How Can I Stop My Recombinant Proteins from Forming Aggregates

Protein Chemistry
1

hi

When expressing recombinant proteins, I often end up with insoluble aggregates. I’ve tried changing expression hosts and temperatures, but solubility is still an issue.

What strategies have you found most effective to improve protein solubility?

Regards

2

Hi Harman

Protein solubility during recombinant expression is a common challenge—especially in bacterial systems like E. coli. As a first step, You can use lower IPTG Conc.(0.05–0.2 mM); too much can overwhelm the system.

You can also prolong the expression time and use longer expression duration at lower temperatures (e.g., overnight at 16°C).

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3

Harman

Sometimes Chaperones help fold difficult proteins. You can co-express with GroEL/GroES, DnaK/DnaJ/GrpE, or trigger factor using plasmids (e.g., Takara pG-KJE8). These work best in E. coli strains that are optimized for chaperone expression.

If your experiment with E. coli fails consistently, try using alternate expression systems like

  • Yeast: Pichia pastoris often improves solubility for eukaryotic proteins.

  • Insect cells: Baculovirus systems allow proper folding and PTMs.

  • Mammalian cells: Ideal for complex proteins needing native modifications.

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4

Thanks Gagan and Navneet

We have tried different concentrations of IPTG from .02mM to . 0.3mM. However, there is no change in the protein status albeit the yield changes with concentration.

We shifted to Insect Cell Lines from Bacteria but the problem is still persistent. I will try coexpression with a Chaperone and update.

Any other suggestions?

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5

You have to tackle this step by step. Next step would be to use the Fusion Tags Adding solubility-enhancing tags can dramatically improve solubility

MBP (Maltose-Binding Protein) – One of the most effective solubility-enhancing tags.

GST (Glutathione S-transferase) – Also improves solubility and aids in purification.

SUMO / NusA / Trx (Thioredoxin) – Helpful for proper folding.

Make sure to use cleavable linkers so you can remove those later.

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6

Hi

Do you require the protein for its functional assay? If not you can solubilize protein using 6M guarding-HCl and then gradually resold by dialysis or dilution.

You can use Arginine, glycerol, Triton X 100 to help refolding the protein

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