inclusions from pET vector expresion

Les Klimczak lklimcza at mail2.sas.upenn.edu
Tue May 9 13:20:14 EST 1995


In article <hraaxl.6.000AFBF8 at marc.cri.nz>,  <hraaxl at marc.cri.nz> wrote:
>Dear protein expression in bugs sufferers
>
>I'm after ideas. We have a viral non-structural protein expressed nicely using 
>a pET vector. Problem is we get quite variable lysis of the bugs. I'll give 

>6   resuspend in 50 ml: 50mM Tris, pH 8, 1 mM edta, 0.1 M NaCl, 0.1 mM PMSF, 
>200 ug/ml lysozyme, 20 ug/ml DNAase, 20 ug/ml RNAase. Incubate room temp 30 
>min to 3 h.

	It seems that sonication may not be efficient in such a large 
volume. You can lyse your bacteria in about 1/50th of the culture volume
(this would be 4 ml in this particular prep). Please note that sonicator 
probes have upper limits of the volumes they can handle - in particular 
the microprobe, which is standard on many pieces of equipment, cannot 
efficiently sonicate more than 1 ml - you need a larger probe for 
anything larger (with a 5 mm thick probe, you can handle about 15-20 ml). If 
your volumes are even larger than that, French pressure cell is the answer.
	Also, there seems to be little justification for such a prolonged
lysis (sonicated DNA will not interfere); it may decrease your yields and
even inclusion bodies may start getting proteolyzed. 5 min with 20 ug/ml
lysozyme should do the job. 

>8   pellet and resuspend in 5 ml wash buffer: 50 mM Tris pH 8, 0.1 mM NaCl, 10 
>mM EDTA, 1 % triton x100

	Your pellet will be more compact if you centrifuge for 30 min at 
100,000 g - it will be easier to handle in subsequent washes.

>Anyone do freeze/thaw to crack open the cells? Any good? How do you get 

	Freeze/thaw works good, but it will take forever with a large volume.
(For a working protocol similar to yours, you may check Plant Cell 7, 105).

	Good luck

	Les


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Les J. Klimczak               |  INTERNET: lklimcza at pennsas.upenn.edu
Department of Biology         |
University of Pennsylvania    |  Tel.:     (215) 898 3673
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