A miniaturized, high-throughput buffer-centric method for protein solubility screening

Efficient access to soluble recombinant proteins remains a major bottleneck in biochemical and structural studies. We describe a buffer centric, fully miniaturized, 96-well plate workflow to map protein solubility across a large number of conditions in a single working day. Liquid-nitrogen-frozen E. coli pellets are cryogenically bead-milled with stainless-steel beads at sub-zero temperature, retaining the native intracellular milieu while ensuring uniform disruption. The resulting dry powder can therefore be extracted with any buffer of choice, enabling systematic exploration of pH, ionic strength, detergents, and chaotropes. Protein solubility is quantified by a one-microliter chemiluminescent anti-His dot-blot. Using a His-Ruby2 reporter panel, we show that dot-blot intensity can be used as a quantitative proxy of protein solubility. We also provide experimentally supported guidelines on the influence of those buffer reagents on subsequent steps of protein production, SDS-PAGE and Ni-NTA purification. This workflow is compatible with upstream genetic solubility-enhancement strategies, and enables direct transition to scale-up, the same day that soluble hits are identified. Because the entire workflow uses standard molecular-biology equipment and inexpensive consumables, it can be readily adopted or automated in any laboratory.