Sortase-mediated protein engineering for the study of host-pathogen interactions

描述(由申请人提供)：索尔特酶是一种细菌酶，能够利用各种供体和受体分子进行蛋白质转酰化。SrtA类索酸酶在适当工程的蛋白质底物或合成肽序列中识别LPXTG序列，它在苏氨酸和甘氨酸残基之间切割，伴随着酰基酶中间体的形成。这种中间体然后通过亲核攻击来分解，使用合成的伯胺或适当配备甘氨酸或丙氨酸的蛋白质作为N-末端。利用分类酶作为工具，将开发新的蛋白质标记策略，使蛋白质修饰不能通过遗传手段实现，例如两个不同的多肽的C末端到C末端的融合。这些方法将应用于合成具有免疫学意义的重组蛋白，如II类MHC产物、T细胞受体胞外区和抗体F(Ab)片段，以期创建这些蛋白的标记版本，用于检测和分离其相关的对应物，或计数携带它们的受体的细胞。第二类MHC四聚体的生产仍然很繁琐，拟议的方法有可能极大地简化这些用于跟踪致病和保护性T细胞反应的关键诊断工具的生产。可以修饰的底物的范围将通过开发使用不同特异性的分类酶的正交标记策略来扩大，无论是在它们的肽识别序列中还是在它们接受某些类型的亲核试剂的能力上。这不仅将通过对金黄色葡萄球菌和化脓性链球菌的SRT A酶进行定点突变来实现，还将通过使用其他类别的分类酶(例如来自金黄色葡萄球菌或炭疽杆菌的SrtB)来实现。最后，我们将把这项技术应用到流感粒子生物发生的问题上，这一过程到目前为止还无法实时观察，但可以使用这里提出的标记策略来可视化，作为一种可能的手段，以确定可能作为干预目标的离散步骤。拟议研究的意义在于开发了一种方法，能够对不能以遗传方式安装的实体的蛋白质进行特定部位的修饰。 与公共卫生的相关性：将开发新的化学酶方法，以促进诊断工具的生成，这些工具可用于跟踪免疫反应，以防止感染性病原体和引起自身免疫的免疫反应。类似的蛋白质修饰策略将被应用于研究流感病毒颗粒是如何组装并从受感染细胞中释放出来的。提出的化学和生物学的结合将为疾病的诊断和治疗带来新的可能性。

DESCRIPTION (provided by applicant): Sortases are bacterial enzymes capable of protein transacylation, using a wide variety of donor and acceptor molecules. The SrtA class of sortase recognizes an LPXTG sequence in a suitably engineered protein substrate or in a synthetic peptide sequence, which it cleaves between the Thr and Gly residues with concomitant formation of an acyl-enzyme intermediate. This intermediate is then resolved by nucleophilic attack, using synthetic primary amines or proteins suitably equipped with Gly or Ala as the N- terminus. Using sortases as tools, new protein labeling strategies will be developed that enable protein modifications not attainable by genetic means, such as the C-terminus to C-terminus fusion of two distinct polypeptides. These methods will be applied to the synthesis of recombinant proteins of immunological interest such as Class II MHC products, T cell receptor ectodomains and antibody F(ab) fragments, with a view to create labeled versions of these proteins that can be used for detection and isolation of their relevant counterstructures, or to enumerate the cells that bear receptors for them. The production of Class II MHC tetramers remains cumbersome, and the proposed methods have the potential of dramatically simplifying the production of these key diagnostic tools used to track pathogenic and protective T cell responses alike. The range of substrates that can be modified will be extended through the development of orthogonal labeling strategies that employ sortases of different specificities, either in their peptide recognition sequence or in their ability to accept certain types of nucleophile. This will be accomplished not only through site-directed mutagenesis of the Srt A enzymes of Staphylococcus aureus and Streptococcus pyogenes, but also through the use of other classes of sortases (e.g. SrtB from S. aureus or B. anthracis). Finally, we shall apply this technology to the question of flu particle biogenesis, a process that has so far defied observation in real time, but that may be visualized using the labeling strategies proposed here as a possible means to identify discrete steps that might serve as targets for intervention. The significance of the proposed studies lies in the development of methods that will enable the site-specific modification of proteins with entities that cannot be installed genetically. PUBLIC HEALTH RELEVANCE: New chemoenzymatic methods will be developed to facilitate the generation of diagnostic tools that can be used to track immune responses that protect against infectious agents as well as those that cause autoimmunity. Similar protein modification strategies will be applied to study how flu virus particles are assembled and released from the infected cell. The proposed combination of chemistry and biology will generate new possibilities for the diagnosis and treatment of disease.