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Use of CE6 phage

Stallion vader at eskimo.com
Sat Jul 23 15:07:56 EST 1994


Hi there,

I recently acquired some CE6 phage from Novagen to do protein expression 
of a fairly toxic protein that has been torturing me for some time.  The 
.2 ml aliquot that I bought was basically an innoculum from which to 
make stocks.  My first attempt with their low MOI protocol ended with the 
culture lysing after about 30 min :-(

There were two reasons for this failure:  the titer of the CE6 stock was 
really twice what they stated, and their protocol left out what they call 
20X Phage Salts (CaCl2, FeCl3, glucose) that should be added to final 
concentration of 1X at the time of infection.  Anyway, the next attempt 
went much better and I attained a lysate with a titer of about 3 X 109 
pfu/ml.

So I did an induction using my CE6 stock and using Novagen's protocol:
*******************************
The use of minimal medium permits labeling of target proein with 
methionine S35.  If a rich medium is preferred, LB or ZY can be 
substitued.  The addition of glucose (Step 2 below) is not necessary in 
rich media.

1.  Grow the host cells (here TOP 10' from Invitrogen) containing the 
target plasmid in M9 (here LB) supplemented with 0.2% maltose and 
antibiotic (here ampicillin 100 ug/ml).

2.  When the OD600 of the culture reaches 0.3, add glucose to a final 
concentration of 4mg/ml (only needed with M9 medium, as noted above).

3.  Continue growing the cells for 1 to 2 hr, or until the OD600 is 
between 0.6 and 1.0.

4.  Add MgSO4 to a final concentration of 10mM and add the CE6 stock to a 
final concentration of 2-4 x 109 pfu/ml.

5.  Continue growing the infected cells for 3 hr.

6.  Harvest cells by centrifugation and purifty targetg protein by 
desired method.

Notes:
a.  The concentration of cells at the end of Step 3 should be about 5x108/ml.

b.  The MOI should be between 5 and 10 to ensure that almost every cell 
is infected, but to prevent the inhibition of protein synthesis which is 
observed at higher MOI.
*********************************

This produced much more protein than I had previously been getting when 
using BL21 (DE3) or BL21 (DE3) pLysS and IPTG.  Still, there are some 
questions that I had.  I have pored over Sambrook and also Studier and 
Moffatt's paper, "Use of Bacteriophage T7 RNA Polymrease to Direct 
Selective High-level Expression of Cloned Genes" J. Mol. Biol. (1986) 
189, 113-130 (BTW a very nice read) but I still have some questions.  

I would appreciate responses to *any* of the questions below either by 
posting in this group or via email.
Thanks!
 --------------------
1.  What is your favorite phage stock preparation protocol?

2.  Do you purify your lysate by doing a PEG precipatation/CSCL 
spindown in an effort to either concentrate their stock or purify it, or 
both?  After making my CE6 stock (using Novagen's protocol) I had a 
lysate of about 3 x 109 pfu/ml which necessitated I add equal volumes of 
culture to CE6 to attain an MOI of about 4.  Ideally I would have 
preferred a titer in the 10E10 range since you have to induce at an MOI 
of 5-10. 

3.  What is your favorite method to store your CE6 stock?  

4.  What is your favorite broth (non-protein labeling) in which to grow 
your cells that you will be inducing?

5.  Is there any benefit in adding glucose to the culture at an O.D. of 
.3 @ 600 nm when using a rich broth such as ZY or LB?  When growing in M9 
+ maltose Novagen recommends adding glucose at this time but their tech 
help didn't know why they didn't recommend it for the richer broths.

6.  After inducing with CE6 is there normally any cell lysis that takes 
place?  (I know there *shouldn't* be any but any personal experiences 
that indicate there is some due to CE6 and *not* the result of the 
induced protein?)

7.  What is your favorite host strain to keep your plasmid in which you 
will be inducing?  Any strains to watch out for?

8.  Has anyone used TOP 10 F' cells (from Invitrogen) for the host strain 
in which to do the CE6 induction and what were their experiences with its 
performance? (sorry but I don't have the genotype right here but it is *not* 
deficient for the ompT and lon proteases.  This did not *appear* to make a 
difference when I ran the protein on a SDS gel to check it, but I am 
uncertain if it will show an influence in the subsequent purification steps).

9.   For S35 methionine protein labeling of your induced protein, what is 
your favorite protocol?  Also, is the background reduction offered by a 
met auxotroph such as B834 a worthwhile reason to switch strains?

10.  Do you have any papers to recommend concerning CE6's use?

Thanks again!

Pat Durham
email:  vader at eskimo.com





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