Figure 4
Purification and in vitro characterization of ExoMeg1.
(A) SDS-PAGE analysis of metagenomic protein purification through a HiTrap Q HP. Aliquots (10 μL) of each sample were electrophoresed on a 12% SDS-PAGE, which was stained with Coomassie blue. Lane M, molecular weight marker SpectraBR; Lane 1, purification by HisTrap column; Lanes 2-10, steps of purification by HiTrap Q HP. The protein was eluted with 20% buffer B (lane 6). (B) Identification of 3′-5′exonuclease activity on dsDNA or ds-AP-DNA with increasing concentrations of ExoMeg1. The AP endonuclease activity should result in an 11-mer product. However, ExoMeg1 has shown only the exonuclease activity, generating a 13-mer product. (C) Activity assay versus time of incubation at 37 °C using ds-AP-DNA. (D) Identification of 3′-5′exonuclease activity on the single strand substrate and the effect of salt on the activity of ExoMeg1. Lanes 1 and 6, reactions in the absence of ExoMeg1 and KCl; Lanes 2 and 7, reactions in the presence of ExoMeg1 but absence of KCl; Lanes 3 and 8, reactions including ExoMeg1 and 50 mM KCl; Lanes 4 and 9, reactions including EXOMEG1 and 200 mM KCl; lanes 5 and 10, reactions containing 10 mM EDTA. (E) Analysis of salt effects on ExoMeg1 activity using increasing concentrations of KCl (50, 100, 250, 500 and 1000 mM). (F) Mg2 + dependence of ExoMeg1 activity. The reactions contain increasing concentrations of MgCl2 (0, 5, 10 and 15 mM). The ExoMeg1 activity is totally inhibited by the absence of MgCl2 (lane 2). (G) Effect of EDTA on ExoMeg1 activity. The reactions contain increasing concentrations of EDTA (0, 1, 5 and 10 mM). The progression of 3′-5′ exonuclease activity is gradually inhibited by EDTA. (H) AP endonuclease assay using a 30-mer labeled at the 3′ end with increasing concentrations of ExoMeg1. Commercial APE1 enzyme (NEB Biolabs) was used as the positive control (lane 2).
