Protein expression: insoluble protein

1. Ranked causes

   1. Missing chaperone / solubility tag for this domainMBP/SUMO or chaperone co-expression solubilises the same ORF.
   2. Induction temperature / rate too aggressive for foldingLowering induction temperature (e.g. 37→18°C) moves product to the soluble fraction.
   3. Lysis buffer incompatible (pH, salt, detergent) with this proteinBuffer screen shifts the soluble band without changing induction.
   4. Over-expression level drives aggregation (plasmid / inducer too strong)Weaker promoter or lower inducer increases soluble yield.

2. Recovery steps

   1. Induce at lower temperature (e.g. 16–20°C) for a longer timeSlower folding often rescues otherwise insoluble products.
   2. Add a solubility tag or co-express chaperones; re-test soluble fractionMaterial redesign when temperature alone is insufficient.
   3. Screen lysis buffer pH / salt / detergent on small aliquotsSome “insoluble” pellets are buffer artifacts.

3. Controls first

   • Did a soluble positive-control protein behave normally in the same lysis while this ORF stayed insoluble?If yes: Folding / solubility problem for this construct — not a global lysis failure.
   • Is total (lysate) expression visible while the soluble fraction has no product?If yes: Folding / solubility problem — not a no-expression case.

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