fusion protein expression vectors

Laura J. Weislo mcp at canctr.mc.duke.edu
Wed Jan 20 10:40:18 EST 1993


In article <1jhuaqINNd81 at gazette.bcm.tmc.edu>, brianb at bcm.tmc.edu (Brian J. Baumgartner) writes:
>We would like to create fusion proteins for synthesis of antibodies.  We
>must be able to cleave our desired peptide from the fusion product and
>affinity purify the fusion.  Several companies make these types of
>prokaryotic expression vectors.  We would appreciate any comments on which
>vector has proven the most effective for you.  No sales reps please!
>
>Thanks, 
>
>Brian Baumgartner
>Dept. of Biochemistry
>Baylor College of Medicine
>Houston, Tx. 77030
>e-mail: brianb at mbcr.bcm.tmc.edu

I've tried the pET16b vector from Novagen that fuses your
protein to a poly HIS tag for purification and allows cleavage
with factor Xa.  Expression is via T7 polymerase when you
transform the plasmid into a lysogen carrying T7 pol gene.
The only problem is you constantly have to test to make
sure you aren't losing plasmid or T7 pol expression/. I
had a problem with losing expression over time, but it
may not have been inherent in the vector. 
 NEBL sells a
vector pMAL2 that fuses proteins to maltose binding protein
and allows cleavage with Xa as well.  This vector has the 
advantage of also having a 3' B-gal sequence that allows
blue/white selection for your initial subcloning.  Both
systems have affinity resins that are available as a kit, 
and are supposed to be easy to use, although I haven't gotten
past the expression problems as of yet!  If you don't care
about functional protein, whioch you may not just for Ab
production, you may not have to worry as much as I have about
the expression...
 One last point, the poly HIS tag is used
as an affinity tag for immobilized Ni++ ions, and if your
protein normally binds iron or zinc, this could be a problem.
Also, superoxide dismutase is a contaminant in purification
via this method from e. coli.
Hope this helps!!! 

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	!!!!			Laura J. Weislo
	 ^^			Duke University
	 @@			Dept. of Chemistry
	  )			Weisl001 at dukemc.bitnet
	 0			Weisl001 at mc.duke.edu
	"YOU'RE MY FORTUNE COOKIE PRIZE" - BEAT HAPPENING
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