Affinity capture of a mammalian DNA polymerase beta by inhibitors immobilized to resins used in solid-phase organic synthesis.
The application of resins normally used in solid-phase organic synthesis to the affinity capture of a mammalian DNA polymerase beta (pol beta) is reported. Lithocholic acid (LCA), an inhibitor of pol beta, was immobilized on various solid supports, and the batch affinity purification of pol beta from a mixture of proteins using these LCA-immobilized resins was examined. Of the resins tested, TentaGel was the most effective at purifying pol beta and at resisting nonspecific absorption of proteins. The immobilized LCA recognized pol beta specifically, which resulted in pol beta binding to the resin. Using the LCA-immobilized resin, it was possible to purify pol beta from a mixture of proteins. Furthermore, it was possible to concentrate pol beta from a crude nuclear extract of human T lymphoma Molt4 cells. To facilitate the immobilization of compounds on TentaGel resins, we also designed and prepared photoaffinity beads containing a photoreactive group at the free termini of the TentaGel resin. The pol beta inhibitors LCA, C18-beta-SQDG, and epolactaene were immobilized on the photoaffinity beads by photoreaction. The batch affinity purification of pol beta from a protein mixture could be also achieved with these beads.
Kuramochi
Kouji
K
Genome and Drug Research Center, Tokyo University of Science, Noda, Chiba 278-8510, Japan.
Haruyama
Tetsuya
T
Takeuchi
Ryo
R
Sunoki
Takashi
T
Watanabe
Madoka
M
Oshige
Masahiko
M
Kobayashi
Susumu
S
Sakaguchi
Kengo
K
Sugawara
Fumio
F
D002846:Chromatography, Affinity; D019951:DNA Polymerase beta / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry*; D006801:Humans; D008095:Lithocholic Acid / Q000737:chemistry* / Q000378:metabolism; D016399:Lymphoma, T-Cell / Q000378:metabolism; D008956:Models, Chemical; D010777:Photochemistry; D011485:Protein Binding; D012116:Resins, Plant / Q000737:chemistry*; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D012116:Resins, Plant; D008095:Lithocholic Acid; D019951:DNA Polymerase beta