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I am struggling with the expression of the certain protein. It seems that it is not properly folded and thus, it is not active. I tried to express it at the lower temperature and for the longer time, but it did not help. Can anyone give me a clue what I can try to do?

The size of the protein is around 55 kDa. I know that I have some soluble protein as I run western blot. The protein contain His-taq on the C-terminus so I started with Ni-NTA column chromatography. Steps of purification:

  1. Incubation the lysate with resin (30 min/4°C)
  2. Unbound proteins were collected as the ‘Flow-through’ fraction
  3. Wash 1 with buffer: 50 mM NaH2PO4, 1M NaCl, 10 mM Imidazole
  4. Wash 2 with 10% buffer B in buffer A
  5. Elution with buffer B: 50 mM NaH2PO4, 1M NaCl, 250 mM Imidazole

Because the protein is co-purified with something else in size around 57 kDa (which might be a chaperone protein) I tried also: 1. add ATP to first wash in concentartion of 2.5 mM and incubate column for 1h/4C 2. add ATP to first wash in concentartion of 2.5 mM and incubate column overnight in 4C 3. add ATP to first wash in concentartion of 2.5 mM and incubate column overnight in room temperature 4. add ATP and glycerol to first wash in concentartion of 2.5 mM for ATP and 4M for glycerol and incubate column for 2h in 4add ATP to first wash in concentartion of 2.5 mM and incubate column overnight in 4C

All the method end up with two close to each other bands (only one of them is my protein) and protein is not active.

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What kind of protein? Is this a membrane proteine? Probably even a helical? Or is it toxic? What conditions did you try? –  Chris Jun 16 at 20:48
    
DNA binding protein. I tried express it in 37°C /4 hrs, 20°C/4hrs and 20°C /overnight –  Ala Jun 16 at 20:53
    
You'll have to add a lot more detail if you want to have a chance for any proper advice. All the known properties of the protein as well as your exact expression and purification protocols at minimum. Otherwise you'll only get some guesses and vague advice. –  Mad Scientist Jun 16 at 21:07
    
Thank you, I edited my question. –  Ala Jun 16 at 21:38

1 Answer 1

If you have over-expressed an eukaryotic full length protein or the enzymatic part, then E. coli does not necessarily provide a good environment for its folding so you need to express it in the same system which the original protein came from, e.g. mammalian or Drosophila etc. E. coli over expression is mostly used to generate wast amounts of protein for the purposes of protein sequence based Ab generation amongst other things. Look at this review for some of the solutions it offers for proteins not folding correctly in E. coli when over-expressed (http://www.biomedcentral.com/1472-6750/4/32).

The remainder of your question is a duplicate of this question (Ni-NTA purification, problem with the chaperone protein), which I have provided an extensive answer/comments to!

In addition, since the lack of enzyme functionality is a potential indication of errors in its state/3D structure, it is however not a definitive reason. Could you provide some explanations as to what experiments you have conducted to reach that conclusion and how this second point about chaperone eluding with your protein relevant to the first and the last section of your question?

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Also, to make it easier on yourself. If it is a soluble protein, use a leader sequence that translocates out of the cell. Then there will be no need for lysis –  jwillis0720 Jul 19 at 10:06

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