Facile Generation of Protein-Protein Conjugates Using Enzymatic Oxidative Coupling Reactions

项目总结/摘要 嵌合蛋白质-蛋白质缀合物为免疫治疗提供了多种成功的平台， 靶向药物递送、细胞生物学研究和疫苗开发。然而，许多理想的结构 不能单独使用遗传方法产生，并且使用化学方法将两种蛋白质靶向偶联， 方法仍然非常具有挑战性。在该计划中，将探索一种新的方法，用于快速和现场- 使用天然氨基酸的蛋白质的特异性偶联。酪氨酸酶将用于氧化溶剂- 蛋白质和肽底物上暴露的酪氨酸残基，产生邻醌， 策略性地放置在其他蛋白质中的半胱氨酸残基。初步数据证实，这种方法 可以在室温下在1小时内从单个蛋白质生成复杂的多功能构建体 尽管在这些反应中固有高度的空间相互作用。酪氨酸酶是 便宜，可以固定在固体支持物上以便于去除，并且仅需要外源氧 来运作。从蛋白质的N-或C-末端位置延伸的酪氨酸残基容易被氧化， 但内部酪氨酸残基在反应期间不受影响。半胱氨酸残基可以放在任何地方 在第二蛋白质靶的表面上。由于这些特征，该方法在其简单性方面独树一帜 和灵活性，使复杂的，多结构域的建设，因此，它将大大扩大范围， 生物缀合物可以用于生物技术应用。 拟议研究的第一个具体目标将探索细胞穿透肽的附着 scFv构建体作为有用的模型蛋白，目的是增加它们穿过细胞膜的能力 进入细胞质半胱氨酸将被引入到scFv表面上的几个位置，并且细胞 使用酪氨酸酶方法连接带有暴露的酪氨酸残基的进入肽。的摄取和 这些构建体的细胞内定位将使用96孔板测定、光学显微镜和 流式细胞仪 具体目标2将探索使用这种方法生产多功能蛋白质， 免疫疗法的应用。酪氨酸标签将被引入一组生物分子的关键位置， 包括scFv结构域和IgG抗体。酪氨酸酶活化后，药物货物分子 将评估与这些位点偶联的不对称性，并且蛋白质-蛋白质偶联化学的不对称性 将被探索用于生产双特异性和三特异性细胞增殖物。这一目标的成功将 提供了一种高效的方法来制备和筛选这些有治疗价值的构建体。 第三个具体目标将集中在确定新的酪氨酸酶具有理想的性质， 对这些酶进行工程改造以实现不同的含酪氨酸序列的活化。

PROJECT SUMMARY/ABSTRACT Chimeric protein-protein conjugates provide a wide variety of successful platforms for immunotherapy, targeted drug delivery, cell biology studies, and vaccine development. However, many desirable constructs cannot be produced using genetic methods alone, and the targeted coupling of two proteins using chemical methods is still very challenging. In this program, a new approach will be explored for the rapid and site- specific coupling of proteins using native amino acids. Tyrosinase enzymes will be used to oxidize solvent- exposed tyrosine residues on protein and peptide substrates to generate ortho-quinones that react rapidly with strategically placed cysteine residues in other proteins. Preliminary data have confirmed that this approach can generate complex, multifunctional constructs from individual proteins in under 1 h at room temperature despite the high degree of steric interactions that are inherent in these reactions. The tyrosinase enzymes are inexpensive, can be immobilized on solid supports to facilitate removal, and require only adventitious oxygen to function. Tyrosine residues that extend from the N- or C-terminal positions on proteins are oxidized readily, but internal tyrosine residues are unaffected during the reactions. The cysteine residues can be placed anywhere on the surface of the second protein target. Due to these features, this method stands alone in its simplicity and flexibility for making complex, multidomain constructs, and thus it will greatly expand the range of bioconjugates that can be accessed for biotechnology applications. The first Specific Aim of the proposed research will explore the attachment of cell penetrating peptides to scFv constructs as useful model proteins, with the goal of increasing their ability to cross cell membranes and access the cytoplasm. Cysteines will be introduced into several locations on the scFv surfaces, and cell entry peptides bearing exposed tyrosine residues will be attached using the tyrosinase method. The uptake and intracellular localization of these constructs will be evaluated using 96-well plate assays, light microscopy and flow cytometry. Specific Aim 2 will explore the use of this approach for producing multifunctional proteins for immunotherapy applications. Tyrosine tags will be introduced in strategic locations on a panel of biomolecules, including scFv domains and IgG antibodies. Following tyrosinase activation, the ability of drug cargo molecules to couple to these sites will be evaluated, and the asymmetric nature of the protein-protein coupling chemistry will be explored for the production of bispecific and trispecific cell engagers. The success of this Aim would provide a highly efficient way to make and screen these therapeutically valuable constructs. The third Specific Aim will focus on identifying new tyrosinase enzymes with desirable properties, and the engineering of these enzymes to achieve the activation of different tyrosine-containing sequences.