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Expression of protein with pI 8.2

Expression of protein with pI 8.2 - Recombinant Protein Forum

Expression of protein with pI 8.2 - Recombinant Protein Forum. Discuss purification, expression, and isolation of recombinant and cloned proteins here.


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Old 03-03-2012, 12:01 PM
Pipette Filler
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Default Expression of protein with pI 8.2



Hi,

I am trying to purify a protein whose pI is in the physiological range, which is generally the pH range for most buffers and growth media used in the lab.

Does anyone have any tips on how I could purify this protein.

I actually tried to express and purify this protein, but ended up isolating a bacterial protein artifact! From the results of my first analytical purification, it seems that because the protein is expressed in LB (which I guess has a pH near 7) it aggregates soon after expression.

Should I change the media or adjust the pH of LB with buffer? What pH should be used so that the bacteria can grow happily and my protein doesn't aggregate?
Would a change in pH of only the lysis buffer be sufficient rather than changing the bacterial growth pH?

The expression and purification buffers I used last time were as follows:

Growth medium : LB
Induction : 1mM IPTG
Lysis buffer: 20mM Tris-HCl pH 8.0, 100mM NaCl, 1mM DTT, 1mM EDTA, MgCl2, PMSF, lysozyme, TritonX, DNase

Any help, suggestions and comments are welcome!
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Old 10-18-2012, 09:58 PM
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Default Re: Expression of protein with pI 8.2

I don't know that the pI is the issue here. Even if you change the pH of the media the cell maintains a certain homeostatic pH in the cytoplasm in order to survive. I've isolated proteins with pI close to this and had no problems with it. Have you tried isolating soluble and insoluble fractions? If is aggregating inside the cell then most of your protein will be in the form of inclusion bodies which you might just have to deal with. There are steps you could take to increase the solubility of your protein however (lower induction temperature and lower IPTG concentrations). If your protein has disulfide bonds you might want to try changing your host (T7 shuffles)
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bacterial culture , expression , media ph , protein , protein pi , purification


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