The Production Optimization of Recombinant Cryptochrome DASH1 of Green Algae Volvox carteri in Escherichia coli: Investigation of Solubility and Binding to DNA
2019
Mahdavi, Saeed | Razeghi, Jafar | Pazhouhandeh, Maghsoud | Kianianmomeni, Arash | Movafeghi, Ali
Objective Optogenetics, as a new scientific branch, uses the photoreceptors to conduct basic and applied research. Identification of the novel photoreceptors and their characteristics is a way to develop the optogenetics. Cryptochrome/Photolyase protein family DASH crypthochromes have DNA repair ability and play role in signaling pathways. Current research goal was the adequate production of the Volvox predicted DASH1 cryptochrome protein (VcCryDASH1) to use in spectroscopy studies as well as DNA repair assay. Materials and methods First, the coding sequence of VcCryDASH1 was isolated from Volvox carteri cDNA library using the specific primers and cloned into pGEX-2TK expression vector. The recombinant protein production was analyzed using two different strains of E. coli under different incubation times, using IPTG to induce the protein expression and then its solubility was investigated by SDS-PAGE. DNA binding ability of the protein was studied by alignment method. Results The appropriate production of the protein obtained in Rosetta strain at 32°C under more than 3 hours incubation. Approximately, 50 percent of the protein was insoluble under 1 and 3 hours incubations. VcCryDASH1 alignment analysis showed that there is high percentage of amino acids in this protein with DNA binding ability. Conclusion These results revealed that under optimum conditions, VcCryDASH1 has dual solubility but our approach can produce the adequate quantity of the protein in the Rosetta. Also, because of having conserved amino acids, the protein can bind to DNA, nominating it as a possible resource to design the DNA- light mediator molecules in optogenetics.
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