Progress in understanding complex biological systems depends on characterizing the underlying interactions of biomolecules, in particular proteins. While the DNA sequencing of an increasing number of organisms has identified their open reading frames (ORF), the possibilities to study the behaviour of the corresponding proteins in the living cell and to characterize multi-protein interactions in vivo and in vitro are limited. Most strategies that aim at realizing this objective are based on the construction of a fusion protein that, upon changes in the environment of the coupled protein, elicits a physical, physiological or chemical response. Examples include the yeast-two hybrid system, split-ubiquitin and green fluorescent protein (GFP) fusion proteins. However, all these techniques have various limitations or disadvantages.
German Patent Application No: 199 03 895 A (Kai Johnsson) describes an ELISA assay for the detection of O6-alkylguanine-DNA alkyltransferase (AGT). The mutagenic and carcinogenic effects of electrophiles such as N-methyl-N-nitrosourea are mainly due to the O6-alkylation of guanine in DNA. To protect themselves against DNA-alkylation, mammals and bacteria possess a protein, O6-alkylguanine-DNA alkyltransferases (AGT) which repairs these lesions [Pegg et al., 1995]. AGT transfers the alkyl group in a SN2 reaction to one of its own cysteines, resulting in an irreversibly alkylated enzyme. As overexpression of AGT in tumour cells enables them to acquire drug resistance, particularly to alkylating drugs such as procarbazine, dacarbazine, temozolomide and bis-2-chloroethyl-N-nitrosourea, inhibitors of AGT have been proposed for use as sensitisers in chemotherapy [Pegg et al., 1995]. DE 199 03 895 A discloses an assay for measuring levels of AGT which relies on the reaction between biotinylated O6-alkylguanine-derivatives and AGT which leads to biotinylation of the AGT. This in turn allows the separation of the AGT on a streptavidin coated plate and its detection, e.g. in an ELISA assay. The assay is suggested for monitoring the level of AGT in tumour tissue, adjusting treatment using AGT inhibitors as sensitisers in chemotherapy and for use in screening for AGT inhibitors.
Damoiseaux, Keppler and Johnsson (ChemBiochem., 4: 285-287, 2001) discloses the modified O6-alkylated guanine derivatives incorporated into oligodeoxyribonucleotides for use as of chemical probes for labelling AGT, again to facilitate detecting the levels of this enzyme in cancer cells to aid in research and in chemotherapy. Two types of variant AGT substrates and an assay for AGT in which it is labelled with biotin (the same as that described in DE 199 03 895 A) are disclosed. In addition, the use of these O6-alkylated derivatives in the directed evolution of the AGT is suggested.