GST-GFP Purification Problem

immunechem immune at
Sat Feb 13 20:12:15 EST 1999

GSH has chelation properties like diacetic acids. your GFP may be metal 
containing protein (I don't know). The major protein bindin force is 
hydrophobic, hydrogen-bonding n ionic exchange. The chelation is 
somewhat different from ionic or salt bridge. If it is true, you have to 
 add EDTA and EGTA to the elution buffer (free GSH). If it is 
hydrophobic binding more strong, then add detergent. If H-bonding, 
acid/base maybe needed in the elution buffer. 

If you don't need activity of the protein, try glycine buffer (pH 2.5) 
may help.

In article <7a4mmt$ona$1 at>, 
klenchin at says...
>In article <36C58FEE.1B965825 at>, 
>moyerowl at wrote:
>>We are trying to purify a fusion protein consisting of GST with GFP
>>fused to its C-terminus.  The protein seems to be expressed fairly 
>>and is soluble.  The supernatant is green due to the high 
>>of GFP.  When sepharose-GSH beads are added, the supernatant loses its
>>green color and now the beads are green.  So far, so good.  Now the 
>>part.  We can't seem to elute the fusion protein even with very high 
>>concentrations.  Other GST fusion proteins have been purified by us
>>using this same protocol.  Any advice/suggestions/wild guesses would 
>>much appreciated.
>Gosh, whatever happened to a common sense? 
>If there is no affinity elution under standard conditions, then the 
>true affinity binding is too strong or there is another mode of binding
>involved. Then one wants to either weaken affinity interaction or 
>additonal binding mode. In either case, there are essentially only two
>types of interactions involved: Ionic and hydrophobic. Naturally, 
>high salt or detergents would be the first thing to do. Simple, right?
>        - Dima

More information about the Methods mailing list