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:
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.
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
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
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.