Molecular Techniques and Methods

Purification of His6-tagged Recombinant Protein
from Plant Tissues in Native Condition

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION




MATERIALS AND SOLUTIONS

Lysis Buffer (pH 8.0) (100 ml)
50 mM Tris-HClpH8.0 ----------------------------------------- 5 ml of 1 M Tris-HClpH8.0
10% Glycerol ------------------------------------------------ 20 ml of 50% Glycerol
200 mM CaCl2 ---------------------------------------------- 20 ml of 1 M CaCl2
4 uM Sodium-cacodylate ------------------------------------- 100 ul of 4 mM Sodium-cacodylate
1 mM DTT --------------------------------------------------- 200 ul of 0.5 M DTT
1 mM EGTA -------------------------------------------------- 200 ul of 0.5 M EGTA
0.01% Triton x-100 ----------------------------------------- 100 ul of 10% Triton x-100
DW -------------------------------------------------------- 55 ml
  • Keep at 4oC.
  • Add 0.5 ml of Protease Inhibitor cocktail (P-9599, Sigma) just prior to use.


    Binding Buffer (100 ml)
    50 mM Sodium phosphate pH 8.0 --------------------------- 5 ml of 1 M Sodium phosphate
    500 mM NaCl ------------------------------------------------ 10 ml of 5 M NaCl
    10% Glycerol --------------------------------------------------- 20 ml Glycerol
    Deionized H2O ------------------------------------------------ 85 ml


    Elution Buffer (100 ml)
    50 mM Sodium phosphate pH 7.0--------------------------- 5 ml of 1 M Sodium phosphate
    500 mM NaCl ------------------------------------------------ 10 ml of 5 M NaCl
    0.5 M Immidazole ------------------------------------------------ 10 ml of 1 M Immidazole
    10% Glycerol --------------------------------------------------- 20 ml Glycerol
    Deionized H2O ------------------------------------------------ 75 ml


    Regeneration Buffer (100 ml)
    6M Guanidine-HCl ------------------------------------------------- 57 g
    0.2 M Acetic acid ----------------------------------------------------- 3.2 ml of 6.3 M Acetic acid
    Deionized H2O ------------------------------------------------------- 40 ml


    Equilibration Buffer (100 ml)
    50 mM sodium phosphate pH 8.0--------------------------- 5 ml of 1 M Sodium phosphate
    0.5 M NaCl ----------------------------------------------------- 10 ml of 5 M NaCl
    Deionized H2O ------------------------------------------------ 89 ml


    Protein Wash Buffer (50 ml)
    100 mM Tris-HClpH8.0 --------------------------------------------- 5 ml of 1 M Tris-HCl
    1 mM EDTA ---------------------------------------------------------- 100 ul of 0.5 M EDTA
    8% Sucrose (0.35 M) ---------------------------------------------- 4 g
    10% Glycerol --------------------------------------------------------- 10 ml of 50% Glycerol
    DW --------------------------------------------------------------------- 35 ml
  • Add 200 uM PMSF ------------------------------------------------- 50 ul of 200 mM PMSF


    Protein Storage Buffer (10 ml)
    10 mM Tris-HClpH8.0 --------------------------------------------- 100 ul of 1 M Tris-HCl
    50 mM NaCl -------------------------------------------------------- 100 ul of 5 M NaCl
    1 mM DTT ---------------------------------------------------------- 100 ul of 0.1 M DTT
    0.1 mM EDTA ---------------------------------------------------------- 2 ul of 0.5 M EDTA
    200 ug/ml BSA -------------------------------------------------------- BSA
    50% Glycerol --------------------------------------------------------- 5 ml of 100% Glycerol
    DW --------------------------------------------------------------------- 5 ml
  • Add cold UDP-glucose (final conc. 0.2 mM) to stabilize the UGT enzyme activity.




    PROCEDURES

    1. Grind 1 g plant tissue in liquid nitrogen.

    2. Add 10 ml of Lysis Buffer immediately.
    Add 2 g Polyvinylpolypyrrolidone and 72 ul 2-mercaptoethanol.

    3. Polytron for 30 seconds in ice. 3 times.

    4. Centrifuge at 14,000g, 4oC for 10 minutes.

    5. Collect the supernatant.
  • Careful - do not disturb the whitish gel-like pellet since it is very soft.
  • Insoluble polysaccharides form a whitish gel-like layer on top of the tissue debris, which forms a dark green pellet.

    6. Centrifuge at 14,000g, 4oC for 10 minutes.

    7. Collect the supernatant.

    8. Prepare chelating resin (Ni-NTA resin or Chelating Sepherose) as follow.
  • Wash with 5 vol. of water.
  • Equilibrate in 10 vol. of Lysis Buffer.
  • Stir at room temperature for 60 min.
  • Remove Lysis Buffer by settle down the resin.

    9. Add 5 ml of ice-cold 50% Ni-NTA resin and NaCl to a final conc. of 0.5M to the supernatant.

    10. Load resin into a 2.5 x 10 cm chromatography column.

    11. Wash column with 10 column volumes of Binding Buffer until the A280 of the flow-through is less than 0.01.

    12. Elute the protein with a 5 column vol of Elution Buffer. Collect 1 ml fractions and analyze by SDS-PAGE.

    13. Concentrate protein by size fractionation column (ex., microcon, centricon, etc.).

    14. Wash the enzyme preparation by adding two volumes of Enzyme Wash Buffer.

    15. Collect enzyme and equilibrate in Enzyme Storage Buffer.

    16. Store enzyme at -20oC.

    17. Measure protein concentration by BCA method.

    18. For denaturation condition, add equal volume of SDS-PAGE Sample Buffer to 5-10 ul of protein.
    Heat protein solution for 5 minutes at 95oC.
  • For native condition, follow the native condition gel electriphoresis.

    19. Run SDS-PAGE.




    NOTES

  • 2-Mercaptoethanol does not interfere with the purification procedure, but stronger reducing agents such as dithlothreitol should be avoided because the metal ions will be reduced.

  • Keep the pH above 7.0 at all times.
  • Most proteins will be eluted by Wash Buffer at pH4.5, although many 6xHis proteins (particularly monomers) can be eluted at a higher pH.



    Re-generation of used Ni-NTA Resin
    (1) Ni-NTA resin can be used 3-5 times maximum.
    (2) Wash the resin with 2 vol. Regeneration Buffer.
    (3) Wash the resin with 2 vol. H2O.
    (4) Wash the resin with 3 vol. 2% SDS.
    (5) Wash the resin with 1 vol. 25% Et-OH.
    (6) Wash the resin with 1 vol. 50% Et-OH.
    (7) Wash the resin with 1 vol. 75% Et-OH.
    (8) Wash the resin with 5 vol. 100% Et-OH.
    (9) Wash the resin with 1 vol. 75% Et-OH.
    (10) Wash the resin with 1 vol. 50% Et-OH.
    (11) Wash the resin with 1 vol. 25% Et-OH.
    (12) Wash the resin with 1 vol. H2O.
    (13) Wash the resin with 5 vol. 100 mM EDTApH8.0.
    (14) Wash the resin with 2 vol. H2O.
    (15) Wash the resin with 2 vol. 100 mM NiSO4.
    (16) Wash the resin with 2 vol. H2O.
    (17) Wash the resin with 2 vol. Regeneration Buffer.
    (18) Equilibrate with 2 vol. Equilibration Buffer.

  • The ethanol gradient is desgined to prevent precipitation of protein on the Ni-NTA resin, and should not be omitted.





    KIT INFORMATION



    REFERENCES

    Kangcheng Ruan1, Chunhe Xu, Tingting Li, Jiong Li, Reinhard Lange and Claude Balny (2003) The thermodynamic analysis of protein stabilization by sucrose and glycerol against pressure-induced unfolding. The typical example of the 33-kDa protein from spinach photosystem II. Eur. J. Biochem. 270, 1654-1661.




  • Please send your comment on this protocol to "editor@MolecularInfo.com".

  • Home
    MT&M
    Online Journal
    Hot Articles
    Order Products
    Classified