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 (K(D)) of 0.25 nM. Steady-state enzyme kinetics showed an apparent K(m) of 5.3 nM and k(cat) of 0.2 min(-1) of MPG for the hypoxanthine (Hx) cleavage reaction. Moreover, hMPG had an optimal activity at pH 7.5 and 100mM KCl. Unlike the previous re...Continue Reading
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Citations
Jul 21, 2009·DNA Repair·Sanjay AdhikariRabindra Roy
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