Recombinant proteins extracted from E. coli were purified twice using a Ni-NTA column and an antibody-coupled agarose bead column. After final purification, the protein is in a 10% solution of 1M tris-hcl pH8 + 90% solution of 100mM glycine-hcl pH3. The recombinant protein was dialyzed three times for 2 hours against PBS pH 7.4.
After dialysis, the solution became cloudy and aggregated clumps were observed.
The predicted properties of this protein are soluble, pi value: 7.7.
What are the probable causes of protein aggregation at this time?
1. In fact, it is an insoluble protein.
2. The pI value of the protein (7.7) and the pH value of the buffer (7.4) are similar.
Are there Anything else?
In addition, when protein aggregation is resolved by adding glycerol or adjusting pH, is there any effect on protein activity?
And, When adjusting the pH of a buffer for solubility of the protein, is acidic or basic better? What is the appropriate range of pH values?
I'm going to process this protein into the cells.
Dear Jaeyoung Park
just some questions to better undestand the issu:
Is it your protein fused with an antibody Fc?
if i undertand well, you do not observe any precipitation after IMAC, why you are observing precipitation during the dialisys performed after the 2nd purification step (which i suppose is a proteinG or proteinA purificiation step) where you elute with glycin pH3 in a contained carryng the Tris concentrated solution to neutralize the ph? is it correct?
pI 7.7 is refer to the entire Fc-protein complex or only to the protein portion?
best
Manuele
Not fused protein, it's just single protein, and not antibodies.
The proteins are neutralized by 1M tris-Hcl (pH 8) immediately after eluted with 100mM glycine (pH 3).
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