Functional expression of 8-hydroxy-5-deazaflavin-dependent DNA photolyase from Anacystis nidulans in Streptomyces coelicolor.
The gene encoding Anacystis nidulans 5-deazaflavin-dependent photolyase (phr) was inserted into the Streptomyces vector pIJ385 to form a transcriptional fusion with the neomycin resistance (aph) gene. The resulting plasmid, pANPL, was introduced into Streptomyces coelicolor, a host which exhibits no detectable photolyase activity and provides 5-deazaflavins. Transformants expressed functional photolyase and could be cultured at much higher cell densities than A. nidulans. A two-step affinity protocol was used to purify photolyase to homogeneity. High-pressure liquid chromatographic analysis established the presence of 5-deazaflavin cofactors in the enzyme, showing that this expression system allows heterologous production of 5-deazaflavin-class photolyases.
Mayerl
F
F
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts.
Piret
J
J
Kiener
A
A
Walsh
C T
CT
Yasui
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D000458:Cyanobacteria / Q000201:enzymology / Q000235:genetics*; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D005796:Genes; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic
D011993:Recombinant Fusion Proteins; C065834:8-hydroxy-5-deazaflavin-dependent DNA photolyase; D004255:Deoxyribodipyrimidine Photo-Lyase