Isolation and purification of proteins using heparin affinity column chromatography.

(Japanese)

Proteins found in the cytoskeleton fractions often aggregate so strongly that their separation in ordinary column chromatography cannot be applied.  For instance, when a gel filtration column was used, most of the proteins appeared in the void fraction together under native conditions, while under denatured conditions such as in the presence of urea in turn no valid separations are made by scattering the proteins over the fractions eluted.  However, removal of the denaturing agents will cause aggregation again.    These aggregated proteins can be solubilized under high salt conditions, and sometimes an ion-exchange column chromatography is tried after being dialyzed.  But, again we encounter re-precipitation of proteins.   We also sometimes encounter massive amount of ribosomal protein contamination derived from disintegration of ribosomes during conventional isolation procedures.  

To isolate these nasty proteins, we developed an effective method using heparin affinity column chromatography. This method was hinted from the fact that the cytoskeleton and the associated factors are sensitive to low levels of heparin (3).

The method was successfully applied for isolation of an apyrase and some other proteins from pea cytoskeleton fractions (6, 7).   When we use our buffer system (CSB and CDB) (4), there is no apparent disintegration of cellular particles such as ribosomes (5), and this greatly favor reduction of contamination of those proteins and also makes method simpler.  Ribosomes can be removed by ultracentrifugation in the presence of CDB prior to application to a heparin column or separated by  heparin affinity column itself as their intact form.

heparin01.jpg (26022 バイト)

Protein isolated in this example (etiolated pea stem cell cytoskeleton)

Proteins once separated by the heparin affinity column chromatography are generally far less aggregative than before, and can be successfully applied to further purification protocols by conventional way, such as gel filtration and ion exchange column chromatography (Shibata et al 1999 (6); Davies et al., 2001(7)

In combination with our high speed SDS-PAGE, isolation and analysis of proteins can be performed within a short period of time (7).

 

CSB:  Cytoskeleton Stabilizing Buffer (5 mM Hepes-KOH (pH 7.5), 10 mM Mg(OAc)2, 2 mM EGTA, 0.1-1 mM PMSF).  When necessary, appropriate concentrations of a neutral detergent are included (0.5% PTE, TX100 etc).   Abe et al 1991 (2)

CDB: Cytoskeleton Depolymerization Buffer (200 mM Tris-HCl (pH8.5), 450 mM KOAc, 25 mM Mg(OAc)2, 2% PTE (a neutral detergent).  Abe et al., 1995 (3)

PTE (a neutral detergent):   Polyoxyethylene-13-tridecylehter (Abe and Davies 1985 (1)

References

1.  Abe, S. and E. Davies. Quantitative isolation of undegraded polysomes from  aged pea tissue in the absence of contaminants and artefacts. Plant Cell Physiology 26: 1499-1509 (1985).

2. Abe S, Davies E. Isolation of F-actin from pea stems: Evidence from fluorescence microscopy. Protoplasma 163: 51-61.(1991)

3. Abe S, Ito Y, Davies E. Isolation of a heparin sensitive, ribosome sedimenting factor from the cytoskeleton fractions of peas and corn. Plant Physiology and Biochemistry 33(4): 463-470 (1995).

4. Davies E, Abe S. Methods for isolation and analysis of polyribosomes  (Chapter 15). In Methods in Plant Cell Biology, Part B in Methods in Cell Biology,  vol 50 (David Berl-Hahn ed), pp. 209-222, Academic press Inc, USA (1995)ISBN 0-120-273872-1.
 
5.
Abe S and Davies E. Methods for isolation and analysis of the cytoskeleton (Chapter 16). In Methods in Plant Cell Biology, Part B in Methods in Cell  Biology, vol 50 (David Berl-Hahn ed), pp. 223-236, Academic press Inc, USA (1995), ISBN 0-120-273872-1
(1995).

6.  K. Shibata, Y. Morita, S Abe, B. Stankovic, and E. Davies: Apyrase from pea stems: Isolation, purification, characterization and identification of an NTPase from the cytoskeleton fraction of pea stem tissue.  Plant Physiology and Biochemistry, 37(12):1-8, 1999.

7. Davies E, Stankovic B, Azama K, Shibata K, Abe S  Novel components of   the plant cytoskeleton: A beginning to plant "cytomics "Plant Science. Invited Review, Plant Science (160)2 (2001) pp. 185-196.

 


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