I am looking for suggestions or comments from protein purification community, especially people who work with Con-A column chromatography.
I am used Con-A Sepharose and Con-A agarose beads (initial work to choose best resin) to purify a recombinant human protein expressed in tobacco plants. I carried out the purification protocols recommended by the product user manuals and .80% of protein of interest stays in the column (binding to the beads), won't elute with high or low pH, + or - NaCl, + or - BSA.
My question is how to improve protein recovery from the column. I know each protein is different and conditions need to be standardized. What is the best way to start with.
Have you tried alpha-D-methylmannoside or alpha-D-methylglucoside for elution?
Concanavalin A is a lectin from the jack-bean, Canavalia ensiformis. It binds mannose residues in glycoproteins. For application, see for example http://research.biology.arizona.edu/mosquito/willott/proj/labpro/Prot/Purif/AffinChro/ConA.html or doi:10.1007/978-1-4939-6412-3_23
Since you are using Con A as a matrix that means that your protein is a glycoprotein ,to elute your protein from the column you need to use alpha methyl-mannoside at high concentration, rather than changing the ionic tf
so that it will replace your protein in the column.
Good luck
Thank you Guys for the response.
Yes, I used both alpha-D-methylmannoside or alpha-D-methylglucoside at 0.9M concentration (that was recommended) for elution.
Later I reduced the concentration to 250 mM and 500 mM respectively (according to J Chromatogr A 2007, 1154: 308-318). Still no improvement.
I read some very old publications from 70s - 80s when the ConA chromatography was developed (I can provide links if you wish). And they observed a tendency of ConA to bind sometimes nonspecificaly by hydrophobic interactions. So I suggest adding glycerol or ethylen glycol to the solutions. These you can get rid of by dialysis afterwards.
Or maybe detergents: CHAPS or Triton-X100.
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