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 number 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 genes 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, renatured, and purified to near homogeneity by ion exchange. Methyltransferase activity assays of hMGMT delta C demonstrate that the reconstituted protein has substantial DNA repair activity, though somewhat less than full-length hMGMT that had been expressed and purified in a soluble form. Mass spectrometry of a mixture of proteolytic fragments confirmed the protein sequence and indicated no detectable oxidation of the active site cysteine. The protein was determined to be monomeric by gel filtration chromatography, and circular dichroism spectra for renatured hMGMT delta C and fully soluble hMGMT are consistent with the renatured protein preparation being fully folded. Refolded hMGMT delta C had a curious propensity to form large aggregates in a time-dependent manner when injected into a dynamic light scattering instrument; this aggregation behavior was not observed for hMGMT purified in a soluble form. Differences in susceptibility to aggregation may account for differences in methyltransfer activity. Yields of purified protein were approximately 5 mg/liter of culture.
Brown
L R
LR
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185, USA.
Deng
J
J
Noll
D M
DM
Mori
N
N
Clarke
N D
ND
D000595:Amino Acid Sequence; D001483:Base Sequence; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase