On Fri, 14 Feb 1997, Bart K. Kwieciszewski wrote:
> 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)
Thats what I found too. High IPTG(>=1mM final conc.) can lead to an increased
amount of insoluble protein. I usually use 0.1mM IPTG.
> 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
> suggestions?
Apparently the bugs can loose your plasmid if you let them grow too long.
I have used one month old plates stored at 4deg C -no problem.
> 2. The strain I use is BL-21. Did anyone try expression in
other
> strains? DH5Alpha gives extremely low levels of expression for me
In my hands DH5a also gives low yields and increased degradation. I
prefer Bl21.
.
> 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?
I started also with single protease inhibitors that I mixed to a
cocktail(ap, Leu, Pep, Pefabloc). Now I use protease inhibitor
tablets(from BM), and they work better in my hands. BUT they are expensive!>
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?
EDTA is also used to inhibit some metal dependent proteases. that's
probably why it increases the yield of the full lenght product. Its
sometimes included in protease inhibitor cocktails.>
> 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
> first.
>> 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
>>Hope that helps
Toumy
>
