pGEX/GST gene fusion problems-> HELP!

Jeanne McAdara mcadara at scripps.edu
Mon Aug 26 16:34:14 EST 1996


Paul N Hengen wrote:
> 
> Mark Gosink (mmgosink at facstaff.wisc.edu) wrote:(snip)
> > suggested we also use lower incubation temperatures (i.e. 30C or even
> > ~22C) at times. (snip)
> > Do you have any idea why this works? Is it that the enzymes or undefined
> proteases are less active at this temperature, or is it that they are not
> expressed at this temperature?
> 

My understanding is that at high temperatures, or with high amounts of inducer 
around, the recombinant protein is being expressed so rapidly that it doesn't 
fold properly.  This causes formation of insoluble aggregates of the 
improperly folded molecules (inclusion bodies).  Also, many organisms have 
specific protease systems whose functions are to destroy misfolded proteins.  
By reducing the temperature or adding smaller amounts of inducer (or reducing 
the induction time) you give proteins a better chance at folding into their 
native structures and avoiding these pitfalls.  You get lower yields but 
better product.

Jeanne McAdara
The Scripps Research Institute
mcadara at scripps.edu



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