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Expression of protein with pI 8.2

expression purification protein pI bacterial culture buffer pH

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#1 eeuzc

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Posted 03 March 2012 - 04:53 AM

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 (to 7.0) 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!

#2 mdfenko

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Posted 05 March 2012 - 01:12 PM

since your protein's pI is 8.2, you should bring your lysis buffer pH closer to neutral to ensure that the protein doesn't come out of solution (assuming that the protein is not in inclusion bodies).
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