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Preparative Nondenaturing Gel Electrophoresis to Purify NADP-Specific Glutamate Dehydrogenase From Chlorella, Rev B

er Stain Plus kit.


Results
Chlorella sorokiniana NADP-specific GDH was purified an additional 8.5-fold for a final 375-fold purification using the Model 491 prep cell (see Table). Nondenaturing preparative electrophoresis of the partially purified NADP-GDH sample was performed under conditions determined to be optimal for analytical native slab gel electrophoresis. A total of 11.5 mg of extremely pure GDH protein was recovered with <2% loss of activity during the procedure. The resolution of the nondenaturing gel in the Model 491 prep cell was sufficient to separate multiple peaks of NADP-GDH activity corresponding to the multiple isoenzymes of the GDH isoenzymes. Analytical SDS-PAGE of the final purified product revealed a single 53 kD band corresponding to the NADP-GDH -subunit and detected no contaminating proteins. The highly purified NADP-GDH protein was recovered in sufficient quantity in its native form to allow NH2- and COOH-terminal sequencing, antigen affinity column production, and the production of hightiter polyclonal and monoclonal antibodies.

These results suggest that the Model 491 prep cell system is amenable to large-scale purification of partially purified proteins in their native, active form. The high resolution of this technique, evidenced by its ability to separate closely migrating isoenzymes, should allow separation of multiple isoenzymes for the purpose of kinetic, biochemical, and immunochemical analyses.


Acknowledgment
We thank Dr LO Ingram for the use of the Model 491 prep cell.


References
Baker AL and Schmidt RR, Intracellular dist
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