Pinpoint XA expression system

T. S. Pillay tpillay at ucsd.edu
Thu Feb 24 02:10:06 EST 1994


Subject: Pinpoint XA expression system
From: danismit, danismit at dante.nmsu.edu.
Date: 21 Feb 1994 05:45:55 GMT
In article <2k9hqjINNae8 at dns1.NMSU.Edu> danismit,
danismit at dante.nmsu.edu. writes:
>We've been trying to get this expression system working in our lab for
>about a month but have not gotten any positive results.  We've checked
>the constructs and the protein is in reading frame.  We see the
>naturally biotin labeled protein of E.Coli but can't seem to find our
>protein of interest.  The same insert placed into a PET22B his tag
>expression vector works well.  Our current thought is we are not using
>a good expression strain.  Tried DH5alpha and XL1 BLue.  ANy
>suggestions would be helpful.
>
>
>Thanks
>Dan Smith
>New Mexico State Univ

Dan
I've had some success with the Pinpoint Xa system.  I was attracted to it
because of the biotinylation tag which provides a  "handle" for purifying
as well as detection.   
I cloned a 1200 base pair insert into Xa cut with Nru1 and Bgl II (not
the easiest way to do things-I think I was being a bit masochistic-but
there are reasons for that -the Nru I site is at the Xa cleavage junction
hence one has the potential for cleaving the biotin tag and being left
with the protein of interest without any extra amino acids).
Well to cut a long story short-  DH5 alpha is not a good strain because
it does not have the lac repressor hence one cannot see induction easily
and toxixc proteins will not be repressed (DH5alpha can be used with pGex
because the plasmid carries the lac 1q gene).  I've expressed my
constructs in XL1 blue (which has lac 1q) and in  JM 109 (also lac1 q)
successfully.  The way I screen is to use antibiotin-peroxidase  rather
than strepavidin-peroxidase ( and ECL detection or similar).    This
gives cleaner blots and also has the potential for stripping and
reprobing.  I run the Xa-CAT control in parallel (this comes with the
kit)  so that I know everythings working.  

I have noticed the following:  Staining gels with Coomassie is not
reliable -even though the Coomassie looks negative with Xa,  you can
still have massive overexpression when you do Western blotting.  The
Xa-CAT control is expressed very well in XL1 blue and JM109, even the
basal expression is very high, probably because it's a high copy plasmid
and there's not enough lac1 q around.  My construct has an Mr of 80 kDa
on SDS-PAGE.  Basal expression is low but detectable and goes up
massively with IPTG.  Also I grow cultures in broth with 2% glucose  to
maximise catabolite repression before induction.
Preliminary evidence indicates that the protein I've expressed has
enzymatic activity in vivo and in vitro-  so the system can be made to
work.  
If you need more info-feel free to contact me. 

T. S. Pillay (tpillay at ucds.edu/  tel:  619-552-8585 ext 7148  fax: 
534-7181)



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