Expression, Purification And Biological Activity Of E.coli DNA Photolyase

Lesions will be induced by ultraviolet light in DNA, which will cause growth delay, mutagenesis, even death of organism. DNA photolyase can repair these lesions by absorbing the 350-450 nm photon. Escherichia coli DNA photolyase has two chromophore cofactors. One is the flavin adenine dinucleotide (FAD), and the second is methenyltetrahydropteroylployglutamate (MTHF). FAD chromophore is essential for active catalysis, which is in fully reduced state in vivo. MTHF chromophore acts as absorbing and transferring energe to FAD.In this paper, we studied the fed-batch production, purification and activity assay of DNA photolyase, and DNA repair property of DNA photolyase on cell and in invo level.Part One. Expression and purification of photolayseE.coli phr gene was cloned into pET-22b(+), and the reconstructed plasmid, pET-22b(+)::phr(N+X) was obtained. Because the pET-22b(+) vector carries an optional C-terminal 6×Hisitine-tag coding sequence, photolyase was expressed as His-tagged fusion protein. Upon induction of BL21(DE3)/pET-22b(+)::phr(N+X) with IPTG (1mM), His-tagged photolyase was overproduced to 20% of total cellular proteins, and about 1/3 was soluble. After one-step purification with Ni²⁺-Chelating Sepharose Fast Flow, His-tagged photolyase was obtained with the purity of approximately 95%. The UV-Vis scanning and fluorescence spectral properties of photolyase showed that the purified photolyase was combined with two chromophors, FADH and MTHF.Part Two. Fed-batch fermentation of BL21(DE3)/pET22b(+)::phr(N+X)...