Activity assay of His-tagged E. coli DNA photolyase by RP-HPLC and SE-HPLC.
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.
Mu
Wanmeng
W
School of Life Sciences, University of Science and Technology of China, Hefei 230026, PR China.
Zhang
Dongfang
D
Xu
Lei
L
Luo
Zhaofeng
Z
Wang
Yuzhen
Y
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006639:Histidine; D007700:Kinetics; D015335:Molecular Probes; D011740:Pyrimidine Dimers / Q000032:analysis / Q000378:metabolism; D011993:Recombinant Fusion Proteins
D029968:Escherichia coli Proteins; D015335:Molecular Probes; D011740:Pyrimidine Dimers; D011993:Recombinant Fusion Proteins; D006639:Histidine; D004255:Deoxyribodipyrimidine Photo-Lyase