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Enhancing the Efficiency and Scope of Sortase-Mediated Ligations

提高分选酶介导的连接的效率和范围

基本信息

批准号：
9098333
负责人：
John M Antos
金额：
$ 35.52万
依托单位：
WESTERN WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-01 至 2020-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9098333
关键词：
Academia Address Adhesives Affinity Amino Acids Antibodies Attention Basic Science Binding Biochemical Reaction Biocompatible Materials Biological Biomedical Research Carbohydrates Chemicals Chemistry Complex Data Development Diagnostic Enzymes Equilibrium Evaluation Foundations Goals Homologous Gene In Situ In Vitro Industry Investigation Ligation Lysine Masks Mediating Medical Medicine Metabolic Diseases Metals Methodology Methods Mind Modification Molecular Structure Nucleic Acids Organic Chemistry Outcome Peptides Polymers Positioning Attribute Post-Translational Protein Processing Process Property Protein Engineering Proteins Proteus Reaction Reagent Recombinant Proteins Recombinants Reporting Research Side Site Streptococcus oralis Streptococcus suis Technology Therapeutic Traction Viral Viral Cancer Virus Diseases Work base combat design functional group human disease improved in vitro activity innovation macromolecule novel novel strategies particle peptide structure polypeptide public health relevance skills sortase stem synthetic peptide therapeutic development tool transacylation

项目摘要

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.

描述（由申请人提供）：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，非常需要构建修饰蛋白质的技术。近年来，由于酶具有显着的位点选择性，使用酶来安装非天然蛋白质修饰已受到关注。在为此目的报道的酶中，细菌分选酶因其选择性以及安装广泛化学修饰的能力而引起了广泛关注。虽然该技术前景广阔，但由于其固有的可逆性而受到限制化学性质的问题，以及常用分选酶无法有效修饰蛋白质靶标末端以外的位点。规避这些限制对于进一步完善基于分选酶的技术至关重要。解决这些问题也将代表我们长期努力利用分选酶反应性构建蛋白质衍生物以用于一系列治疗、诊断、生物材料和基础研究应用的重要一步。具体来说，该提案的目标是制定以下策略：1) 控制分选酶催化的连接反应的平衡；2) 通过受控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，我们将探索将掩蔽金属结合肽掺入分选酶底物基序中。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种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，以及其在合成具有使用潜力的独特多肽中的应用作为医用粘合剂。其次，我们将利用天然存在的分选酶同源物的反应性作为有效生成与亲核赖氨酸残基的异肽键的手段。使用合成肽和重组蛋白靶标的组合，我们将表征猪链球菌和口腔链球菌的分选酶同源物在体外选择性生成异肽键的能力。该提案的创新之处在于金属肽复合物在阻断反应可逆性方面的新颖应用，以及使用从未在蛋白质修饰化学背景下研究过的天然存在的分选酶同系物。拟议的研究也很重要，因为它将大大提高基于分选酶的策略的效率和范围。总的来说，这些研究将为强大的新蛋白质修饰方法奠定基础，并将增强我们对分选酶反应性及其在蛋白质修饰化学中的实施的基本理解。