Patent ID: 8623627

Claim:
A recombinant alkylcytosine transferase (ACT) comprising a protein having at least one cysteine capable of reacting with an O 2 -benzylcytosine, thereby transferring the benzyl substituent to the mercapto function of the cysteine at least as fast as on reaction with an O 6 -benzylguanine under identical conditions; and wherein (i) the ACT protein has the amino acid sequence of SEQ ID NO:1 or SEQ ID. No 12; or (ii) the ACT protein differs from SEQ ID. No 1 by one or more amino acid substitutions selected from the groups consisting of: R114A, S131V, E148Q, G157W, M159R; R114S, S131T, D135T, E148D, G157P, M159E; R114N, S131N, G157A, M159S; R114A, S131T, D135S, G157K, M159E; R114E, S131R, D135A, G157E, M159E; R114S, S131V, E148Q, G157L, M159R; R114E, S131N, D135N, G157T, M159F; and wherein the variant differs from SEQ ID NO: 1 or SEQ ID NO: 12 in one, two or three amino acids in positions other than positions 114, 131, 135, 148, 157, and 159; or wherein (iii) the variant differs from SEQ ID. No 1 or 12 by an amino acid in position 60 is M or I; an amino acid in position 114 is A, E, N, R or S; an amino acid in position 121 is A or V; an amino acid in position 131 is N, S, T or V; an amino acid in position 135 is D, N or T; an amino acid in position 148 is D, E, Q or V; an amino acid in position 153 is L or S; an amino acid in position 157 is A, G, L, T, P or W; and an amino acid in position 159 is E, F, M, R, S or L; or (iv) the variant differs from SEQ ID: 12 by one, two or three amino acids at a position other than positions 60, 114, 121, 131, 135, 148, 153, 157, and 159.