Dear Hossein Samiei Abianeh

which is the pI of the expressed construct? which is the concentration of your sample?

Did you performed a SEC of this sample to see if you have a single peak?

It seems that you protein is at least partially aggregate since 30nm is really huge for a 40-50KDa protein (if you consider that an human igG1 mab that is 150Kda is generally around 12-13nm in DLS)

You can try to use different buffers in terms of ph and ionic strenght and see if it change something or not.

I do not think that the trx presence justify this difference in MW, even if you can certanilly try to remove it, if in your construct is present a digestion site for a protease (ad TEV or Fatt.-xa) and check if something change.

best

Manuele

Dear Manuele Martinelli

The theoretical pI of my expressed construct is around 9.3, and I’m running the DLS experiments in 100 mM carbonate buffer at pH ~9.5, which is slightly above the pI. Do you think this could be promoting aggregation of the protein?

As for the protein concentration, the initial DLS measurements were performed at 700 µg/mL. To rule out concentration-dependent aggregation, I also tested a diluted sample at 150 µg/mL, and although the PDI decreased slightly to ~27%, the hydrodynamic diameter remained largely unchanged, suggesting the presence of stable oligomers or aggregates rather than reversible concentration-dependent self-association.

Olá, Hossein Samiei Abianeh Hossein! think the pH of your buffer is too close to the pI of the protein.

Two things:

i) after purification, do you see any turbidity in the protein solution?

ii) are you centrifuging your sample before doing the DLS analysis?

I had the same problem as you (I think): I didn't achieve a PDI lower than 0.2, and my hydrodynamic radius was around 7 nm (I work with a 39 kDa protein, which is a tetramer).

If you haven't done a centrifugation, try it!

@Bruna Ukrainski

Dear Bruna Ukrainski,

Thank you for your advice.

I performed the purification under denaturation conditions using urea. Following that, I dialyzed the sample under a gradual gradient, and any aggregation was removed by centrifugation at high speed. Despite these conditions, the size of the particles was around 30 nm.

Currently, I am in the process of adjusting the extraction buffer, changing the pH from 9.5 to 7. I hope this modification will yield better results, and I will share the updated data with you here once I have it.

Best regards,