Need Help in Purification

K.D. James mbkdj at s-crim1.dl.ac.uk
Sat Jul 8 13:26:53 EST 1995


JT31 (jt31 at aol.com) wrote:
: I am currently in the process of purifying an NADP Dehydrogenase from E.
: coli. My first procedure is Ammonium Sulfate fractionation....My protein
: precipitates at approximately 30% salt conc., however the activity is much
: lower (about a 35% loss). Even after desalting through a Sephadex G-10
: column there is no increase in activity. Normally after that I put the
: semi-crude extract through a Ion exchange process using a DEAE Celluolose
: resin. Again the procedure works out find, the enzyme is succesfully
: eluted (Using 300mM Tris HCl pH 7.2), However More activity is lost.
: Finally After running the extract through a Cibacron Blue resin, and
: eluting with approx 1.2M NaCl, the porotein is successfully retrieved but
: again more activity is lost! All procedures are performed in a cold room
: as well. Does anyone have any hints, tips, or tricks for keeping this
: enzyme active??? I would greatly appreciate the Info.

: Dominic Ricciardi

 I have been purifying NAD dehyrogenases from Gram negatives using dye-ligand 
columns (inc Cibacron blue) and DEAE cellulose. The following questions spring
to mind:
        Is your loss of activity due to poor stability or poor recovery from
      the columns? My enzymes can suffer from the former, so adding 2mM DTT
      or 20% glycerol (depends on the enzyme) can help retain activity.

        As far as I'm aware, Cibacron Blue is good for both NAD and NADP
      linked enzymes, but have you tried Procion Red? I'm sure this has
      a high affinity for NADP linked types. Also, if you have to raise
      the NaCl concentration to 1.2M to recover your enzyme there is the 
      possibility of using a lower concentration to elute unwanted protein
      while leaving yours bound, followed by a more biospecific elution 
      with NAD/P. Of course, cofactor elutions may well be something you've
      already tried. (You can get Procion Red HE-3B as Matrex Gel Red A
      from Amicon.)

 Currently I use a Cibacron Blue column as a first step, passing cell extracts
straight on to it (okay if your matrix can be regenerated harshly- I use a 
6% cross-linked agarose bead type which can be cleaned with 8M urea). This 
followed by a DEAE Sephacel step, eluting with an NaCl gradient.
 I'm also always interested in protein purification tips...

   Keith James

 (No connection with Amicon etc.)





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