Molecular Techniques and Methods

Purification of Recombinant Proteins from Inclusion Bodies

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

The over-production of proteins in E. coli often leads to inclusion body formation. This is due to aggregation of partially folded proteins which accumulate rapidly in the cytoplasm of E. coli. Inclusion body formation is not restricted to foreign proteins and even endogenous soluble proteins may form inclusion bodies when over-produced. Pure inclusion bodies can be prepared by low speed centrifugation after cell lysis. It is important to lyse the cells as completely as possible to avoid contaminations with other cellular components. The low speed pellet also contains part of the membrane fraction, which can be removed by Triton X-100.

Inclusion bodies are soluble only in strong denaturants such as urea, guanidine-HCl, strong base, and acetonitrile. If an over-produced protein is soluble in urea, it can be purified readily by ion-exchange chromatography in the presence of 8 M urea. Cyanate is slowly formed from urea and carbamylates uncharged amino groups of proteins. Hence prolonged exposure of the protein to urea must be avoided at high pH. While some aggregated proteins are soluble in 8 M urea (more readily at higher pH), others are often insoluble and can be pelleted by low speed centrifugation. In such cases, proteins cannot be purified by ion-exchange chromatography. This often happens when the recombinant protein is strongly basic.




MATERIALS AND SOLUTIONS

Lysis Buffer (100 ml)
50 mM Tris-HCl (pH 8.0) ------------------------------- 5 ml of 1 M Tris-HCl
25% Sucrose (w/v) -------------------------------------- 25 g of Sucrose
1 mM EDTA -------------------------------------------- 0.2 ml of 0.5 M EDTA
Add deionized H2O to make a final volume of ---------- 100 ml


10 X Lysozyme Solution (10 ml)
Lysozyme ----------------------------------------------- 10 mg
Lysis Buffer --------------------------------------------- 10 ml


10 X DNase I Solution (1 ml)
DNase I ------------------------------------------------- 100 mg
100 mM MgCl2 ------------------------------------------ 0.1 ml of 1 M MgCl2
10 mM MnCl2 ------------------------------------------- 0.01 ml of 1 M MnCl2
Add deionized H2O to make a final volume of ---------- 1 ml


Detergent Buffer (100 ml)
0.2 M NaCl --------------------------------------------- 4 ml of 5 M NaCl
1% Deoxycholic acid (w/v) ------------------------------ 1 g
1% Nonidet P-40 (v/v) ---------------------------------- 1 ml
Add deionized H2O to make a final volume of ---------- 100 ml


Triton X-100/ EDTA Solution (100 ml)
1% Triton X-100 (v/v) ----------------------------------- 1 ml
1 mM EDTA --------------------------------------------- 0.2 ml of 0.5 M EDTA
Deionized H2O ------------------------------------------- 98.8 ml


8 M Urea-Buffer (100 ml)
8 M Urea ------------------------------------------------ 48 g of Urea
50 mM Tris-HCl (pH 8.0) ------------------------------- 5 ml of 1 M Tris-HCl
1 mM EDTA -------------------------------------------- 0.2 ml of 0.5 M EDTA
Add deionized H2O to make a final volume of ---------- 100 ml


SDS Sample Buffer (100 ml)
0.125 M Tris-Phosphate (pH 6.8) ------------------------ 25 ml 0.5 M Tris-Phosphate
20% Glycerol -------------------------------------------- 20 ml of 100% Glycerol
4% SDS ------------------------------------------------- 40 ml 10% SDS
0.1% Bromophenol blue --------------------------------- 2 ml of 5% Bromophenol blue
Deionized H2O ------------------------------------------ 13 ml




PROCEDURES

Isolation of Inclusion Bodies from Total Cell Extracts:

1. Harvest E. coli cells by centrifugation at 2,000g for 5 minutes.

2. Resuspend E. coli cells in the same volume of Lysis Buffer.

3. Add 1/10th volume of 10 X Lysozyme Solution.

4. Incubate the cell suspension on ice for 30 minutes and then freeze at -20oC.

5. Thaw the suspension by immersing the tube in water. As the solution is thawed it becomes viscous.

6. Add 1/10th volume of 10 X DNase I Solution to the suspension.

7. Incubate the cell suspension at room temperature.
  • The viscosity of the solution decreases as DNA is digested by DNase I.
  • An indication of how well the DNA has been digested is obtained by pouring the solution from one tube to another.
  • Once the solution is fluid rather than gelatinous the digestion is sufficiently complete.

    8. Add an equal volume of Detergent Buffer to the lysate.

    9. Centrifuge at 5,000g for 10 minutes.

    10. Remove supernatant by pipette without disturbing the white tight pellet (i.e., inclusion bodies) and upper jelly-like layer (membrane proteins).

    11. Resuspend the pellet in the Triton X-100/EDTA Solution and spin down the inclusion bodies.
  • Repeat this procedure until the jelly-like layer is no longer seen.
  • (Triton X-100 washes remove most of the membrane proteins.)


    Solubilization of Inclusion Bodies:

    12. Resuspend a small amount of inclusion bodies in 8 M Urea-Buffer.
    (a) Keep 100 ul of the suspension aside for the analysis by SDS-PAGE.

    13. Centrifuge 1 ml of the suspension for 10 minutes at 25,000g (80,000 rpm.).
    (b) Transfer the supernatant to another tube.

    14.
    (c) Resuspend the pellet in 1 ml of 8 M Urea-Buffer.

    15. Separately mix the 100 ul suspension of
    (a), (b), and (c) from steps 12, 13, and 14 with 100 ul SDS Sample Buffer.

    16. Analyse proteins by SDS-PAGE. If the protein is found in the supernatant
    (b), it is soluble in urea and can be purified by ion-exchange chromatography in the presence of urea.


    Solubilization of Inclusion Bodies in Urea for Ion-Exchange Chromatography:

    17. Solubilize inclusion bodies in at least 10 volumes of 8 M Urea-Buffer. Dissolve the inclusion body pellet by using a pipette.

    18. Centrifuge the solution at 48,000g (20,000 rpm) at 4oC.
  • A large jelly-like pellet is normally obtained.
  • Collect the supernatant containing solubilized inclusion bodies in a new tube.

    19. Resuspend the pellet in 8 M Urea-Buffer.

    20. Centrifuge the solution at 48,000g (20,000 rpm) at 4oC.
  • Collect the supernatant containing solubilized inclusion bodies in a new tube.

    21. Analyse the supernatants from steps 18 and 20 by SDS-PAGE.

    22. Retain the pooled supernatants for further purification by ion-exchange chromatography.




    NOTES




    KIT INFORMATION




    REFERENCES

  • Jessen, T.H., Komiyama, N.H., Tame, J., Pagnier, J., Shih, D., Luisi, B., Fermi, G., and Nagai, K., 1993, In "Methods in enzymology (ed. J. Everse, K.D. Vandegrift, and Winslow, R.M.)", Vol. 231, pp.347-64. Academic Press, London.


  • Marston, F.A.O., Lowe, P.A., Doel, M.T., Shoemaker, J.M., Whiter, S., and Angal, S., 1984, BioTechnology, 2, 800.


  • Nagai, K. and Thogersen, H.C., 1987, In Methods in Enzymology (ed. R. Wu and Grossman, L.), Vol. 153, pp.461-81. Academic Press, London.


  • Pavletich, N.P. and Pabo, C., 1991, Science, 252, 809.

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

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