To finally ensure that there are
To finally ensure that there are no other alternatives to the CHIR-124 australia of exonuclease activity from Artemis we quantified the endonuclease and exonuclease activities throughout the purification and the results are presented in Table 1. These results clearly demonstrate that following fractionation on a HAP column, 100% of the endonuclease activity is recovered in the HAP FT resulting in nearly a 30-fold purification while only 2.5% of the exonuclease activity is retained in that fraction with no increase in specific activity despite the loss of 96% of the total protein. Consistent with this data, analysis of the Artemis polypeptide as determined by western blotting in conjunction with analysis of exonuclease activity was performed on the HAP FT and elution pools of protein from a separate preparation. The percent of total antibody reactivity or exonuclease activity resolved in the two pools of protein was determined and the results demonstrate that greater than 90% of Artemis protein loaded onto the HAP column was recovered in the HAP flow-through material, while less than 10% was recovered in the gradient elution pool while greater than 90% of the exonuclease was identified in the elution pool and less than 10% in the FT pool of protein (Supplemental Fig. 3). These results indicate that the exonuclease found to co-purify with [His]6-Artemis under certain conditions can be separated away from [His]6-Artemis under other conditions (as described above), and therefore is probably a prominent exonuclease that has a similar affinity as Artemis for certain column conditions, but is not intrinsic to the Artemis polypeptide.
Discussion Separation of the nuclease activities, as presented in this paper, was achieved with multiple protein purification preparations. However, it is important to note that the separation of exonuclease activity from Artemis did vary between protein preparations. As we continued to improve our purification procedures, specific changes, albeit small, in the protocol resulted in subtle differences in separation of exonuclease activity from endonuclease activity. In separating [His]6-Artemis over the HAP column, we found that greater separation of activity was achieved on a 5mL HAP column compared to a 2mL HAP column, despite more than enough protein-binding capacity on the 2mL column. However, the residual exonuclease activity that flowed through the 2mL column could be separated from Artemis by re-running the flow-through on a second HAP column (data not shown). This suggests that saturation of the hydroxyapatite column with exonuclease activity, at least to a certain level, can occur, leading to sub-optimal separation. These variations are largely a result of the specifics of our protocol, and can be impacted by numerous factors, including specificity of the matrix used, MacroPrep Ceramic Hydroxyapatite (Biorad), or relatively high pH (7.85) of the buffer used in the purification. Interestingly, we did observe a nominal amount of Artemis in many of the fractions collected from the HAP column, including the wash and elution. The diminutive levels of [His]6-Artemis were often only observed by Western blot analysis. This phenomenon was also observed during fractionation over the nickel–agarose column, indicating a certain degree of spreading of the fusion protein during all fractionation steps.