Construction and overexpression of a synthetic gene for human DNA methylguanine methyltransferase

Renaturation and rapid purification of the protein

Lesley R. Brown, Jing Deng, David M. Noll, Noriko Mori, Neil D. Clarke

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)337-345
Number of pages9
JournalProtein Expression and Purification
Volume9
Issue number3
DOIs
StatePublished - Apr 1997

Fingerprint

Synthetic Genes
Methyltransferases
Purification
Genes
DNA
Proteins
Agglomeration
Mutagenesis
Insertional Mutagenesis
Ion Exchange
Dynamic light scattering
Circular Dichroism
Chromatography
Codon
DNA Repair
Escherichia coli
Gel Chromatography
Mass spectrometry
Cysteine
Assays

ASJC Scopus subject areas

  • Biochemistry

Cite this

Construction and overexpression of a synthetic gene for human DNA methylguanine methyltransferase : Renaturation and rapid purification of the protein. / Brown, Lesley R.; Deng, Jing; Noll, David M.; Mori, Noriko; Clarke, Neil D.

In: Protein Expression and Purification, Vol. 9, No. 3, 04.1997, p. 337-345.

Research output: Contribution to journalArticle

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