GST-fusion system hints and questions

Bart K. Kwieciszewski riverflo at u.washington.edu
Fri Feb 14 12:46:43 EST 1997

Hi there,
	I have been working on purification of proteins using the 
GST-fusion system for a while, and would like to compile some hints and
advice on how to get the highest protein yields.  Here are some hints that
I have tested that increase the yields for me (and some of them can be
very obvious, so please don't get mad :) )

	1.  Growing the E.Coli at 30 degrees Celsius or below.  I am yet
to try lower temperatures, but I've read that it might increase the yield
of the full lenght protein as compared to its degradation products.
	2.  Higher levels of IPTG increased the expression of the protein
for me, but some people said that it might also increase the amount of
inclusion bodies... who knows.
	3.  Every step of harvest and purification done on ice.
	4.  Keeping the induction time short (around 2 hrs)

I have to explain that I am working on the GST-NS5A fusion protein, which
is expressed in very low amounts and being EXTREMELY unstable, ie. being
eaten up by SOME proteases.  The best I have done so far is to get a yield
of about 300-400ug of the purified protein from a liter of culture, and of
that about 30-50ug would be the full lenght product.  This is where my
questions come:

	1.  I have observed an unproportionally high level of degraded
protein that is GST.  Did others observed this too?  Any suggestions of
why that might be?  Someone suggested to me that it's the unfusion do to
the fact that I have been using bacterial stocks older than 2 months.  Any
	2.  The strain I use is BL-21.  Did anyone try expression in other
strains?  DH5Alpha gives extremely low levels of expression for me.
	3.  What about protease inhibitors?  I have been using a mixture
of aprotinin, leupeptin and PMSF, but some people suggested that aprotinin
and leupeptin might be hurting the preps, and not helping at all (their
explanation being - these are eukaryotics protease inhibitors).  What
protease inhibitors work well?  Benzamidine?  Sodium metabisulfate?  E-64?
	4.  Last but not least - what's the deal with EDTA and washing the
pellet?  Someone suggested to me that this increases the yield of the full
length product?  Does it?

I am posting all these questions, because I have been struggling with this
system for a while, without significant improvement in the results and I
can't just go out buy all these supplies (limited budget) and try all
these new conditions (limited time) without having some confirmation

Thank you very much for any hints and information you might provide,

With regards,

Bart K. Kwieciszewski
University of Washington
Regional Primate Research Center
Department of Microbiology
HSB H-331

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