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

描述(申请人提供)：高通量分析对于全面的功能蛋白质组研究是必不可少的。包括人类在内的完整基因组的成功完成，再次推动了这些技术的发展。对于实现这一目标来说，非常重要的是开发用于检测和鉴定特定蛋白质的新的蛋白质捕获工具。这些新的捕捉剂应对热和蛋白质降解稳定，具有高亲和力，易于生产，并呈现低交叉反应。为了应对这一挑战，我们建议使用基于细胞的环肽文库来选择针对特定蛋白质靶标的特定环肽序列。环肽是一类新兴的植物来源的主链环化多肽(约28-37个氨基酸)，具有一个不稳定的二硫键(3个二硫键)，具有不寻常的打结结构。它们独特的环状主干拓扑结构和三个二硫键的打结排列使它们对热和酶降解非常稳定。环类化合物具有一系列生物学功能，如子宫紧张性活性、抑制胰酶和神经降压素结合、细胞毒性、抗HIV、抗微生物和杀虫活性。综上所述，这些特性使环肽成为理想的分子支架，用于发现针对特定生物分子靶标的稳定的高亲和力配体，从而取代传统上用作蛋白质捕获试剂的不稳定的基于抗体的支架。我们最近展示了在活的细菌细胞中产生环肽文库的有趣的可能性。可以使用基于细胞的报告器(基于FRET或基于致死性)在细胞内筛选生物产生的文库，以选择能够使用诸如荧光激活细胞分选(FACS)等高通量方法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定的环肽序列固定到适当的固体载体上。特别令人感兴趣的是利用蛋白质反式剪接将这些微量蛋白质固定在固体载体上。这种方法允许从混合物中对特定的蛋白质/多肽(例如线性化的环肽)进行定点和无痕迹的固定化，而不需要纯化或重新浓缩待固定化的配体。更重要的是，这种方法可以与无细胞表达系统对接，从而快速、高通量地制备基于环肽的特定微阵列。 与公共卫生相关：这项建议提出了一种创新的方法，用于筛选和选择一种新型的极其稳定的蛋白质捕捉剂，并结合一种新的多功能配体固定化方法，以快速生产基于环肽的微阵列。我们预计，这些技术将在总体上对科学产生重大影响，但也将为世界各地的个别患者和门诊诊所提供临床蛋白质组学的诊断和预后好处。

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 cyclotide based 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.