SUMMARY: bacteria for pGEX exp.

Morris F. Manolson mm6y+ at andrew.cmu.edu
Sun Oct 31 15:53:51 EST 1993


Dear Netters, what follows is a summary of responses to my question on which
bacterial strain to use for expressing a pGEX fusion protein that is quickly
being degraded.  The most common response I received was for the
bacterial strain BL21 which is lon- (protease deficient).  There were
also two posting refering to lon- strains (k12 CAG629) FREE from NEB (if
you buy something else first, mind you).  Several people also suggested
inducing with IPTG at a lower temp., ie: 24 or 30 degrees C instead of
the 37 degrees normally used.  Thanks one and all for your helpfull
suggestions!  Morrie

Morris F. Manolson
Div. of Cell Biology
Hospital for Sick Children
555 University Ave.
Toronto, Ont., Canada, M5G 1X8
TEL:   416-813-6662 (office)
       416-813-5594 (lab)
FAX:   416-813-5028
EMAIL: morrie at resunix.ri.sickkids.on.ca

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What temperature, time and IPTG concentration do you induce at? 
I've found that at 30 degreesC, 30 min incubation, and 0.5mM IPTG
followed by rapidly chilling the cells on ice,
I was able to recover a GST fusion that was almost undetectable
with other conditions.  You may have tried this already of course,
please let me know what other tips you may pick up regarding
lon- cells.
Cheers Shoumo 
Shoumo Bhattacharya
MRC Molecular Medicine Group
Royal Postgraduate Medical School
Hammersmith Hospital, London UK

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You might want to try BL21.  I believe it was developed by John Studier's 
group at the Brookhaven National Lab.  
 
Good luck,
 
Alan @ Pharmacia

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In my lab we use BL21 strain. The same one that is supplied with the pET 
expression system by Novagen, 565 Science Dr.,Madison, WI 53711 USA. The 
strain is lon-. We used it with much success. However, there are 3 types 
of this strain. BL21 which the non-expression host for the pET system. 
BL21(DE3) and BL21(DE3)pLysS are the expression hosts for the pET system.
We used the non-expression host for our expression of proteins with pGEX.
 
Hope this helps.
Blessings to you and your work
Genie
 
--
|Jin Ngee, Chia			Chemical Carcinogenesis Laboratory	   |
|(Genie, the OligoMan)		Institute of Molecular and Cell Biology	   |
|mcblab47 at leonis.nus.sg		National University of Singapore	   |
|Tel: (065)772-3797		Kent Ridge Crescent			   |
|Fax: (065)779-1117		Singapore 0511				   |

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-Strains CAG629 and CAG626 are available from New England BioLabs, free
with
a restriction enzyme order or for the cost of shipping without an order.  I
use CAG 629, which is temperature sensitive.  Plasmids must be passaged
through a strain such as JM109 to be methylated before transforming into
CAG626 or CAG629, but when this is done, standard transformation protocols
work nicely.
 
Good luck.
 
Anne Savitt
Department of Microbiology
SUNY Stony Brook
Stony Brook, NY   11794-5222
email:  asavitt at sunysb.edu

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-We have used the TOPP strains of E.coli from Stratagene with some
considerable
success.  These are non-K12 coli, and, as stated in the Stratagene catalogue,
different TOPP strains benefit the expression of different proteins.  In the
case of GST fusions we have made, sometimes you can enhance expression levels
and other times you can reduce breakdown.
Their only drawback is that they are very hard to make significantly
competant (if you buy them competent from Stratagene they only guarantee a 
competence of >10^4 colonies/microgram): so do your cloning in something else
and then transfer to the TOPPs to optimise expression.
It's easy to test six strains, and the effects can be dramatic.
 
Regards - Anthony.
 
 
Anthony Davies                            adavies at cgl.ucsf.edu
Department of Pathology			  tel:(415)-476 8340
University of California, San Francisco	  fax:(415)-476 9672  

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-I've recently acquired a cell strain E.coli k12 CAG629 from NEB (free!).
Genotype : lon rpoHam ; hence is lon- and has temp-sensitive amber supressor.
Maybe you could try using this.
 
Sor-Cheng Lee
Lees at uk.ac.afrc.jii
John Innes Institute
U.K.

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-We have used BL21(DE3)/pLysS (self-lysing, lon-) for use with pET14b
and TG1 
with a pTrc99A derivative.



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