PMID:15916808: Difference between revisions
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Revision as of 03:00, 20 February 2009
Mu, W, Zhang, D, Xu, L, Luo, Z and Wang, Y (2005) Activity assay of His-tagged E. coli DNA photolyase by RP-HPLC and SE-HPLC.J. Biochem. Biophys. Methods 63:111-24
| Abstract | Escherichia coli DNA photolyase was expressed as C-terminal 6x histidine-fused protein. Purification of His-tagged E. coli DNA photolyase was developed using immobilized metal affinity chromatography with Chelating Sepharose Fast Flow. By one-step affinity chromatography, approximate 4.6 mg DNA photolyase was obtained from 400 ml E. coli culture. The purified His-tagged enzyme was combined with two chromophors, FADH and MTHF. Using the oligonucleotide containing cyclobutane pyrimidine dimer as substrate, both reversed-phase high-performance liquid chromatography and size-exclusion high-performance liquid chromatography were developed to measure the enzyme activity. The enzyme was found to be able to repair the cyclobutane pyrimidine dimer with the turnover rate of 2.4 dimers/photolyase molecule/min. |
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