c.surridge
Czuee Morey
Saturday, 03 Mar 2012 12:50 UTC
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!
c.surridge
Dave Dilyx
04 Mar 2012 | 05:28
>Would a change in pH (to 7.0) of only the lysis buffer be sufficient
This is likely your best bet. You’ll need to establish a lysis buffer that yields your target protein in useful amounts and non- aggregated form for further purification. Here’s a reference for a lysis scouting protocol: http://journals.iucr.org/f/issues/2011/09/00/en5458/en5458.pdf ( hi/ low salt, hi, mid, low pH, with/ without detergent etc.)
If your protein still keeps crashing out during further purification you may need to screen more parameters and assay for aggregation. There’s a quick protein solubility screening kit with protocol that does just that: http://www.dilyx.com/protein-solubilization-kit
And finally, if your protein sample is already crashed out, there’s a simple way to test if the precipitation is reversible; protocol here: http://www.dilyx.com/protein-aggregate-solubilization-kit/ . This procedure may help you identify pH, salt, organic, additive etc. that can rescue your entire protein preparation.
c.surridge
Elizabeth McEwen
09 Mar 2012 | 08:07
Thanks for your valuable input Dave. Cannot say about the OP but it definitely helped me a lot! :)