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

ABSTRACT Efficient access to soluble recombinant proteins remains a major bottleneck in biochemical and structural studies. We describe an aqueous, solvent-centric, fully miniaturized 96-well workflow to screen extraction conditions that preserve soluble recombinant protein during lysis and clarification in a single working day. Liquid-nitrogen-frozen E. coli pellets are cryogenically bead-milled with stainless-steel beads, retaining the native intracellular milieu while ensuring uniform disruption. The resulting wet, frozen cell powder can therefore be extracted with user-defined solvent, enabling systematic exploration of pH, ionic strength, detergents, and chaotropes. Protein solubility is assessed by a 1 µL chromogenic anti-His dot-blot. We demonstrate the use of the protocol by solubilizing a set of highly challenging de novo-generated proteins and show that dot-blot intensity provides a practical semi-quantitative proxy for successful extraction of soluble proteins. We also provide experimentally supported guidelines on the influence of solvent reagents on subsequent steps of protein production, SDS-PAGE, and Ni-NTA purification. This workflow is compatible with upstream genetic solubility-enhancement, chassis- and cultivation-based strategies and enables direct transition from screening hits to scale-up. Because the workflow uses standard molecular biology equipment and inexpensive consumables, it can be readily adopted or automated in most laboratories. Competing Interest Statement The authors have declared no competing interest. Footnotes In the revised version, we strengthen our central claim by adding two new application datasets that go well beyond the original mRuby2 benchmarking: A panel of terpene synthases spanning all domains of life (bacteria, archaea, and eukaryotes), including functional validation for one model enzyme (new Figure 3). A set of de novo-designed, aggregation-prone proteins to test performance on difficult targets (new Figure 4). In addition to these new datasets demonstrating the protocol's value on realistic and challenging cases, we also tested and quantified how extraction-solvent composition influences the anti-histidine dot-blot readout, and we provide practical guidance for using the assay in a semi-quantitative manner (new Figure 2). ABBREVIATIONS - SDS-PAGE - sodium dodecyl sulfate–polyacrylamide gel electrophoresis - Ni-NTA - nickel Nitriloacetic acid - IPTG - Isopropyl β-D-1-thiogalactopyranoside - DMSO - Dimethyl sulfoxide - HEPES - 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid - PBS - Phosphate-buffered saline