Cell-based screening and selection of cyclotide-based capture reagents for protei

基于细胞的蛋白质筛选和基于环肽的捕获试剂的选择

• 批准号: 7938817
• 负责人: Julio A Camarero
• 金额: $ 40.1万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7938817
• 关键词: Affinity; Ambulatory Care Facilities; Amino Acids; Antibodies; Binding; Biological Process; Cells; Characteristics; Clinical; Detection; Development; Diagnostic; Family; Fluorescence Resonance Energy Transfer; Fluorescence-Activated Cell Sorting; Genome; Goals; Human; Immobilization; Individual; Libraries; Life; Ligands; Methods; Molecular; Neurotensin; Patients; Plants; Production; Proteins; Proteome; Proteomics; Proteus; Reagent; Reporter; Research; Science; Screening procedure; Site; Solid; Structure; System; Technology; Trans-Splicing; Trypsin; Vertebral column; antimicrobial; base; cross reactivity; cytotoxicity; disulfide bond; high throughput screening; innovation; interest; polypeptide; prognostic; public health relevance; response; scaffold; technique development; tool

DESCRIPTION (provided by applicant): High-throughput assays are indispensable for comprehensive functional proteome research. The development of these techniques has been promoted again by the successful completion of complete genomes, including the human. Of great importance for achieving this goal is the development of new protein capture tools for the detection and identification of specific proteins. These new set of capture reagents should be stable to thermal and proteolytic degradation, have high affinity, easy to produce and present low cross-reactivity. In response to this challenge, we propose the use of cell-based libraries of cyclotides for selecting specific cyclotide sequences against particular protein targets. Cyclotides are a new emerging family of plant-derived backbone-cyclized polypeptides (~28-37 amino acids long) that share a disulfidestabilized core (3 disulfide bonds) characterized by an unusual knotted structure. Their unique circular backbone topology and knotted arrangement of three disulfide bonds makes them exceptionally stable to thermal and enzymatic degradation. Cyclotides have been associated with a range of biological functions such as uterotonic activity, inhibition of trypsin and neurotensin binding, cytotoxicity, anti-HIV, antimicrobial, and insecticidal activity. Together, these characteristics make cyclotides ideal candidates to be used as molecular scaffolds for the discovery of stable high affinity ligands against particular biomolecular targets thus replacing the less stable antibody-based scaffold, which have been traditionally used as the protein capture reagent of choice. We have recently demonstrated the intriguing possibility of generating libraries of cyclotides inside living bacterial cells. Biologically-generated libraries can be screened inside the cell using cell-based reporters (both based on FRET or lethality based) for the selection of particular cyclotide sequences able to bind a particular protein target using high throughput methods such as fluorescence-activated cell Sorting (FACS). Selected cyclotide sequences can be then immobilized using a micro-array format onto appropriate solid supports. Of particular interest is the use of protein trans-splicing for the immobilization of these micro-proteins onto solid supports. This approach allows the site-specific and traceless immobilization of particular proteins/polypeptides (for example linearized cyclotides) from mixtures without any need to purify or re-concentrate the ligand to be immobilized. More importantly, this approach can be interfaced with cell-free expression systems for the rapid and high throughput of specific cyclotidebased microarrays. PUBLIC HEALTH RELEVANCE:: This proposal presents an innovative approach for the screening and selection on a new type of extremely stable protein capture reagents in combination with a new versatile way of ligand immobilization for the rapid production of cyclotide-based micro-arrays. We anticipate that these technologies will have a large impact in science in general but will also provide the diagnostic and prognostic benefits of clinical proteomics to individual patients and outpatient clinics around the world.

描述（由申请人提供）：高通量测定对于全面的功能蛋白质组研究是不可或缺的。包括人类在内的完整基因组的成功完成再次促进了这些技术的发展。实现这一目标的重要性在于开发新的蛋白质捕获工具，用于检测和鉴定特定蛋白质。这些新的捕获试剂组应该对热和蛋白水解降解稳定，具有高亲和力，易于生产并呈现低交叉反应性。为了应对这一挑战，我们建议使用基于细胞的cyclotides库来选择针对特定蛋白质靶标的特定cyclotide序列。环肽是一个新出现的植物源性骨架环化多肽家族（约28-37个氨基酸长），其具有以不寻常的打结结构为特征的二硫去稳定化核心（3个二硫键）。其独特的环状骨架拓扑结构和三个二硫键的打结排列使其对热和酶降解非常稳定。环肽类化合物具有一系列生物学功能，如子宫收缩活性、抑制胰蛋白酶和神经降压素结合、细胞毒性、抗HIV、抗微生物和杀虫活性。总之，这些特征使得环肽成为理想的候选物，用作分子支架，用于发现针对特定生物分子靶标的稳定的高亲和力配体，从而取代不太稳定的基于抗体的支架，其传统上用作蛋白质捕获试剂的选择。我们最近已经证明了在活细菌细胞内产生环肽文库的有趣可能性。可以使用基于细胞的报道分子（两者都基于FRET或基于致死性）在细胞内筛选生物学产生的文库，以使用高通量方法如荧光激活细胞分选（FACS）选择能够结合特定蛋白质靶标的特定环肽序列。然后可以使用微阵列形式将选择的环肽序列固定到适当的固体支持物上。特别感兴趣的是使用蛋白质反式剪接将这些微蛋白固定到固体支持物上。该方法允许从混合物中位点特异性和无痕迹地固定特定蛋白质/多肽（例如线性化的环肽），而不需要纯化或再浓缩待固定的配体。更重要的是，这种方法可以与无细胞表达系统接口，用于快速和高通量的特定的基于环肽的微阵列。 公共卫生相关性：该提案提出了一种创新的方法，用于筛选和选择一种新型的非常稳定的蛋白质捕获试剂，并结合一种新的通用的配体固定方法，用于快速生产基于环肽的微阵列。我们预计，这些技术将对科学产生巨大影响，但也将为世界各地的个体患者和门诊诊所提供临床蛋白质组学的诊断和预后益处。