Nucleic Acids Research, 1992, Vol. 20, No. 22 6081-6090
© 1992
MOLECULAR BIOLOGY |
Inactive O6-methylguanine-DNA methyltransferase in human cells
Imperial Cancer Research Fund, Clare Hall Laboratories South Mimms, Herts EN6 3LD, UK
* To whom correspondence should be addressed
Received May 27, 1992. Revised October 8, 1992. Accepted October 8, 1992.
A plasmid encoding a recombinant human O6-methylguanine-DNA methyltransferase (MGMT) fused to a fragment of the bacteriophage XN protein has been constructed. The fusion protein retained methyltransferase activity when expressed at high levels in E.coli and was purified to essential homogeneity by a simple procedure. Antisera raised against the purified fusion protein recognized MGMT in western blots of extracts of human cells. For most cell lines, there was a quantitative relation between the amount of immunologically detectable MGMT protein and enzyme activity. However, four cell lines contained detectable MGMT protein despite having no measurable methyltransferase activity. Additionally, a HeLa line contained considerably more immunoreactive MGMT protein than could be accounted for by its methyltransferase activity. Thus, some cells contain significant amounts of inactive MGMT. Preliminary characterization of the inactive protein in HeLaS3 cells indicated that it has some properties in common with MGMT methylated at the active cysteine residue.
+Present address: Donner Laboratory, Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720, USA