A miniaturized, high-throughput aqueous solvent-centric method for protein solubility screening
This article has been Reviewed by the following groups
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
- Evaluated articles (Arcadia Science)
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.
Article activity feed
-
extraction buffers are dispensed
what volume of extraction buffer do you use here? Information on the ratio of 'dried pellet material' to extraction buffer would be helpful here.
-
dot-blot signal
What are the advantages of the dot blot over a conventional sds-page analysis? SDS-PAGE likely takes the same amount of time as a dot blot, but potentially gives you more information about each sample.
-
provided in
This might just be an artifact of biorxiv, but I don't see a table description anywhere. What were the adverse reactions for the SDS-PAGE? Did the gel just not run straight or stain evenly? Also, what are the units for purification yield?
-
suffice
If you are growing the cells in a single flask, at which step do you split up the pellet into a 96-well plate?
-
d than optimized IPTG
It makes sense that you use the auto-induction for more high throughput purposes. After finding an optimal condition and scaling up, would you recommend switching to IPTG? Would you expect any changes?
-
