Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro

Methods. 2004 Apr;32(4):437-44. doi: 10.1016/j.ymeth.2003.10.007.

Abstract

The in vivo and in vitro labeling of fusion proteins with synthetic molecules capable of probing and controlling protein function has the potential to become an important method in functional genomics and proteomics. We have recently introduced an approach for the specific labeling of fusion proteins, which is based on the generation of fusion proteins with the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) and the irreversible reaction of hAGT with O6-benzylguanine derivatives. Here, we report optimized protocols for the synthesis of O6-benzylguanine derivatives and the use of such derivatives for the labeling of different hAGT fusion proteins in vivo and in vitro.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Blotting, Western
  • Estrogen Receptor alpha / genetics
  • Estrogen Receptor alpha / metabolism
  • Fluorescein / chemistry
  • Fluorescein / metabolism
  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / metabolism
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Guanine / analogs & derivatives*
  • Guanine / chemistry
  • Guanine / metabolism*
  • HeLa Cells
  • Humans
  • Methotrexate / analogs & derivatives
  • Methotrexate / chemistry
  • Methotrexate / metabolism
  • Microscopy, Fluorescence
  • Molecular Structure
  • O(6)-Methylguanine-DNA Methyltransferase / chemistry
  • O(6)-Methylguanine-DNA Methyltransferase / genetics
  • O(6)-Methylguanine-DNA Methyltransferase / metabolism*
  • Polymerase Chain Reaction
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / drug effects
  • Recombinant Fusion Proteins / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Serine Endopeptidases / genetics
  • Serine Endopeptidases / metabolism
  • Staining and Labeling / methods*
  • Subcellular Fractions / metabolism
  • Tamoxifen / analogs & derivatives*
  • Tamoxifen / pharmacology

Substances

  • Bacterial Proteins
  • Estrogen Receptor alpha
  • Fluorescent Dyes
  • Fungal Proteins
  • LexA protein, Bacteria
  • Recombinant Fusion Proteins
  • O(6)-benzylguanine
  • Tamoxifen
  • afimoxifene
  • Guanine
  • O(6)-Methylguanine-DNA Methyltransferase
  • Serine Endopeptidases
  • Fluorescein
  • Methotrexate