RIKEN Center for Sustainable Resource Science
Chemical Biology Group (Archives)

Outlines:

Affinity purification of small-molecule binding protein has been one of the most important techniques in forward chemical genetics. Once hit compound(s) which induce a specific phenotype in a cellular system are identified, the next step is to identify the molecular target.
Recently, we reported a coupling method that enables the introduction of a variety of small molecules onto glass slides through a photoaffinity reaction. By using this method, aryldiazirine groups covalently attached to glass slides are transformed upon irradiation with UV light into highly reactive carbenes, which in turn bind to or insert irreversibly into a proximal small molecule in a manner that is independent of the functional groups. This method, referred to as photo-cross-linking, has proven useful in the construction of small-molecule microarrays. It is expected that a similar chemical approach could be used to introduce small molecules onto affinity gel, such as onto agarose beads.
We have prepared PALC beads and demonstrated that photo-cross-linked small-molecule agarose beads can be used to identify and purify the binding protein. It was of note that complex small molecules such as FK506, rapamycin, and cyclosporin A could be introduced on the affinity matrix by using a uniform and straightforward procedure.

Identification of M-GFN-binding proteins. (A) Model structure of M-GFN beads. (B) Detection of the coprecipitated proteins for M-GFN beads from RAW264 cell lysates. RAW264 cell lysates were precleared with control beads and incubated with M-GFN beads. The reacted beads were washed, and the eluted proteins were subjected to SDS/PAGE and visualized by CBB staining. The coprecipitated proteins for M-GFN beads were identified as described in Materials and Methods. (C-E) Purified His-tagged SGTA (C), GLO1 (D), or SCP2 (E) protein was incubated with control beads and M-GFN beads in the presence or absence of M-GFN as a competitor. The reacted beads were washed, and the eluted proteins were immunoblotted with anti-Xpress Ab, which allows detection of recombinant His-tagged proteins containing the N-terminal leader peptide (Xpress epitope). C, Control beads; M, M-GFN beads.(Ref:2)

References:

1. N. Kanoh, K. Honda, S. Simizu, M. Muroi, H. Osada.
   Photo-crosslinked small molecule affinity matrix for facilitating forward and reverse chemical genetics.
   Angew. Chem. Int. Ed., 44, 3559-3562 (2005).

2. M. Kawatani, H. Okumura, K. Honda, N. Kanoh, M. Muroi, N. Dohmae, M. Takami, M. Kitagawa, Y. Futamura, M. Imoto, H. Osada.
   The Identification of an osteoclastogenesis inhibitor through the inhibition of glyoxalase I.
   Proc. Natl. Acad. Sci. U S A., 105, 10691-11696 (2008).