Expression, purification and characterization of codon-optimized human N-methylpurine-DNA glycosylase from Escherichia coli.
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 reports by others, this newly purified full-length hMPG is appreciably stable at high temperature, such as 50 degrees 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.
Adhikari
Sanjay
S
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, LL level, S-122, 3800 Reservoir Road, NW, Washington, DC 20057, USA.
Manthena
Praveen Varma
PV
Uren
Aykut
A
Roy
Rabindra
R
D001483:Base Sequence; D003062:Codon / Q000235:genetics*; D045647:DNA Glycosylases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D019271:Hypoxanthine / Q000378:metabolism; D007700:Kinetics; D020349:Surface Plasmon Resonance
D003062:Codon; D019271:Hypoxanthine; D045647:DNA Glycosylases; C076131:DNA-3-methyladenine glycosidase II