Expression, purification and characterization of codon-optimized human N-methylpurine-DNA glycosylase from Escherichia coli

Sanjay Adhikari, Praveen Varma Manthena, Aykut Üren, Rabindra Roy

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, initiates excision repair of several N-alkylpurine adducts, deaminated and lipid peroxidation-induced purine adducts. MPG from human and mouse has previously been cloned and expressed. However, due to the poor expression level in Escherichia coli (E. coli) and multi-step purification process of full-length MPG, most successful attempts have been limited by extremely poor yield and stability. Here, we have optimized the codons within the first five residues of human MPG (hMPG) to the best used codons for E. coli and expressed full-length hMPG in large amounts. This high expression level in conjunction with a strikingly high isoelectric point (9.65) of hMPG, in fact, helped purify the enzyme in a single step. A previously well-characterized monoclonal antibody having an epitope in the N-terminal tail could detect this codon-optimized hMPG protein. Surface plasmon resonance studies showed an equilibrium binding constant (KD) of 0.25 nM. Steady-state enzyme kinetics showed an apparent Km of 5.3 nM and kcat of 0.2 min-1 of MPG for the hypoxanthine (Hx) cleavage reaction. Moreover, hMPG had an optimal activity at pH 7.5 and 100 mM KCl. Unlike the previous reports by others, this newly purified full-length hMPG is appreciably stable at high temperature, such as 50 °C. Thus, this study indicates that this improved expression and purification system will facilitate large scale production and purification of a stable human MPG protein for further biochemical, biophysical and structure-function analysis.

Original languageEnglish (US)
Pages (from-to)257-262
Number of pages6
JournalProtein Expression and Purification
Volume58
Issue number2
DOIs
StatePublished - Apr 2008

ASJC Scopus subject areas

  • Biotechnology

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