Efficient ASK‐assisted system for expression and purification of plant F‐box proteins

Abstract

Ubiquitin-mediated protein degradation plays an essential role in plant growth and development as well as responses to environmental and endogenous signals. F-box protein is one of the key components of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex, which recruit specific substrate proteins for subsequent ubiquitination and 26S proteasome-mediated degradation to regulate developmental processes and signaling networks. However, it is not easy to obtain purified F-box proteins with high activity due to their unstable protein structures. Here, we found that Arabidopsis SKP-like proteins (ASKs) can significantly improve soluble expression of F-box proteins and maintain their bioactivity. We established an efficient ASK-assisted method to express and purify plant F-box proteins. The method meets a broad range of criteria required for the biochemical analysis or protein crystallization of plant F-box proteins.

Extended JAZ degron sequence for plant hormone binding in jasmonate co-receptor of tomato SlCOI1-SlJAZ

“…Arabidopsis ASK protein was co-expressed to improve the stability of SlCOI1. 26 As expected, SlJAZ1-3/5-8 but not FLAG-SlJAZ9-11 pull-down the GST-SlCOI1 in the presence of 100 nM JA-Ile (Figure 2a). However, SlJAZ4/13 (which incorporates the same canonical JAZ degron LPIARR as SlJAZ1-3) could not pull-down the GST-SlCOI1 under the same condition (Figures 1b and 2a).…”

Rice DWARF14 acts as an unconventional hormone receptor for strigolactone

“…The full-length O. sativa D3 or A. thaliana MAX2 was fused with His 6 and co-expressed with ASK1, which stabilizes F-box proteins ( Yan et al , 2013 ; Yao et al , 2016 ; Li et al, 2017 ), in sf9 insect cells. After purification by Ni-NTA (Novagen) affinity chromatography, the His-D3–ASK1 or His-MAX2–ASK1 complex was eluted and further purified by HiTrap Q followed by Superdex 200 10/300 in a buffer containing 20 mM MES, pH 6.5, 150 mM NaCl, and 5 mM DTT.…”

Dynamic Perception of Jasmonates by the F-Box Protein COI1

“…JAZ1, TIR1, COI1,and ASK1 (which stabilizes the COI1 protein [Yan et al, 2013]) were cloned into pFast-FH (which includes a FLAG tag inserted into pFast-HTB, Life Technologies). Bacmid preparation and insect cell transfection were conducted according to previous studies (Li et al, 2017).…”