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

描述（由申请人提供）：高通量分析对于全面的功能蛋白质组研究是必不可少的。包括人类在内的全基因组的成功完成再次促进了这些技术的发展。为了实现这一目标，开发新的蛋白质捕获工具来检测和鉴定特定蛋白质是非常重要的。这些新型捕获试剂应具有耐热性和蛋白水解降解稳定性好、亲和力高、易于生产、交叉反应性低等特点。为了应对这一挑战，我们建议使用基于细胞的环核苷酸文库来选择针对特定蛋白质靶点的特定环核苷酸序列。环肽是一类新兴的植物衍生的骨架环化多肽（约28-37个氨基酸长），具有一个不寻常的结状结构，具有二硫不稳定的核心（3个二硫键）。它们独特的圆形骨架拓扑结构和三个二硫键的打结排列使它们在热降解和酶降解方面异常稳定。环肽具有一系列生物学功能，如子宫扩张活性、抑制胰蛋白酶和神经紧张素的结合、细胞毒性、抗hiv、抗菌和杀虫活性。综上所述，这些特性使环核苷酸成为分子支架的理想候选者，用于发现针对特定生物分子靶标的稳定高亲和力配体，从而取代传统上用作蛋白质捕获试剂的不太稳定的基于抗体的支架。我们最近展示了在活细菌细胞内产生环核苷酸文库的有趣可能性。生物生成的文库可以在细胞内使用基于细胞的报告器（基于FRET或基于致死性）进行筛选，以便使用高通量方法（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核苷酸序列。然后可以使用微阵列格式将选定的环核苷酸序列固定在适当的固体支架上。特别令人感兴趣的是使用蛋白质反式剪接将这些微蛋白质固定在固体载体上。这种方法允许从混合物中固定特定的蛋白质/多肽（例如线性化的环肽），而不需要纯化或重新浓缩要固定的配体。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环肽基微阵列。

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.