Biochemical and Temperature-Related Expression and Solubility of Domain-Truncated BPM1 Variants in Escherichia coli

BPM1, a representative of the plant MATH-BTB protein family comprises three conserved domains—MATH, BTB, and BACK—that facilitate diverse protein–protein interactions central to developmental processes. However, recombinant production of BPM1 and its variants in Escherichia coli are frequently constrained by low solubility and poor stability. In this study, we systematically optimized E. coli-based expression strategies to enable soluble production and purification of domain-truncated BPM1 variants (BPM1ΔBTB, BPM1ΔMATH, and BPM1ΔBACK). A combinatorial approach was employed: varying induction temperature, medium composition, affinity tag selection, bacterial strain, and solubility-enhancing supplements. Expression outcomes were highly dependent on specific parameter combinations. Notably, BPM1ΔBTB—previously the most recalcitrant variant—showed a marked solubility improvement when expressed as a GST fusion in E. coli Rosetta (DE3) cultivated in TB medium supplemented with MgCl2. By contrast, BPM1ΔMATH and BPM1ΔBACK displayed enhanced solubility when expressed in BL21 (DE3) cultivated in 4 × YT medium instead Rosetta (DE3) in 2 × YT medium. Constructs with N-terminal His-tags consistently resulted in poor solubility or failed expression. These results establish a framework for producing otherwise insoluble BPM1 variants and highlight a broadly applicable strategy for handling unstable proteins through tailored E. coli expression systems.