Recently I am involved in studying gyrase protein. To make a functional holoenzyme, it should be in A2B2 form. So, I trying to form this complex but need an expertise from you to achieve it. Thank you in advance.
It is only necessary to mix the two purified subunits together in buffer. Ideally, this would be done using equal molar concentrations of the two proteins. However, to account for the possibility that one of the proteins may not be 100% functional, it is worth trying various ratios and testing to see which has the greatest activity. The mixture can be aliquoted and stored at -80.
"E. coli gyrase tetramer was reconstituted by mixing 1 µM of each purified subunit in buffer consisting of 50 mM Tris-HCl (pH 7.5), 100-mM KCl, 2-mM dithiothreitol, 1-mM EDTA, and 20% (v/v) glycerol."
from: Article A Homogeneous, High-Throughput Fluorescence Anisotropy-Based...
This reference also contains the methods for expression and purification of the individual subunits from E. coli.
You could run an analytical size exclusion chromatography column, or perform analytical ultracentrifugation.
In our studies with B. subtilis gyrase, we always used a 4fold molar excess of GyrB with respect to the concentration of GyrA. Since GyrB has no topoisomerase activity on its own, does not bind DNA and (in comparison with A2B2) has a neglectible ATPase activity the excess should not disturb your experiments.
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