ZINC PROTEIN PURIFICATION WITH PDC
USING PDC KIT
Our PDC KIT is also applicable for Zn protein purification while some such proteins bind to Zn chelate column and others do not.
Example : with PDC-Sepharose CL-4B Kit (PDC-4S KIT)
The crude extract (redissolved ammonium sulfate precipitate) containing a mesophilic alkaline protease MW=50,000 daltons (Zn protein) from Pseudomonas aeruginosa IFO 3455 was kindly supplied by J.P. Chessa and E. Planamente, Laboratoire de Biochimie, Institut de Biochimie B6, Sart Tilman 4000 Liège (Belgium).
Sample volume that was loaded onto each column: 1 ml (conc. in alcaline protease: approx. 1 mg/ml).
SDS-PAGE 12% in the presence of beta-mercaptoethanol
(15 µl of sample - stained with Coomassie blue)
|Panel A (Cu-PDC)
||Panel B (Ni-PDC)
Panel C (Zn-PDC)
Panel D (Co-PDC)
Not shown: no binding of the protein
to Ni, Zn & Co-PDC columns
Lane 1: redissolved ammonium sulfate precipitate
Lane 2: buffer A
Lane 3,4,5,6: buffer B
Lane 7: markers (97,400; 66,200; 45,000; 31,000;
21,500 & 14,400 daltons).
Conclusion: Cu-PDC was the only chelate gel allowing the purification of this protease in a single step (Panel A, lane 4,5,6). No binding to Ni-PDC, Zn-PDC and Co-PDC was observed.
PROTOCOL FOR Zn PROTEIN PURIFICATION WITH PDC-MATRIX
1. Selection of the most appropriate Metal chelate column in a primary screening with PDC KIT
The selection of the most appropriate column can be realized in the same manner as for 6x His-tagged proteins. However, the points 4, 5, 6 and 8 sometimes are not necessary.
Once the most appropriate metal chelate column is selected, the optimization of the chromatography conditions can be performed in the following manner:
- The Na2HPO4 50mM, NaCl 150mM pH 8.0 buffer is often used as the loading and washing buffer. The Tris.AcOH 50mM, NaCl 150mM pH 8.0 buffer also can be used as the washing buffer when the affinity of the metal ion-PDC complex for the protein of interest is fairly high.
- The Na2PO4 50mM, NaCl 150mM pH 6.0 buffer is often used as the first elution buffer.
- The second elution can be performed either with NaOAc 50mM, NaCl 150mM pH 4.0 buffer or a gradient of imidazole 0-300 mM pH 7.4.
- It is not recommended to use regenerated columns for the initial protein screening. New columns should be used.
- But the regenerated gel can be used for purification of proteins in the optimization step.
6x His-tagged protein purification with PDC using PDC KIT
General purification of proteins with PDC using PDC KIT