Abstract
Cryptochromes are photolyase-like blue light receptors that are conserved in plants and animals. Although the light-dependent catalytic mechanism of photolyase is well studied, the photochemical mechanism of cryptochromes remains largely unknown. Lack of an appropriate protein expression system to obtain photochemically active cryptochrome holoproteins is a technical obstacle for the study of plant cryptochromes. We report here an easy-to-use method to express and study Arabidopsis cryptochrome in HEK293T cells. Our results indicate that Arabidopsis cryptochromes expressed in HEK293T are photochemically active. We envision a broad use of this method in the functional investigation of plant proteins, especially in the large-scale analyses of photochemical activities of cryptochromes such as blue light-dependent protein–protein interactions.
Highlights
Plants possess several photoreceptors mediating light regulation of gene expression and photophysiologic response (Lin, 2002; Yu et al, 2010)
To develop a polymerase chain reaction (PCR)-product based and easy-to-use expression system, we first investigated whether the linear-DNA based transfection was efficient enough for plant protein expression in HEK293T cells
When transfected with different amount of linear-DNA in 96-well-plate, the AtCRY2–GFP expression level increased with increasing linear-DNA amount, and achieved a maximum value at the linear-DNA amount of 0.2 μg per well, which is comparable with the expression level of plasmid-based transfection at plasmid amount of 0.2 μg per well (Figure 2A)
Summary
Plants possess several photoreceptors mediating light regulation of gene expression and photophysiologic response (Lin, 2002; Yu et al, 2010). We have expressed and purified cryptochromes and their interacting proteins in various expression systems, e.g., Escherichia coli, yeast, and insect cell (Malhotra et al, 1995; Liu et al, 2008; Zuo et al, 2011). All those systems have major drawbacks, which limit their use in a wide range of biochemical analyses. These disadvantages include deficient chromophore, toxicity, low yield, and light independent constitutive activities
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