The Effective And Active Expression Of Homing Pigeon Cryptochrome1

Cryptochromes(Crys)are flavin-containing blue light photoreceptors mediating light regulation of growth and development in plants,circadian clock and rhythms of animals and magnetoreception of avian.Until now,it has been confirmed that Columbia livia Cryptochrome1(ClCry1)is a magnetic sensitive protein.Although the structural and function of plant Crys has been studied in some details,the structural and function of animal Crys remains poorly defined in details because it has been difficult to purify animal Crys with their flavin cofactors,especially for Cry-mediated geomagnetic response of avians.All preliminary works on ClCry1 proteins acquisition was only reported by our group,but they have some deficiencies.Therefore,intensive studies performed with ClCry1 were subject to certain limitations.Here,we chose a variety of expression systems,including eukaryotic heterologous,prokaryotic heterologous and cell-free,to express Cl Cry1 protein.Based on the previous sf9 insect expression system,we constructed eight kinds of expression tools which were suitable for sf21 cell-free,E.coli prokaryotic and cell-free expression systems,respectively.Then we established an expression and purification scheme by exploring and optimizing of various conditions for each system.Finally,using UV/visible absorption spectroscopy,we confirmed that the purified ClCry1 contains flavin adenine dinucleotide(FAD)cofactor.Through the systematic researches,we achieved the active ClCry1 protein with above-mentioned biosynthetic systems,and compared the expression results and the products of each system.All of these works provided the material basis for the studies of the structure and function of ClCry1 protein and analyses of biogeomagnetic navigation mechanism.This is the first report about the acquisition of Cry protein with cell-free system and the comparision ClCry1 proteins from different expression systems.In this paper,we solved the practical problems of low expression and purification of active ClCry1 protein through the analysis,exploration and optimization of expression conditions and purification processes of each system.We overcome the problem,which is difficult to receive the functional whole protein,and got the ClCry1 proteins contained FAD cofactor.