Molecular Techniques and Methods

Processing of Fusion Proteins:
In vitro Re-folding of Proteins

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Proteins produced in E. coli often form inclusion bodies and these proteins can only be solubilized with denaturing reagents (e.g., urea and guanidine-HCl) which cause complete unfolding. These proteins must be re-folded to their native and functional forms in order to carry out biochemical and structural studies. In general, a protein has the lowest free energy in its native conformation and should fold into the native conformation spontaneously. Nevertheless, the in vitro re-folding of proteins is usually not straightforward. The nascent polypeptides probably start folding using short-range interactions as they emerge from the ribosome. Long-range interactions gradually build up to complete the folding process. In vitro the full-length protein could be trapped in a local free energy minimum and that re-folding is not possible unless this cotranslational folding process is re-produced. The major obstacle of spontaneous re-folding is aggregation of proteins. There are no general protocols for re-folding proteins but in all cases proteins are first dissolved in denaturants (e.g., urea and guanidine-HCl), which keep proteins unfolded and prevent aggregation. It is also important to include reducing reagents such as 2-mercaptoethanol and dithiothreitol (DTT) to keep sulfhydryl groups reduced. When denaturants are removed, proteins are allowed to fold. This is normally accomplished by three methods:


Dilution:
Protein in denaturant (e.g., urea or guanidine-HCl solutions) are diluted into a large volume of dilution buffer. The denaturant concentration quickly decreases and proteins are allowed to fold. As protein molecules spend little time in intermediate concentrations of denaturant, the aggregation of unfolded protein or folding intermediates can be avoided.


Dialysis:
Protein concentrations are kept high and the denaturant (e.g., urea and guanidine-HCl) concentration is decreased slowly so that proteins tend to aggregate.


Folding on a Solid Support:
Aggregation of proteins can be avoided by fixing proteins on a solid support. Proteins in urea are applied to an ion-exchange column and as urea is washed from the column proteins are allowed to fold. The proteins are then eluted from the column with a salt gradient.


Whichever of the above methods is used, the pH, ionic strength, organic solvent, other additives, temperature, and the time course of changes in these parameters affect the folding process.

  • The folding process is more complex for proteins with disulfide bridges. Formation of incorrect disulfide bridges must be avoided. Folding is normally carried out in the presence of mixed disulfide reagents such as a mixture of reduced and oxidized glutathione.


  • Folding of proteins with a prosthetic group such as haem, vitamin B12, and flavin is more difficult to achieve. Such proteins are normally folded first in the absence of the prosthetic group and the prosthetic group is added later to the folded polypeptide.





  • MATERIALS AND SOLUTIONS

    Dilution Buffer (1 liter)
    50 mM Tris-HCl (pH 6.0) --------------------- 50 ml of 1 M Tris-HCl
    1 mM EDTA ---------------------------------- 2 ml of 0.5 M EDTA
    1 mM DTT ------------------------------------ 1 ml of 1 M DTT
    Deionized H2O -------------------------------- 947 ml




    PROCEDURES

    1. Dilute the unfolded protein solution in 8 M urea by Dilution Buffer. The final concentration of urea is less than 0.8 M and the folded protein is stable under these conditions.

    2. Purify the reconstituted protein by ion-exchange chromatography.

    3. Alternatively, remove the urea completely by dialysis in cold Phosphate Buffered-Saline (pH 7.4).




    NOTES




    KIT INFORMATION




    REFERENCES

  • Carter, P., Abrahmsdn, L., and Wells, J.A., 1991, Biochemistry, 30, 6142.


  • Jacnicke, R. and Rudolph, R., 1989, In Protein structure: a practical approach (ed. T. E. Creighton), pp.191-223. IRL Press, Oxford.


  • Reinach, F.C., Nagai, K., and Kendrick-Jones, J., 1986, Nature, 322, 80.


  • Varadarajan, R., Szabo, A., and Boxer, S.G., 1989, PNAS, 82, 5681.


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

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