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;它们也非常耐热。通过用来自感兴趣的生物体的细胞质蛋白免疫羊驼，通过PCR选择性扩增存在的Vhh序列，然后以噬菌体展示模式表达作为文库的Vhh，将产生和分离一组代表性的此类Vhh。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，分选酶是一种允许对感兴趣的蛋白质（包括Vhh）进行定量和位点特异性修饰的细菌酶。分选酶识别LPXTG基序，并在Thr和Gly残基之间切割，同时形成硫代酰基中间体。根据需要用荧光团、生物素或其组合修饰的短合成肽解析该硫代酰基中间体，并允许共价、近定量和位点特异性地检测该硫代酰基中间体。 在分选酶切割位点安装这种探针，而不对Vhh抗原结合位点造成化学损伤。这允许快速产生用生物素亲和标记的Vhh。它们可直接用于回收靶抗原，以便于通过亲和纯化和蛋白质组学（LC/MS/MS）分析进行鉴定。实际上，这种无偏倚的方法已经在申请人实验室的试验运行中成功地减少到实践中，导致从用未分级小鼠脾细胞免疫的羊驼构建的文库开始分离识别鼠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组分，以mg量提供。 虽然在申请人的实验室中已经进行了一些酵母工作，但是所提出的项目呈现了对酵母细胞生物学的第一次认真的尝试，但是明显扩展到多细胞真核生物。该方法的一个关键要素是没有要求

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