pET vectors, good or bad?

Jeff Kramer jeff at COMPBIO.MED.WAYNE.EDU
Wed Aug 2 12:30:43 EST 1995


Dr. Browning writes:

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>>Mike Holloway wrote:
--------------------------------------------------------------------------------
>>I'm giving up on the pET system and wondered if anyone else has had 
>>similar experiences.
>>	...stuff deleted...
>>Anyone had experience with pET vectors?
--------------------------------------------------------------------------------
>Jeff Kramer wrote:

>I have spentf years of my life trying to get lipoxygenase expression using
>pET 3a while a grad student working on my PhD.  Even after I got expression, >I
>rarely got anywhere near reproducible amounts of protein.  The specific 
>activity was, thankfully, pretty reproducible. ... stuff deleted...So in 
>short, my advice is, give up on the pET vectors.  I believe NEB now sells 
>them, and I love NEB (no affiliation, etc).  I have never gotten anything 
>from them that wasn't excellant (I got the pET 3a before NEB liscenced the 
>system), but I wouldn't encourage anyone to use pET vectors, if another 
>system is readily available...
----------------------------------------------------------------------------

We have used pET vectors to very successfully express several plant proteins 
(mg amounts with activities equal to the native protein), so I do not think 
pET is to blame for problems.  Some proteins just do not express well, fold 
properly, get modified, etc. in E. coli systems.  If there are problems with 
expression, try another form of the pET vector.  We had one protein that did 
not express well in pET3d, but did in another pET vector!  We also have to 
test different host strains to optimize expression.  However, sometimes it 
is necessary to use fusion expression to get some proteins expressed in E. 
coli or in the worst cases bacculovirus or yeast expression is the only 
choice.  Also, some people use co-expression of groEL and groES chaparone 
proteins to get good expression/activity.

If pET does not work for your protein, try something else.  But don't knock 
a system just because it did not work for your particular protein.
Karen S. Browning, Ph.D.
Department of Chemistry and Biochemistry
University of Texas
Austin, TX  78712

512-471-4562
512-471-8696 FAX
kbrowning at mail.utexas.edu

----- End Included Message -----

Perhaps my opinion here is a bit colored by the years of frustration while
working with the pET system, by I have to stick with what I wrote above.  I am
not saying anyone should stay away from pET vectors based solely on my exper-
ience.  I have been collecting complaints from this very net, however, to send
to my old boss.  I have collecyed about ten complaints so far.  I'd be willing
to bet a few others will have more to say on the subject in response to Mike
Holloway's original query yesterday...

								-Jeff

					Jeffrey A. Kramer, Ph.D.
					Wayne State University Medical Center
					C.S. Mott Center for Human Growth and
						Development
					275 E. Hancock
					Detroit, MI  48201
					jeff at compbio.med.wayne.edu
					http://compbio.med.wayne.edu/~jeff/




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