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Efficient Expression and Purification of Cryptochrome1 from Columbia livia in E. coli

[ Vol. 25 , Issue. 11 ]

Author(s):

Xiaoxia Yuan, Wenhao Wang, Wenjian Wu and Jing Wang*   Pages 986 - 995 ( 10 )

Abstract:


Background: Cryptochrome is a flavin-binding blue-light photoreceptor that functions in growth and development in plants, the circadian clock in animals and navigation in birds. However, a lack of purified cryptochrome has hindered studies of the structure and function of this protein. In this study, we obtained a substantial amount of the Columbia livia Cryptochrome1 (ClCry1) protein by using a prokaryotic expression system. In addition, we performed comprehensive experiments to assess the influence of several factors on the purification and yield of ClCry1, such as the inducer that was used, temperature, duration of expression and type of growth medium. These assays clearly indicated that such factors influenced the purification and yield of ClCry1. Moreover, Flavin Adenine Dinucleotide (FAD) was added during expression and purification of ClCry1, which resulted in production of large amounts of ClCry1 protein with the FAD cofactor from the Escherichia coli (E. coli) heterologous expression system. We believe that this study provides a novel avenue to acquire large amounts of ClCry1 that contains FAD and lays the foundation for studies of the geomagnetic navigation mechanism of Aves.

Objective: In this article, our motivation is to sufficiently acquire functional ClCry1 protein.

Method: In this article, we performed series of experiments to optimize the yields of ClCry1 protein expression by conducting with expression-vectors, variable inducers, temperatures, medias and durations of induction, which also identified the most appropriate conditions for obtaining functional ClCry1. Moreover, we identified a solution for the FAD abscission of ClCry1 by adding additional FAD into the dialysis buffer during the purification.

Results: Following our performed series of experiments, we assessed several crucial parameters, such as inducer, temperature, duration of induction, culture medium and recombinant expression vector. The highest yields of ClCry1 were observed with 0.01 mM IPTG and expressing for 8 h with pET21a-ClCry1 as recombinant expression vectors.

Conclusion: We demonstrated the feasibility of heterologous expression of ClCry1 in E. coli. In addition, we identified a solution for the low yield and FAD abscission of ClCry1 by conducting several experiments with variable inducers, temperatures, medias and durations of induction, which also identified the most appropriate conditions for obtaining functional ClCry1. Moreover, the typical yield was approximately 6 mg of ClCry1 from 2-liter culture, and 50% of the final protein contained the FAD cofactor. These results strongly suggest that our expression strategy is useful.

Keywords:

Columbia livia Cryptochrome1, Escherichia coli system, expression, purification, flavin adenine dinucleotide, photoreceptor.

Affiliation:

Department of Biology and Chemistry, College of Liberal Arts and Sciences, National University of Defense and Technology, 410073, Changsha, Department of Biology and Chemistry, College of Liberal Arts and Sciences, National University of Defense and Technology, 410073, Changsha, Department of Biology and Chemistry, College of Liberal Arts and Sciences, National University of Defense and Technology, 410073, Changsha, Department of Biology and Chemistry, College of Liberal Arts and Sciences, National University of Defense and Technology, 410073, Changsha

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