Directed Evolution of O6-Alkylguanine-DNA Alkyltransferase for Efficient Labeling of Fusion Proteins with Small Molecules In Vivo

Abstract

We report here the generation of mutants of the human O(6)-alkylguanine-DNA alkyltransferase (hAGT) for the efficient in vivo labeling of fusion proteins with synthetic reporter molecules. Libraries of hAGT were displayed on phage, and mutants capable of efficiently reacting with the inhibitor O(6)-benzylguanine were selected based on their ability to irreversibly transfer the benzyl group to a reactive cysteine residue. Using synthetic O(6)-benzylguanine derivatives, the selected mutant proteins allow for a highly efficient covalent labeling of hAGT fusion proteins in vivo and in vitro with small molecules and therefore should become important tools for studying protein function in living cells. In addition to various applications in proteomics, the selected mutants also yield insight into the interaction of the DNA repair protein hAGT with its inhibitor O(6)-benzylguanine.

The periciliary ring in polarized epithelial cells is a hot spot for delivery of the apical protein gp135

“…1 A). The SNAP tag, a modified form of the enzyme O 6 -alkylguanine-DNA alkyltransferase, allows temporally and spatially defined cohorts of proteins to be selectively labeled and detected (Juillerat et al, 2003;Keppler et al, 2004). The tag binds covalently to O 6 -benzylguanine (BG), resulting in the irreversible transfer of the substituted benzyl group to the reactive thiol within the SNAP tag.…”

Analysis of Protein Turnover by Quantitative SNAP‐Based Pulse‐Chase Imaging

“…It acts as a suicide enzyme that catalyzes a covalent binding reaction between itself and the alkyl group that is removed from guanines, thereby restoring DNA integrity but inactivating its own catalytic activity (Pegg, 2000). SNAP, the modified form of hATG, has lost its affinity to DNA but efficiently reacts with soluble O 6 -benzylguanine (BG), of which the benzyl moiety is readily transferred to the SNAP protein ( Figure 1, Juillerat et al, 2003;Keppler et al, 2003). The benzyl rings in BG can be coupled to a large variety of molecules (Keppler et al, , 2004(Keppler et al, , 2006) that include fluorescent moieties as well as non-fluorescent ones (a selection of SNAP substrates is presented in Table 2).…”

Direct Reversal of DNA Alkylation Damage

“…Kai Johnsson's group has pioneered the use of hAGT fusion proteins as in vitro and in vivo biotechnology tools. 135 This group has evolved hAGT mutants that possess high reactivity for transferring large alkyl groups on the O 6 -position of guanine free base to the reactive Cys residue of the protein. 135 Thus, bifunctional small molecules which contain O 6 -BG conjugated through the benzyl group to a probe molecule can be prepared as good substrates for the hAGT mutants.…”

“…135 This group has evolved hAGT mutants that possess high reactivity for transferring large alkyl groups on the O 6 -position of guanine free base to the reactive Cys residue of the protein. 135 Thus, bifunctional small molecules which contain O 6 -BG conjugated through the benzyl group to a probe molecule can be prepared as good substrates for the hAGT mutants. 136 The mutant hAGT can be fused with other proteins of interest.…”