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

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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.