His-Tag Recombinant Protein purification

Protein Chemistry Protein Isolation / Purification
1

Hello,

I am purifying a six-His tagged 50kDa hnRNP K protein from E coli cells using IMAC with Cobalt TALON resin. I am trying to send the proteins for crystallography and it requires a good quality of sample. My problem is that smaller bands are visible on the gel and I don’t know how to get rid of them.
What might the problem and how can I fix it? Can using a whole range of gradients (50, 60, 70, 80, 90, 100, 110mM, etc.), or adding imidazole to the wash buffer improve the specificity of the resin-bound proteins?

Thank you and appreciate your help!

2

Barry It seems like you’re facing contamination from degradation products or non-specific binding during your IMAC purification of His-tagged hnRNP K. Here are some possible explanations and solutions:

Protein Degradation (Proteolysis)

Smaller bands could be proteolytic degradation fragments of hnRNP K. Solution:

  • Add protease inhibitors (e.g., PMSF, EDTA-free protease inhibitor cocktail) to all buffers.
  • Keep purification steps at 4°C and minimize processing time.
  • Reduce freeze-thaw cycles and avoid extended storage at room temperature.

Poor Column Washing

  • Impurities may not be sufficiently washed out before elution.
  • Solution:
    • Extend the wash steps (e.g., wash with 5–10 column volumes instead of 3).
    • Include high-salt buffer washes (e.g., 300–500 mM NaCl) to disrupt weak non-specific interactions.

Co-Purification of Truncated Protein Isoforms

  • hnRNP K may have alternative start sites or degradation-prone regions in E. coli.
  • Solution:
    • If you suspect truncation, confirm the full-length protein by Western blot using an anti-His or specific hnRNP K antibody.
    • If truncation is an issue, consider optimizing E. coli strain selection (e.g., BL21(DE3) pLysS) to reduce degradation.
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3

It also could be a lot of non-specific interactions on the column. As said previously, I’d increase the number of washes. But in addition to that, you could increase the starting concentration if imidazole during binding step to 20 mM, increase wash step concentration as well. You will likely lose protein yields but it will certainly be cleaner.

In addition to this, if you have access to another column, running it on SEC would fix it fairly fast as the separation will be apparent. I’d suggest using an S75 column if your protein of interest is 50 kDa and behaves as a monomer.

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