GST yeast vectors

Christopher Hug chug at CELLBIO.WUSTL.EDU
Fri Oct 15 14:10:53 EST 1993


There seems to be some interest in GST fusion protein vectors that work in
yeast.  Here's a brief description of some that I have made and some of the
results that I have obtained.  The description follows that of galactose
inducible vectors.


Inducible Yeast Expression Vectors and Yeast GST Fusion-Protein Vectors

        1.  High-copy galactose-inducible yeast expression vectors.
        
pRS 423-GAL1/10         (HIS-3) pBJ-243 ATCC# 77449
pRS 424-GAL1/10         (TRP-1) pBJ-244 ATCC# 77450
pRS 425-GAL1/10         (LEU-2) pBJ-245 ATCC# 77451
pRS 426-GAL1/10         (URA-3) pBJ-246 ATCC# 77452
pBS-GAL1/10             (SK-)   pBJ-247 ATCC# 77453
        -These  vectors were constructed by inserting the GAL1/10 divergent
promoter cassette (as an EcoR1-BamH1 fragment) into the EcoR1 and BamH1
polylinker sites of the  pBlueScript derived 2 micron pRS 420 series
vectors (Christianson, Sikorski, Dante, Shero, and Hieter. Gene 110: 119-22
(1992)) to provide high copy galactose inducible yeast vectors with
convenient cloning sites.  Note that no transcription terminators are
provided.  
        -The  GAL1/10 promoter cassette clone was provided by Dr. Mark
Johnston.   The GAL1 promoter is adjacent to the BamH1 site and  the GAL10
promoter is  next to the EcoR1 site; the complete cassette is 685 bp.  
        -The two promoters are of similar strength.
        -Avoid palindromic restriction enzyme sequences between the start
of transcription within the GAL1/10 promoter and the start of translation
by placing the initiating AUG of the insert close to the GAL1/10 cassette
possible.  
        -Check for premature AUG/termination codons 5' to cloned insert.

        2.  Inducible glutathione-S-transferase fusion protein yeast
expression vectors for affinity chromatography on glutathione agarose.

pRS 423-GAL1/10-GST     (HIS-3) pBJ-381 ATCC# 77455
pRS 424-GAL1/10-GST     (TRP-1) pBJ-382 ATCC# 77456
pRS 425-GAL1/10-GST     (LEU-2) pBJ-319 ATCC# 77457
pRS 426-GAL1/10-GST     (URA-3) pBJ-383 ATCC# 77458
pBS-GAL1/10-GST (SK-)   pBJ-384         ATCC# 77459
        -pBJ-319 was constructed by PCR  of the bacterial expression vector
pGEX-3X (Smith and Johnson, Gene 67: 31-40(1988))  using a 5' primer that
incorporated a BamH1 site prior to the initiating AUG and a 3' primer
homologous to sequences following the stop codons of the GST coding
sequence.  Digesting the PCR product with BamH1 yielded a fragment that had
as the 5' end the BamH1 of the primer and as the 3' end the BamH1 site of
the polylinker of pGEX-3X; this fragment was subcloned into the BamH1 site
of pBJ-245, 3' to the  GAL1 promoter and a clone of the correct orientation
was isolated.  Following a partial digest with BamH1 and blunting with T4
polymerase and re-ligating, a complete BamH1- HindIII digest identified a
clone in which the BamH1 site 5' to the initiating AUG of GST was
destroyed, but the 3' BamH1 polylinker site remained intact.   This vector
is pBJ-319.
        -The GAL1/10-GST promoter-fusion protein cassette was removed from
pBJ-319 by digesting with Sac1 and Xho1 and the released fragment  was
inserted into  similarly digested pRS-423, -424, -426, and pBS to provide
vectors with other yeast markers.  pBJ-384  was constructed for  cloning
use.  
        -Cloning sites for these vectors are BamH1 (same frame as pGEX-3X;
ggATCc is 123; note that this is the same frame as the BamH1 site of the
TrpE fusion protein vector pATH-1) and Spe1.  A stop codon occurs in-frame
in the Spe1 restriction site, so 3' sites are not useful.  Note that the
SmaI and EcoR1 sites of pGEX-3X are not present.
        -The factor Xa endopeptidase recognition site of pGEX-3X is
maintained, and precedes the BamH1 cloning site.   
        -The GAL10 promoter can be used  to co-express a second polypeptide
in the same cell. Using a vector similar to pBJ-319, CAP1p co-purified on a
glutathione agarose column  with GST-CAP2p when CAP1 was cloned 3' to GAL10
and CAP2 was cloned in-frame with GST.   Furthermore, proteins that
associate with the expressed fusion protein might co-purify on the
glutathione-agarose, allowing purification of a protein complex.

Christopher Hug    
chug at cellbio.wustl.edu  
 
Department of Cell Biology and Physiology, Box 8228,   Washington
University  School of Medicine,  
660 South Euclid Ave.,    St. Louis, MO  63110

These vectors have been deposited with the ATCC for release in the fall of
1993.           





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