Media problems on Nickel column

Casey M. Finnerty cmf5 at cornell.edu
Thu Jan 30 16:57:11 EST 1997


I'd like to find out how people prepare samples of recombinant 6xHis
protein in insect cell culture medium for separation on a nickel column.

The specifics of my problem are as follows: I'm trying to isolate a 6xHis
tagged secreted protein from serum-free (or reduced serum) insect cell
culture medium. The protein is expressed and secreted from Sf9 cells
infected with my recombinant baculovirus. I'm growing my cells as shaker
cultures in Sf900 medium supplemented with 20% TNMFH. My column is a 5 ml
Pharmacia HiTrap Chelating (IDA) column. I equilibrate and wash the column
with 20 mM Tris pH 7.5 plus 30 mM imidazole, pH 7.5. Elution is a
combination of linear and step gradients with the buffered imidazole. In
small scale experiments I determined that most of the unwanted proteins
(e.g. albumin) eluted between 20 and 30 mM imidazole, and my recombinant
protein eluted around 100-250 mM imidazole. In order to prevent the
occupation of valuable nickel binding sites by unwanted proteins, I add 30
mM imidazole pH 7.5 to the sample. The sample also contains 0.01% sodium
azide.
   My problem is that sample loading causes leaching of the nickel off the
column. My equilibration buffer alone does not cause noticeable nickel
leaching, so I don't think the imidazole is a problem. Gibco (who makes
the medium) tells me that Sf900 does not contain chelators or reducing
agents such as DTT, cysteine or glutathione. After adding the imidazole,
the sample pH is 7.5, so that isn't a problem either. Pharmacia tells me
that neither the surfactants in the medium nor the sodium azide should
strip the nickel off the column. The Pharmacia tech assistant suggested
that glcyine or histidine in the medium may be stripping off the nickel.
I've tried dialyzing the sample into the equilibration buffer, and this
helped but did not eliminate the problem (I may have not fully dialyzed
the sample, however).
   Some have suggested that I switch to a NTA based resin, but I have
trouble believing that a tetradentate chelator will be all that much
better than a tridentate chelator such as IDA. Am I wrong? I am thinking
that I may need to concentrate and buffer exchange my sample, though I'm
worried that too much concentration may cause precipitation of the
surfactants or serum proteins in the medium (and perhaps co-precipitation
of my recombinant protein). Dialysis bags will not be practical, as I hope
to scale up to > 1 L. Does anyone use ultrafiltration cassettes
successfully (and better yet, know of some that don't cost megabucks)?
Thanks.



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