白质设计原则，但也应该有助于 了解这类蛋白质的折叠，并提供 关于配体-受体相互作用的基本知识 免疫系统

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.