usefull all purpose yeast shuttle vectors

robert joseph litt rjl4 at quads.uchicago.edu
Sat May 8 17:35:19 EST 1993


Morrie wanted to know about our favorite expression vectors in yeast.
Right now I am mostly using two different vectors. A GAL1/10 promoter
fragment from pBM150 (Johnston & Davis MCB 4: 1440-1448.) subcloned
into the Sikorski & Hieter vector Morrie mentioned called pRS316 (this
is a URA3, CEN-ARS vector with a pBluescript polylinker, bacterial
origin of replication and the f1 phage origin replication).  This
vector gives high plasmid yields, has many convenient subcloning sites
(sometimes too many) and I have used it for dut ung mutagenesis.  I
also have a version of this on a HIS3 plasmid.  I am beginning to work
with the MET3 promoter that I have obtained from Harry Mountain at the
University of Sheffield.  This promoter should give much lower
expression levels than pGAL and is suppossed to be very tightly
regulated by the presence/absence of methionine.  A good reference for
this promoter would be Gene 34: 269-281.  This promoter may be very
useful for studying a gene when you don't want it overexpressed at GAL
promoter levels.  I haven't done anything with it yet, but Mountain's
published data looks very good, and he has done quite a bit to
characterize the expression of this promoter.

We also use a different construct when we wish to purify proteins
expressed in yeast.  This was from Bob Duschennes' lab.  This contains
a 2micron ori rep, URA3 and leu2-d genes.  The expresssion unit is a
GAL promoter in front of a Glutathione Synthel Transferase (GST) gene
followed by a thrombin cutting site and a polylinker for making in
frame fusions.  The GST gene allows us to purify the fusion proteins
in a single step on a glutathione agarose column.  We have used this
for purifying our protein of interest and a mutant of this protein.
The GST tag seems to stabilize some mutants in the cell.

Bob Litt
rjl4 at midway.uchicago.edu



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