1) Crude Extract #1
2) Ion exchange
3) Phenyl Sepharose
4) S-200 Gel Filtration
5) -
6) Crude Extract #2
Questions or Comments?
E-mail me: vetting@aecom.yu.edu
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Expression
of Recombinant Proteins in E. coli by Autoinduction
(Non-IPTG Expression)
This
method was adapted from a presentation at a structural genomics conference
which was telecast on the web. They use this method to cut back on IPTG
use and to reduce the amount of time one needs to monitor cell growth.
One must use an E. coli strain that is competent to take up and use lactose
such as the MET- strain B834DE3 or its parent strain BL21DE3. Your plasmid
also must be controlled by the lac repressor such as pet23a or pet28a
(our favorite vector). It uses a buffered (phosphate) LB like media (more
tryptone though) and three carbon sources: glucose, glycerol, and lactose.
The organism uses the glucose first until it runs out at about an OD of
0.5. The glucose turns off expression until it is gone. After this expression
is turned on by the lactose. The organism then uses the glycerol and lactose
as carbon sources. I am not sure which the organism will use first, the
glycerol or the lactose, or both.
I have generated ODs of 14 in ordinary baffled shaker flasks at 30º (24
hour grow up) and about 8-12 gm per liter of cell paste. As you can see
from the gel the expression is excellent yielding 1 gram of protein from
4 liters (50 gm of cells). The increase in mass one gets from each liter
allows you to either get more protein per grow up or to scale down the
number of flasks per grow up. The method should be applicable to growth
at any temperature. Highly recommended for grow ups where the protein
is expressed reasonable well. Very convenient since you do not have to
monitor the OD.
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Per
Liter
6 gm Na2HPO4
3 gm KH2PO4
20 gm Tryptone
5 gm Yeast Extract
5 gm NaCl2
pH to 7.2 with NaOH and then autoclave
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Filter
Sterilize the Following Stocks
60 % Glycerol
10 % Glucose
8 % Lactose (not very soluble)
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Immediately prior to grow up (after autoclaving) add the following
to each flask
25 mls of Lactose stock (final concentration 0.2 %)
10 mls of Glycerol stock (final concentration 0.6 %)
5 mls of Glucose stock (final concentration (0.05 %)
Add ampicillin to 50-100 mg/ml
Add one colony to each flask
Shake at desired temperature
Wait 16-24 hours
Harvest
Win Nobel Prize
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