best method to lyse bacteria for recombinant protein purification

Randy Willis willis at gandalf.psf.sickkids.on.ca
Thu May 29 08:40:11 EST 1997


Malathi Hari wrote:
> 
>What is in your opinion and experience the best method to lyse bacteria
> to purify soluble recombinant protein.
> 
>Freeze thaw and Sonication seem to leave behind a large amount of >unlysed bacteria thus increasing loss of preciuos protein.


Malathi,

I am surprised when you say that sonication is leaving quite a bit of
protein behind in your preparations.  If your protein is truly soluble,
we have always found 80-90% is released in the first round of sonication
(typically 8X30sec cycles on ice).  In any event, we do two to four
rounds of sonication with 30' centrifugations at 13-18000g in between. 
I have found empirically, that cells which are too concentrated or too
dilute in the buffer do not sonicate as well but unfortunately cannot
offer you a (g cells)/(unit vol) conversion factor.

Recently, we have begun to combine freeze/thaw with sonication such that
we leave the cell resuspended in a buffer (pH 7-8) containing 0.5mg/ml
lysozyme on ice for 1-2h and then follow this up with a brief
sonication.  It has also been suggested that people add 1% Triton X-100
or some such detergent but this has the nasty side-effect of absorbing
at 280nm.  This has also been hugely successful in our lab.  The only
fear being the possibility of contaminating proteases in the lysozyme
prep, but we take care of this with a melange of inhibitors.

Another technique which we have used but to which we have limited access
is French Press.  This basically forces your sample through an
incredibly tiny aperture, shearing your cells open.  The nice thing
about this is that you can run your sample through many times and, if it
is at 4C, not worry about unnecessary heating.

Finally, there is always grinding frozen cells with a silica powder,
mortar and pestle...unless you really want some mean bitchin' forearms,
I do not recommend this technique.

Good luck,

Randall C Willis, Researcher
Biochemistry Research, Hospital for Sick Children
3522-555 University Ave
Toronto, ON
M5G 1X8  CANADA

416-813-5933 (ph)  -5022 (fax)
willis at gandalf.psf.sickkids.on.ca



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