Generation of purified rek. Ad5 stocks

Dr. Jeney Csaba jencsa at net.sote.hu
Fri Aug 22 04:44:04 EST 1997


wirts000 at GOOFY.ZDV.UNI-MAINZ.DE wrote:

>Hi all,

>I'm searching for good and detailled protocols for the purification of
>recombinant Ad5 viruses amplified in 293 cells. How are the conditions
>for CsCl-Ultracentrifugation or is there a more convenient alternative ?

>Thanks in advance !

>Stefan Wirtz
>1st Medical Department
>University of Mainz, Germany
>Internet : wirts000 at mail.uni-mainz.de

There is a lot of alternativ methods, the best on the basis of my
experience is the following:
infect the cells with at least 100 MOI and harvest them 72 hours later
with centrifugation (5 min 200 g 4 C) discard the supernatant (can be
saved for inoculum, but the good laboratory practice is to use
concentrated, purified stocks for infection) and apply five cycle of
freeze-thaw on the sediment (the supernanant contans a lot of virus,
but it is about 1/100 of the whole virus yield and the large volume
makes it invaluable for preparative purposes).
To open the cells totally and denature most of the proteins in the
prep without adding volume we use freon extraction. One volume of
freon added and shake the mixture for 5 minutes, to separate the
phases centrifuge (5 min 2000g 4 c) (be careful the freon will built
up pressure which can cause infectious vapor spill), the upper layer
contains the virus with other soluble proteins, the interphase
contains the denatured proteins and the lower one the freon.
Pipette out the the upper layer without diturbing the interphase and
extract once again.
The freon should be properly discarded and before that should stored
at 4 C.
We purify the crude lysate with UC applying preformed gradient which
is only ON run. For Beckman SW41Ti rotor we have recipe:
Gravity of CsCl              1.42         1.31         1.2        1.11
% 1.53 g/ml CsCl           80             60            40         20
% 10 mM pH 8.0 TRIS  20             40            60         80

Az 1.53 g/ml gravity CsCl solution is 718.6 mg/ml in 10 mM pH 8.0
TRIS.
Layer 2ml of each gravity of CsCl solutions carefully on the top of
the other and fill up the tube with the crude lysate. Centrifuge 30 K
ON 4C. After it there would be an upper red part, this contains the
proteins of the prep (it contains a lot of adanovirus stuctural
proteins) and between the half a the lower third of the tube there
would be two sharp bands the lower one is full capsid the ussually
fainter upper band is the so called incomplete or empty capsid, but
this is also infectious. We suck out the bands puncturing the tube
with a needle attached to a syringe. A second cycle of UC is worth the
time and for this we dilute the virus with 10 mM pH8.0 TRIS 2 times
and apply on the top of the same gradient.
Add 15% glycerol and store at -80 C. It maybe desirable to remove the
CsCl from the prep, but usually it is not necessary bacause before the
experiment you should dilute it at least 100 times it means that the
final CsCl concentration will be 30 mM and during the storage the CsCl
even has a stabilizing effect.  But if it counts an ON dializis
against 10 mM TRIS pH 8.0 is the practice but it drops the titer.

Good luck and if you have any question e-mail me.

Csaba




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