GST-fusion yield 50mg/0.5 lt culture?

[Didier Thomas]

In article <vurquidi.1195365225A at news.ox.ac.uk>, vurquidi at molbiol.ox.ac.uk
(Virginia Urquidi) wrote:

> I'd appreciate the comments of those with experience in the subject.
> 
> The protein yields for GST fusion proteins according to the Pharmacia
> purification protocols are the following:
> 
>                         50mg    10mg    1mg     50ug
> Culture volume          20lt    4lt     20ml    1ml
> 
> Someone has just said to me that he produced 50mg of pure protein out of
> half a liter culture. Is that feasible?
> 
> Also, the 0.5 lt culture was inoculated with the total of the 50ml overnight
> culture (instead of the recommended 1:100 dilution). Is this sensible?  
> 
> Is the OD600 between 0.5 and 2 adequate for induction? What would happen if
> you induce at much higher cell densities?
> 
> Thanks in advance for your comments
> 
 Virginia,

never expect this kind of yield. 50 mg of gst fusion protein per .5 l of
culture is a joke !!

Induction should be started on an exponentially growing culture (OD ~ .5
is OK) and should last at least 3 hrs.

Temperature : 30 or 37 C depending on your protein.

Keep in mind that the yield depends also on the size of your fusion
protein. The longer the less you get in a bacterial system.

Good luck.

Didier

-- 
Didier Thomas
University of California, Irvine
College of Medicine
Dept. of Biological Chemistry
Med. Sci. I, Room D214
Irvine, CA 92717
USA