TY - JOUR
T1 - Construction and overexpression of a synthetic gene for human DNA methylguanine methyltransferase
T2 - Renaturation and rapid purification of the protein
AU - Brown, Lesley R.
AU - Deng, Jing
AU - Noll, David M.
AU - Mori, Noriko
AU - Clarke, Neil D.
N1 - Funding Information:
We are very grateful to Dr. Phil Potter of St. Jude Children's Research Hospital for providing signi®cant amounts of hMGMT for comparative purposes. We thank Joel Hoskins of the Center for Advanced Research in Biotechnology for synthesis of oligonucleotides. Mass spectral analyses were carried out by Dr. Amina Woods at the Middle Atlantic Mass Spectrometry Laboratory, an NSF Regional Instrumentation Facility. This work was supported by a grant from the National Institutes of Health (CA59492) and by the Markey Center for Macromolecular Structure and Function at Johns Hopkins. N.D.C. was supported in part by an American Cancer Society Junior Faculty Award, and during the initial stages of this work by a National Research Council Research Associateship at the Center for Advanced Research in Biotechnology/National Institute of Standards and Technology.
PY - 1997/4
Y1 - 1997/4
N2 - 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 (hMGMTΔC) 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 (hMGMTΔC) has been successfully denatured, renatured, and purified to near homogeneity by ion exchange. Methyltransferase activity assays of hMGMTΔC 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 hMGMTΔC and fully soluble hMGMT are consistent with the renatured protein preparation being fully folded. Refolded hMGMTΔC 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.
AB - 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 (hMGMTΔC) 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 (hMGMTΔC) has been successfully denatured, renatured, and purified to near homogeneity by ion exchange. Methyltransferase activity assays of hMGMTΔC 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 hMGMTΔC and fully soluble hMGMT are consistent with the renatured protein preparation being fully folded. Refolded hMGMTΔC 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.
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U2 - 10.1006/prep.1996.0719
DO - 10.1006/prep.1996.0719
M3 - Article
C2 - 9126605
AN - SCOPUS:0031127778
SN - 1046-5928
VL - 9
SP - 337
EP - 345
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 3
ER -