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

描述 摘要： VHH的稳定性既不需要二硫键，也不需要糖基化；它们也具有显著的热稳定性。通过用感兴趣生物的细胞质蛋白免疫羊驼，将产生一组具有代表性的VHH，并通过选择性扩增PCR呈现的VHH序列来分离VHH，然后以噬菌体展示模式将VHH作为文库进行表达。在每个VHH的C末端，噬菌体表达载体携带一个对索尔特酶的识别基序，这是一种细菌酶，允许对包括VHH在内的目的蛋白进行定量和定点修饰。索尔特酶识别LPXTG基序，并在苏氨酸和甘氨酸残基之间切割，同时形成硫代酰基中间体。短的合成肽，根据需要用荧光团、生物素或它们的组合进行修饰，分解这种硫代酰基中间体，并允许共价、近定量和位点特异性 将这种探针安装在索酸酶裂解部位，不会对VHH抗原结合部位造成化学损伤。这使得用生物素亲和标记的VHH的快速生产成为可能。它们可直接用于目标抗原的回收，以便于通过亲和纯化和蛋白质组(LC/MS/MS)分析进行鉴定。事实上，这种不偏不倚的方法已经成功地在申请人的实验室进行了试运行，导致分离出一种高亲和力的VHH，它识别小鼠II类MHC产品，从用小鼠脾细胞免疫的羊驼构建的文库开始。下一个设想的目标是酵母核孔的成分，以镁含量提供。虽然申请人的实验室已经完成了一些酵母工作，但拟议的项目是对酵母细胞生物学的第一次认真尝试，但明显扩展到了多细胞真核生物。该方法的一个关键要素是缺乏要求

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