A new strategy to disrupt protein-protein interactions in eukaryotic cells.

破坏真核细胞中蛋白质-蛋白质相互作用的新策略。

• 批准号: 8351788
• 负责人: Hidde L. Ploegh
• 金额: $ 97.5万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8351788
• 关键词: Affinity; Affinity Chromatography; Alpaca; Antigen Targeting; Antigens; Bacteriophages; Binding Sites; Biotin; Cellular biology; Chemicals; Cleaved cell; Cytoplasmic Protein; Elements; Enzymes; Eukaryota; Eukaryotic Cell; Laboratories; Libraries; MHC Class II Genes; Mus; Nuclear Pore; Organism; Phage Display; Post-Translational Protein Processing; Production; Proteomics; Retrieval; Running; Site; Splenocyte; Work; Yeasts; abstracting; disulfide bond; expression vector; fluorophore; glycosylation; interest; liquid chromatography mass spectrometry; protein protein interaction; sortase; synthetic peptide; threonyl-glycine

DESCRIPTION Abstract: Vhh's require neither disulfide bonds nor glycosylation for stability; they are remarkably thermostable as well. By immunizing an alpaca with cytoplasmic proteins from the organism of interest, a representative set of such Vhh's will be generated and isolated by means of selective amplification of the Vhh sequences present by PCR, followed by expression of the Vhh's as a library in phage display mode. At the C-terminus of each Vhh, the phage expression vectors carry a recognition motif for sortase, a bacterial enzyme that allows quantitative and site-specifi modification of proteins of interest, including Vhh's. Sortases recognize an LPXTG motif, and cleave between the Thr and Gly residues with concomitant formation of a thioacyl intermediate. Short synthetic peptides, modified according to need with fluorophores, biotin or combinations of thereof, resolve this thioacyl intermediate and allow covalent, near-quantitative and site-specific installation of such probes at the site of sortase cleavage, without inflicting chemical damage on the Vhh antigen combining site. This allows the rapid production of Vhh's that are affinity tagged with biotin. They can be used directly for retrieval of the target antigen to facilitate its identification by affinity purification and proteomic (LC/MS/MS) analysis. Indeed, this unbiased approach has been successfully reduced to practice in a trial run in the applicant's laboratory, leading to the isolation of a high affinity Vhh that recognizes murine class II MHC products, starting from a library constructed from an alpaca immunized with unfractionated mouse splenocytes. The next targets envisioned are components of the yeast nuclear pore, available in mg amounts. While some yeast work has been performed in the applicant's laboratory, the proposed project presents the first serious foray into yeast cell biology, but with obvious extensions into multicellular eukaryotes. A key element to the approach is the lack of a requirem

描述 抽象的： Vhh 既不需要二硫键，也不需要糖基化来保持稳定性；它们也非常耐热。通过使用来自感兴趣生物体的细胞质蛋白对羊驼进行免疫，将产生一组代表性的此类 Vhh，并通过 PCR 选择性扩增 Vhh 序列来分离，然后在噬菌体展示模式下将 Vhh 表达为文库。在每个 Vhh 的 C 末端，噬菌体表达载体携带分选酶的识别基序，分选酶是一种细菌酶，可以对感兴趣的蛋白质（包括 Vhh 的蛋白质）进行定量和位点特异性修饰。分选酶识别 LPXTG 基序，并在 Thr 和 Gly 残基之间裂解，同时形成硫代酰基中间体。根据需要用荧光团、生物素或其组合进行修饰的短合成肽可解析这种硫酰基中间体，并允许共价、近定量和位点特异性 在分选酶切割位点安装此类探针，不会对 Vhh 抗原结合位点造成化学损伤。这使得可以快速生产带有生物素亲和标记的 Vhh。它们可直接用于目标抗原的检索，以便通过亲和纯化和蛋白质组 (LC/MS/MS) 分析对其进行识别。事实上，这种公正的方法已在申请人实验室的试运行中成功地付诸实践，从用未分级小鼠脾细胞免疫的羊驼构建的文库开始，分离出可识别小鼠 II 类 MHC 产物的高亲和力 Vhh 。设想的下一个目标是酵母核孔的成分，以毫克为单位。 虽然申请人的实验室已经进行了一些酵母工作，但拟议的项目首次认真涉足酵母细胞生物学，但明显扩展到多细胞真核生物。该方法的一个关键要素是缺乏要求