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

描述（由申请人提供）：分选酶是一种细菌酶，能够使用多种供体和受体分子进行蛋白质转酰基化。SrtA类分选酶识别适当工程蛋白底物或合成肽序列中的LPXTG序列，并在Thr和Gly残基之间切割，同时形成酰基酶中间体。这种中间体然后被亲核攻击分解，使用合成的伯胺或适当配备Gly或Ala作为N端的蛋白质。利用分选酶作为工具，将开发新的蛋白质标记策略，使通过遗传手段无法实现的蛋白质修饰成为可能，例如两种不同多肽的c端到c端融合。这些方法将用于合成具有免疫学意义的重组蛋白，如II类MHC产物、T细胞受体外结构域和抗体F（ab）片段，目的是创建这些蛋白的标记版本，可用于检测和分离其相关反结构，或枚举承担其受体的细胞。II类MHC四聚体的生产仍然很麻烦，所提出的方法有可能极大地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。可以修饰的底物范围将通过正交标记策略的发展而扩大，该策略采用不同特异性的排序酶，无论是在其肽识别序列中还是在其接受某些类型亲核试剂的能力中。这不仅可以通过对金黄色葡萄球菌和化脓性链球菌的Srt A酶的定点诱变来实现，还可以通过使用其他类别的分选酶（例如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颗粒生物发生的问题上，这是一个迄今为止无法实时观察的过程，但可以使用这里提出的标记策略来可视化，作为识别可能作为干预目标的离散步骤的可能手段。提出的研究的意义在于开发方法，这些方法将使不能通过遗传安装的实体对蛋白质进行位点特异性修饰。

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.