Structure‐guided optimization of SLY1 expression and purification in Escherichia coli

The SCFSLY1 ubiquitin ligase complex is a key regulator of gibberellin signaling, mediating the degradation of DELLA proteins through targeted ubiquitination. To enable detailed biochemical and structural studies of this complex, efficient recombinant production of SLY1 and its adaptor ASK1 is essential. In this study, we optimized the co-expression and purification of the ASK1-SLY1 complex in Escherichia coli, focusing on molecular determinants that influence solubility, stability, and protein interaction. Hydrophobicity analyses and AlphaFold structural modeling of ASK1 and SLY1 identified prominent hydrophobic surfaces, particularly around helix α7 of SLY1, which are involved in DELLA binding and potentially driving aggregation during purification. Based on these insights, we adopted a co-expression strategy with an MBP tag fused at the N-terminal end of one partner, which significantly enhanced solubility and enabled successful isolation of the intact ASK1-SLY1 complex. Overall, this work presents an optimized protocol for recombinant production of the ASK1-SLY1 complex and provides structural rationale for overcoming key challenges in its expression and purification.