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recombinant protein is not in the supernatant after celllysis

recombinant protein is not in the supernatant after celllysis - Recombinant Protein Forum

recombinant protein is not in the supernatant after celllysis - Recombinant Protein Forum. Discuss purification, expression, and isolation of recombinant and cloned proteins here.


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Old 12-13-2009, 10:17 AM
Pipette Filler
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Unhappy recombinant protein is not in the supernatant after celllysis



Hello,

I tried different methods for celllysis after the expression of the recombinant protein (human interleukin-8). The expression works well but I don't have the protein in my supernantent after celllysis (E.coli).

I combine tris or phosphate buffer with 50mM NaCl and 0,2mg/ml Lysozyme (+1mM EDTA). I add proteaseinhibitors and DNase. After the 1.30h incubation on ice the samples are put into a sonication-waterbath for 30 min. After sonicationbath I make a frozen/thaw cycle with liquid nitrogen (30 min).
the supernatant doesn't have the recombinant protein. It's always in the pellet of the cellysat.
What is wrong?
A colleague told me that it needed higher salt concentration but the authors in many papers do also use between 40-200 mM NaCl. When i use more salt i can't use it for the cationexchanger. During or after Desalting it is going to precipitate. My protein should be soluble!
I guess i have inclusion bodies but I don't want to purify them - it's to difficult!!!

Please help me! I've been working on that sh... since april and i am totally frustrated.
I'll be happy for every answer.

thanks
Trypsin
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Old 04-04-2010, 03:51 PM
Pipette Filler
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Default Re: recombinant protein is not in the supernatant after celllysis

I went through the same frustrations. You may simply need to accept that your protein is insoluble...at least under the current induction conditions. Purifying IBs is not that bad, if you don't need large quantities then magnetic bead purification is super easy. You just need to do the dialysis afterwards. If you really want to try to produce soluble IL-8, you could trying reducing the culture temp to 30 and cut the concentration of the inducer in half, or 1/4. Hope this helps
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