Expr of fusion proteins in E. coli

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
Sun Mar 28 11:37:00 EST 1993


-------------Dr. Leonard N. Bloksberg 
Writes---------------------------------------
A short while back someone asked a question about expressing fusion proteins
in E. coli which sparked a long and interesting discussion about the subject.
Someone recomended a method described in:

        A novel strategy for production of a highly expressed recombinant
protein in as active form.
        by Blackwell, J. and Horgan R.  FEBS 295(1,2,3)10-1
                            <Stuff deleted>

        Well, I tried this method to get a better folded potato ADP-glucose
pyrophosphorylase from my E. coli.  I got a 5 fold increase in yield of 
activity per mg total protein, and higher yields of total mg protein.  
Unfortunately, it is still not as heat stable as the wild type protein, but
I now think that is because we cut the leader peptide out for E. coli 
expression.  Conditions that we now use are exactly as described in the paper,

        LB with 0.1x NaCl
        1M Sorbitol
        2.5mM Glycine Betaine

        Pre-Growth at 37dC
        Protein Induction at 25dC

Thanks to all netters who participated in this discussion.  While I was not 
the original poster, I was glad I was reading the net that day to catch this
little tidbit.  This little boost was all I needed to allow me to use a 6l
prep in fernbachs instead of the 100l fermentor for purification of my 
site directed mutants to characterize kinetics.  That amounts to a massive
savings of time and money for our lab.  Thanks and good luck to all others
who may want to try this little trick.

        Dr. Leonard N. Bloksberg
        PreissJ at clvax1.cl.msu.edu
        Dept. of Biochemistry
        Michigan State University
------------------------------------------------------------------------------
----
Hi Leonard!
    I posted the original message listing the  
Blackwell, J. and Horgan R. reference.  I would like to
let it be known that a good friend of mine Dr. Kiran Madura
,now working at Cal Tech, put me onto this paper.  I tried it 
and it worked Vwell.  Thanks again Kiran !!   
________________________________________
Dr. Dan Gietz
Assistant Professor
Department of Human Genetics
University of Manitoba
770 Bannatyne Ave, Rm 250
Winnipeg, Man, Canada
R3E 0W3
GIETZ at BLDGHSC.LAN1.UMANITOBA.CA
_________________________________________





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