GST fusions - please, help

Cornelius Krasel krasel at wpxx02.toxi.uni-wuerzburg.de
Sun Oct 22 15:26:02 EST 1995


ijiwaru at wheel.dcn.davis.ca.us wrote:
> In article <46bbgc$16vi at news.doit.wisc.edu>, klenchin at macc.wisc.edu (Dima
> Klenchin) wrote:

> > I need to overexpress 70K protein as a GST fusion. It is cloned into
> > Pharmacia's pGEX-2T. As usually, expression level and solubility is a
> > problem. 
> > 
> > 1. What strain should be used? Pharmacia recommends BL21 that we don't have
> > right now. Does it really offer any advantages over, say, JM109? Is
> > anything else better available?

> BL-21 is good because of the lack of lon protease (if I remember
> correctly).

It also has some other proteases missing. AFAIK BL-21 is not one of the
common E. coli K-12 strains. JM109 is not protease-deficient, but there
are K-12 strains which are. I am currently playing around with PR745
which is available from NEB. It is lon minus and stabilizes our protein
(when I express it in JM109 I get only GST).

A funny thing with BL21 seems to be that it might be necessary to
screen clones for expression levels. I am expressing another protein
in BL21 (actually BL21(DE3)pLysS, but I don't think that matters);
I tested four clones which expressed the protein in widely different
ranges.

I am currently testing whether BL21 or PR745 is the better host for
my fusion protein -- I don't know yet (I even have no clue if I can
expect very different expression levels).

> > 2. I will need to remove thrombin after cleavage. Is there any
> > highly specific affinity matrix that will stick thrombin selectively?

I use a S sepharose column to which the thrombin binds but my protein
flows through. This will not necessarily work for your protein.

> > 3. Increasing solubility. I know of the following cheap tricks: low IPTG 
> > concentration/room temperature growth. I remember someone's suggestion
> > to heat shock bacteria before induction at 25C.

I also fiddle around with this kind of stuff and, so far, have not
obtained dramatic improvements (although I had hoped for it) ... :-(

--Cornelius.

--
/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany   email: phak004 at rzbox.uni-wuerzburg.de  SP3 */
/* "Science is the game we play with God to find out what His rules are."  */



More information about the Methods mailing list