GST-protiens

Curt Ashendel ashendel at aclcb.purdue.edu
Mon Jul 25 15:20:00 EST 1994


On 25 Jul 1994 18:08:24 GMT, 
David L. Haviland, Ph.D.  <HAVILAND at KIDS.WUSTL.EDU> wrote:

>Just out of curiosity, what sort of yeild/ liter have people been obtaining 
>using GST-fusion vectors?  Right now we're averaging about 5ug/Liter and 
>that seem horribly low.  Is production of one's favorite fusion protien 
>linked to any particular bug?

Time for someone to write a FAQ on this and post it periodically.

There must be over 100 posts you can find on this subject in the 
Bionet-archive at the IU site (searchable via gopher, WAIS, and www, I 
think).  Also, many published papers indicate their yields.

But to answer the question: 

We generally get 100 to 250 mg of plain GST from one liter, but no more 
than 1/4 of that with fusions.  The yield can be 5 mg/liter or lower for 
really large (>80 kD) fusion proteins.  The *generel* rule is that the 
larger  the protein, the lower the yield.  This is because instability, 
susceptability to proteases, and folding difficulties increase with size.  
Some small proteins can be a problem also, depending on sequence, ability 
to fold into the native conformation, and solubility and stability of the
native conformation.  

Some ideas to get higher yields:

  Induce at lower temperatures (22 to 25C)
  Use protease deficient bacteria (BL21 and derivatives)
  Work quickly and include protease inhibitors
  Load your resin by application to a column, rather than batch mode   
  Lyse your cells in 20 to 50 volumes of buffer rather than 5 to 10
  Optimize the time, temp, and [inducer] used with induction - sometimes 
     shorter times or lower concs give higher yields.
  Make sure your lysis is efficient (consider BL21-pLysS or pLysE cells)
  

Good luck.


Curt Ashendel
Purdue University
West Lafayette, IN
ashendel at aclcb.purdue.edu



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