Reconstitution of E. coli DNA Polymerase III Complex
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Corresponding Organization : MRC London Institute of Medical Sciences
Other organizations : University of North Texas, MRC Laboratory of Molecular Biology
Variable analysis
- Enhanced clamp binding mutants of polymerase and exonuclease with amino acid changes: for the polymerase in residues QADMF to QLDLF from 920-924, and for the exonuclease QTSMAF to QLSLPL from 182-187
- Protein expression and purification of Pol IIIα, β, and ε subunits
- All proteins were expressed in
E. coli BL21 (DE3) - Pol III core complexes consisting of α, β and ε were assembled at equistoi-chiometric ratio and then chromatographically separated by gel filtration (Superdex 75 column)
- θ mutant E41C was created to incorporate a Cys residue for maleimide labeling
- The θ subunit containing a E41C mutation was labeled with Cy5 maleimide (GE Healthcare) following manufacturer instructions
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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