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.