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;它们具有显著的热稳定性， 好.通过用来自感兴趣的生物体的细胞质蛋白免疫羊驼， 通过选择性扩增存在的Vhh序列来产生和分离这种Vhh， PCR，随后以噬菌体展示模式表达作为文库的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，分选酶是一种细菌酶， 以及对感兴趣的蛋白质（包括Vhh）的位点特异性修饰。分选酶识别LPXTG基序，并且 在Thr和Gly残基之间裂解，同时形成硫代酰基中间体。短合成 根据需要用荧光团、生物素或其组合修饰的肽， 中间体，并允许共价，近定量和位点特异性安装这样的探针在位点 分选酶切割，而不对Vhh抗原结合位点造成化学损伤。这使得快速 产生用生物素亲和标记的Vhh。它们可以直接用于检索目标 抗原，以便于通过亲和纯化和蛋白质组学（LC/MS/MS）分析对其进行鉴定。这确 公正的方法已成功地减少到实践中的试验运行在申请人的实验室，导致 涉及从文库开始分离识别鼠II类MHC产物的高亲和力Vhh， 构建自用未分级小鼠脾细胞免疫的羊驼。我们设想的下一个目标 是酵母核孔的组分，以mg量提供。 虽然一些酵母工作已经在申请人的实验室进行，拟议的项目提出， 这是第一次对酵母细胞生物学的认真尝试，但显然扩展到了多细胞真核生物。一个关键 该方法的一个要素是不需要免疫化学工具，如二抗 以快速鉴定分离的Vhh特异性的靶抗原。生物素安装的便利性 和荧光团单独或组合，通过分选酶反应也使得能够使用 作为纯化工具或用于通过细胞荧光测定法或荧光显微镜观察手柄。 此外，因为Vhh是单结构域，所以它们可以在基因上融合并表达为Vhh的融合伴侣。 其他感兴趣的蛋白质（例如，用于靶抗原定位研究的GFP;用于选择性靶抗原定位研究的泛素连接酶;以及用于靶抗原定位研究的泛素连接酶）。 靶抗原的降解;与感兴趣的蛋白质融合以使其选择性结合 蛋白质与Vhh靶抗原）作为真核生物合成生物学的一步。

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-specific 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 requirement for immunochemical tools such as secondary antibodies to rapidly identify the target antigens for which the isolated Vhh’s are specific. The ease of installation of biotin and fluorophores, alone or in combination, by means of the sortase reaction also enables the use of any Vhh of interest as a purification tool or a handle for visualization by cytofluorimetry or fluorescence microscopy. Moreover, because Vhh are single domains, they can be genetically fused and expressed as fusion partners of other proteins of interest (e.g. GFP for localization studies of the target antigen; ubiquitin ligases for selective degradation of the target antigen; fusion to a protein of interest to bring about selective association of that protein with the Vhh target antigen) as a step towards synthetic biology in eukaryotes.