CONSTRUCTION AND OVEREXPRESSION OF A SYNTHETIC GENE FOR HUMAN DNA METHYLGUANINE METHYLTRANSFERASE - RENATURATION AND RAPID PURIFICATION OF THE PROTEIN

A synthetic gene was constructed that encodes human DNA methylguanine methyltransferase (hMGMT). The synthetic gene was designed with a numb er of unique restriction sites to facilitate cassette mutagenesis and to reflect the preferences found among genes in Escherichia coli. Both the full-length gene and a gene for a functional variant (hMGMT Delta C) that lacks the C-terminal 28 codons were constructed, and the gene s were overexpressed using a T7 RNA polymerase promoter. The proteins are made in the form of insoluble aggregates but the truncated form of the protein (hMGMT Delta C) has been successfully denatured, renature d, and purified to near homogeneity by ion exchange. Methyltransferase activity assays of hMGMT Delta C demonstrate that the reconstituted p rotein has substantial DNA repair activity, though somewhat less than full-length hMGMT that had been expressed and purified in a soluble fo rm. Mass spectrometry of a mixture of proteolytic fragments confirmed the protein sequence and indicated no detectable oxidation of the acti ve site cysteine. The protein was determined to be monomeric by gel fi ltration chromatography, and circular dichroism spectra for renatured hMGMt Delta C and fully soluble hMGMT are consistent with the renature d protein preparation being fully folded. Refolded hMGMt Delta C had a curious propensity to form large aggregates in a time-dependent manne r when injected into a dynamic light scattering instrument; this aggre gation behavior was not observed for hMGMT purified in a soluble form, Differences in susceptibility to aggregation may account for differen ces in methyltransfer activity. Yields of purified protein were approx imately 5 mg/liter of culture. (C) 1997 Academic Press.