how to decrease the degradation of protein in purificaton

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    • #12259
      wangzhy
      Participant

      Hello !
      I am working on purification of a human protein in Ecoli, about 100 kD . his-taged. I have tried so many method to decrease the degradation of protein in purification ,such as , low temp induce, all process on ice , protein protease inbihitor,but the degradation is still severe.So I will appreciate that if you anyone can give me a suggesion to decrease the degradation. Thank you very much.

    • #94760
      JackBean
      Participant

      How do you know, it is degraded? Are you sure, it is produced at all? That it’s not put into inclusion bodies? Do you have correct activity measurement (including reall substrates etc.)?

    • #94859
      wangzhy
      Participant
      quote JackBean:

      How do you know, it is degraded? Are you sure, it is produced at all? That it’s not put into inclusion bodies? Do you have correct activity measurement (including reall substrates etc.)?

      thanks for your reply, I have checked by Westernblotting and found it is degradation exactly.

    • #100412
      fenglinbest
      Participant

      Proteins smaller than the target may result from premature truncation during translation or from proteolysis. If these smaller proteins include the fusion tag, they will copurify. Truncation may result from frameshift or ribosomal dissociation when translating through rare codons or through secondary structure in the transcript.
      Before cloning, minimize the introduction of unfavorable secondary structures in the RNA by checking for potential hairpins at cloning junctions or where codon optimization or mutations are planned.

    • #115799
      chance
      Participant

      We commonly use protease inhibitors, such as PMSF (0.2mM), Leupeptin (1ug/ml), EDTA (1mM), A Pepstatin (1ug/ml), Aprotinin (1ug.ml).
      The above are some protease inhibitors and dosage used in recombinant protein purification experiments. You can mix with several protease inhibitors in the experiment, and the effect may be better. Because different protease inhibitors are targeted to different hydrolytic enzymes.

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