Expression of HsCRY in insect cells and research into its binding state with FAD by NMR

Cryptochrome(CRY)is a kind of protein sensitive to blue light,and the radical pair formedbetween the conserved tryptophan triad and cofactor flavin adenine dinucleotide(FAD)plays a central role in mediating circadian clock and magnetoreception.The full-length 1 761 bp DNA of HsCRY1 was cloned from HeLa cell using RT-PCR.Eukaryotic expression vector HTA-HsCRY1 was constructed and verified through colony PCR and sequencing,and it was then transformed into DH10 Bac to obtain recombinant bacmid.Bacmid-HsCRY1 was obtained through picking white colonies and PCR analysis.Insect sf9 cells were transfected by Bacmid-HsCRY1 to produce target protein.CRY was analyzed by Western Blot and purified by nickel affinity chromatography with 100mmol/L and 200mmol/L imidazole.Three conserved Trp related to electron transfer were predicted by alignment of CRY homologies,and the mutants were also expressed through Bac-Bac baculovirus expression system.The binding state between protein and cofactor flavin adenine dinucleotide was detected by 31 P NMR.Compared with the 31 P spectrum of free FAD,the chemical shift change of FAD binding with HsCRY1 and mutant3(W397)was observed,while no chemical shift was observed for FAD binding with mutant1(W320)and mutant2(W374).These results demonstrate that HsCRY1 and its mutants are successfully expressed through Bac-Bac baculovirus expression system,and that mutation affects the binding state of FAD with protein.