Prokaryotic expression and solubilisation of Arabidopsis ROOT UVB SENSITIVE 1 from inclusion bodies in Escherichia coli
2022
Hou, Xue-Wen | Tong, Hong-Yun | He, Zheng-Hui
The RUS (ROOT UVB SENSITIVE 1) proteins characterized by their unique DUF647 domain are widely distributed in eukaryotes. Their functional roles are largely unknown except for the possible involvement of Arabidopsis RUS1 and RUS2 in early seedling development. To investigate the biochemical roles of the RUS proteins, full length and truncated Arabidopsis RUS1 were seamlessly fused with GFP and cloned into prokaryotic expression vector pQE-100 which allows proteins expressed with an N-terminal 6×His tag. Expression of the full length RUS1-GFP could not be detected after adding the inducer IPTG, while a truncated RUS1-GFP was expressed at high levels and formed inclusion bodies in Escherichia coli. The inclusion bodies were dissolved in a denaturing buffer, and then the truncated RUS1-GFP fusion protein in the supernatant was bound to a Ni-NTA slurry. The bound proteins were eluted after the non-specific binding proteins were washed away. The purified truncated proteins were detected as a single clear band of the expected size in SDS-PAGE, and were further confirmed by the Western blot test. Our results suggest that the impossible expression of the full length RUS1 protein in E. coli can be expressed in truncated form, and inclusion bodies can be effectively solubilized.
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