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

描述（由申请人提供）：分选酶是能够使用多种供体和受体分子进行蛋白质转酰化的细菌酶。SrtA类分选酶识别适当工程化的蛋白质底物或合成肽序列中的LPXTG序列，其在Thr和Gly残基之间切割，同时形成酰基酶中间体。然后使用合成的伯胺或适当地配备有Gly或Ala作为N-末端的蛋白质，通过亲核攻击来拆分该中间体。使用分选酶作为工具，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，如C-末端到C-末端融合的两个不同的多肽。这些方法将应用于合成免疫学感兴趣的重组蛋白质，例如II类MHC产物、T细胞受体胞外域和抗体F（ab）片段，目的是产生这些蛋白质的标记形式，其可用于检测和分离其相关的对应结构，或计数携带其受体的细胞。II类MHC四聚体的生产仍然很麻烦，所提出的方法有可能大大简化这些用于跟踪致病性和保护性T细胞反应的关键诊断工具的生产。可以被修饰的底物的范围将通过开发正交标记策略来扩展，所述正交标记策略采用不同特异性的分选酶，所述特异性在其肽识别序列中或在其接受某些类型的亲核试剂的能力中。这将不仅通过金黄色葡萄球菌和化脓性链球菌的SrtA酶的定点诱变来实现，而且通过使用其它类型的分选酶（例如来自S.金黄色葡萄球菌或B。炭疽菌）。最后，我们将把这项技术应用于流感病毒粒子的生物生成问题，这是一个迄今为止无法在真实的时间内观察到的过程，但可以使用这里提出的标记策略作为一种可能的手段来识别可能作为干预目标的离散步骤。拟议研究的意义在于开发方法，使蛋白质的位点特异性修饰具有不能遗传安装的实体。 公共卫生相关性：将开发新的化学酶方法，以促进诊断工具的产生，这些诊断工具可用于跟踪保护免受感染因子以及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如何组装和从感染细胞释放。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.