Enhancing the Efficiency and Scope of Sortase-Mediated Ligations

 描述(申请人提供)：化学修饰的蛋白质衍生物代表了一类重要的分子，其应用范围从治疗开发到基础生物医学研究。因此，构建修饰蛋白质的技术是非常可取的。近年来，由于酶提供的显著的位点选择性，使用酶来安装非天然蛋白质修饰已经获得了吸引力。在已报道的用于这一目的的酶中，细菌分类酶因其选择性和安装广泛的化学修饰的能力而引起了极大的关注。虽然这项技术前景看好，但由于固有的可逆性，它受到了限制。 化学，以及常用的分类酶不能有效地修饰蛋白质靶标末端以外的位点。绕过这些限制对于进一步改进基于分类酶的技术至关重要。解决这些问题也将代表着我们长期努力的重要一步，即利用索尔塔酶的反应性来构建用于治疗、诊断、生物材料和基础研究应用的Protei衍生物。具体地说，这项建议的目标是开发一种策略，用于1)控制山梨酸酶催化的连接反应的平衡，以及2)通过控制异肽键的形成来靶向内部氨基酸位置。我们将通过两个具体目标来实现这一目标。首先，我们将探索将掩蔽的金属结合肽掺入到索酸酶底物基序中。这将允许通过以下方式控制反应平衡 关键反应副产物的失活。这些研究将包括对这一金属促进策略的严格优化，探索其与全尺寸蛋白质靶标的兼容性，以及它在合成具有潜在用途的独特多肽方面的应用。 作为医用粘合剂。其次，我们将利用自然产生的酸解酶同系物的反应性，作为一种有效地与亲核赖氨酸残基生成异肽键的方法。使用合成肽和重组蛋白靶标的组合，我们将在体外表征来自猪链球菌和口腔链球菌索糖酶同源物选择性产生异肽键的能力。这一提议的创新方面是金属多肽络合物在封闭反应可逆性方面的新应用，以及使用从未在蛋白质修饰化学的背景下研究过的自然产生的排序酶同系物。这项拟议的研究也具有重要意义，因为它将大大提高基于排序酶的策略的效率和范围。总体而言，这些研究将为强有力的新的蛋白质修饰方法提供基础，并将加强我们对山梨酸酶活性及其在蛋白质修饰化学中的实施的基本理解。

 DESCRIPTION (provided by applicant): Chemically modified protein derivatives represent an important class of molecules with applications ranging from therapeutic development to basic biomedical research. Technologies for the construction of modified proteins are therefore highly desirable. The use of enzymes for installing non-natural protein modifications has gained traction in recent years due to the remarkable site-selectivity that enzymes afford. Among enzymes reported for this purpose, bacterial sortases have garnered significant attention because of their selectivity, and their ability to install a wide range of chemical modifications. While promising, this technology is subject to limitations stemming from the inherent reversibility of the chemistry, and the inability of commonly used sortases to efficiently modify sites other than the termini of protein targets. Circumventing these limitations is critical to the further refinement of sortase-based technology. Addressing these issues would also represent an important step in our long-term effort to exploit sortase reactivity for the construction of protei derivatives for a range of therapeutic, diagnostic, biomaterials, and basic research applications. Specifically, the objective of this proposal is to develop strategies for 1) controlling the equilibrium of sortase-catalyzed ligation reactions and for 2) targeting internal amino acid positions through the controlled formation of isopeptide bonds. We will achieve this objective through two specific aims. First, we will explore the incorporation of masked metal binding peptides into sortase substrate motifs. This will allow for control of reaction equilibrium through the deactivation of critical reaction by-products. These studies will include a rigorous optimization of this metal promoted strategy, an exploration of its compatibility with full size protein targets, and its application to the synthesis of unique polypeptides with potential for use as medical adhesives. Second, we will exploit the reactivity of naturally occurring sortase homologs as a means for efficiently generating isopeptide bonds with nucleophilic lysine residues. Using a combination of synthetic peptides and recombinant protein targets, we will characterize the ability of sortase homologs from Streptococcus suis and Streptococcus oralis to selectively generate isopeptide bonds in vitro. The innovative aspects of this proposal are the novel application of metal peptide complexes for blocking reaction reversibility, and the use of naturally occurring sortase homologs that have never been studied in the context of protein modification chemistry. The proposed research is also significant because it will substantially improve the efficiency and scope of sortase-based strategies. Overall, these studies will provide a foundation for powerful new protein modification methods, and will enhance our fundamental understanding of sortase reactivity and its implementation in protein modification chemistry.