Context around the ATG for protein expression

rtemple at NETMAIL.HSCBKLYN.EDU rtemple at NETMAIL.HSCBKLYN.EDU
Thu Nov 14 14:44:41 EST 1996


Shinhan Shiu wrote:
Also, I use at least four independt PCR reactions when I try to clone
the full length protein into the yeast vector. I have to suspect that
the errors during the amplification is the major cause. Does it happen
a lot when you try to clone PCR fragment (not quite a yeast question)?
I use Taq.
______________________________
Hi Shinhan -
As far as  PCR-induced oopses, they happen more than anyone would like to think
when you are heavily using PCR for cloning. I also work in non-yeast
transmembrane protein expression in yeast & if you get a misincorporation at a
vital spot, your expression could be in BIG trouble. As a rule, I try to use PCR
as little as possible for anything that I'm going to be expressing for a
functional assay. And it's always good to resequence something you're going to
base a thesis/paper on anyway! There are "low-error" polymerases (Vent, DeepVent
- New England Biolabs) that may be a little better than Taq, but they aren't
perfect.

Good luck & happy hunting!

Robyn Temple

rtemple at netmail.hscbklyn.edu
State University of New York
Health Science Center at Brooklyn


_______________________________________________________________________________
Subject: Context around the ATG for protein expression
From:    sshiu at students.wisc.edu (Shinhan Shiu) at Internet
Date:    11/14/96  2:46 AM

Hi there,

I am trying to express a plant transmembrane kinase under the control
of GAL1 promoter in yeast but can't detect its expression after
induction with galactose. I had posted one message about it and got
some prompt responses about the induction conditions which seemed to
be alright.

After talking to one of my colleage, we suspect that it's probably
because that the context around the translation start site is not
optimal for expression in yeast. In plant, the -3 should be an A and
-2 is better to be a pyrimidine. Does yeast also have similar kind of
requirement?


Thanks in advance 


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