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

Public Health Relevance

The ability to perturb protein-protein interactions inside cells on a large scale (with the specificity afforded by antigen-antibody interactions) is a new tool to analyze host cell biology in a manner not previously possible. If applied on a large scale, as proposed here, this technology should be considered a complement to genetics and RNA-interference based approaches. This project will provide a new window on the biology of the eukaryotic cell and thus illuminate new aspects of normal physiology and aberrations in disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
NIH Director’s Pioneer Award (NDPA) (DP1)
Project #
4DP1GM106409-05
Application #
9117623
Study Section
Special Emphasis Panel (ZGM1)
Program Officer
Fabian, Miles
Project Start
2012-09-30
Project End
2017-07-31
Budget Start
2016-08-01
Budget End
2017-07-31
Support Year
5
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Whitehead Institute for Biomedical Research
Department
Type
DUNS #
120989983
City
Cambridge
State
MA
Country
United States
Zip Code
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