I need to purify two helicases, both derived from E. coli. One is the replicative helicase DnaB, which is co-expressed with its loading protein DnaC on a dual-expression vector. The other is the ATP-dependent helicase RecQ. DnaB and RecQ carry an N-terminal His-tag, and their purification and storage buffers contain 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 10% glycerol.
For activity assays, I used annealed double-stranded DNA substrates: a 59-bp unlabeled long strand and a 21-bp short strand with a 5'-fluorescent label. The short strand is fully complementary to the central region of the long strand, resulting in 5' and 3' overhangs on the long DNA. After incubating the enzymes with the substrate at 30°C for 30 minutes, I added 10× DNA loading buffer and ran the samples on a 15% native PAGE gel. However, I observed no release of the labeled short strand in either case.
Possible reasons I identified:
Questions:
- Are my hypotheses reasonable?
- Are there other potential issues or suggestions to troubleshoot this?
Years ago, I tried the same experiment with DnaB, with and without DnaC, and I also could not get it to work.
Rapid reannealing of the two strands may be part of the problem, but it also may be that the helicase activity is not expressed except as part of a larger multiprotein complex, such as the replisome.
It may help to add single-stranded binding protein (SSB). The Mg2+ concentration may be an important variable.
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