renaturation of proteins expressed in inclusion bodie

To: Nikolai troianovsk_s at MSDISK.WUSTL.EDU
Thu Apr 20 19:15:13 EST 1995


>Hi,
>Has anyone a reliable procedure (protocol and/or reference) for the
>renaturation of proteins expressed in e. coli as inclusion bodies?
> 
> 
>Stephan Witte
>dept. of immunology
>University of Constance
>stephan.witte at uni-konstanz.de
 Reply:

Refolding

After solubilizing the eukaryotic polypeptides, refolding is effected by
changing the buffer condition, out of the denaturant or other
solubilization agent. This can be achieved by dialysis or by dilution. The
rate at which the concentrations of the solubilization agent and the
recombinant polypeptide are changed can be controlled simultaneously, using
dilution. As for solubilization there are a number of variables which
influence the yield of active protein obtained.
(i) Rate of change from solubilization to refolding environment.
(ii) Purity of the recombinant polypeptide.
(iii) Concentration of the recombinant polypeptide.
(iv) pH.
(v) Ionic components of the solvent.
(vi) Presence or absence of thio reagents.

There are protocols which use two stages to effect refolding; it is thought
that in the first stage the proteins fold into forms approximating their
native state and in the second stage, folding is completed. Examples
include dilution of prochymosin from 8 M urea into buffer at pH 10.7; after
incubation the pH is adjusted to 8.0 (Marston,F.A.O.,et al. 1984
Bio/technol.,2,800). Another approach is to transfer the solubilized
proteins into low concentrations of urea to allow partial refolding
(Builder,S.E,and Ogez,J.R. 1985, US Patent No.4511502).

The purity of the recombinant polypeptide may be important as contaminants can
interfere with refolding. In this respect, isolation of inclusion bodies is
useful and furthermore, it is possible to purify the polypeptides in the
presence of certain solubilization reagents before refolding. Ion-exchange
chromatography can be performed in the presence of urea, even at
concentrations of 8 M. Guanidine hydrochloride is charged, and precludes
the use of ion-exchange chromatography, but not the use of gel filtration.
H.p.l.c. and f.p.l.c. can be used to purify the polypeptides in the
presence of a number of the solubilization conditions; h.p.l.c. in
particular can
be used in conjunction with organic solvents. Other methods which have been
used to purify recombinant polypeptides before refolding are high speed
centrifugation (Builder,S.E,and Ogez,J.R. 1985, US Patent No.4511502) and
organic extraction (Konrad,M.W.,and Lin,L.S. 1984 US Patent No.4450103).

The concentration of the recombinant protein during refolding is critical,
in order to ensure that intramolecular interactions occur in preference to
intermolecular interactions. This is particularly important if the native,
active protein contains intramolecular disulphide bonds. When the
disulphide bonds in the inclusion bodies are disrupted by the use of thiol
reagents or derivatization to S-sulphonates, then the formation of correct
disulphide bonds is promoted by mixtures of reduced and oxidized
glutathione. Both the concentration and ratio of reduced:oxidized reagent
must be optimized, and with the pH of the refolding buffer at 8 or above,
thiol-disulphide interchange occurs rapidly.

It is possible to promote thiol-disulphide interchange at high pH in the
absence of exogenous thiol reagents; the protein itself can act as a source
of free thiol (Marston,F.A.O.,et al. 1984 Bio/technol.,2,800).

During the development of a refolding protocol, the efficiency of refolding
must be assessed to allow optimum conditions to be established. There are a
number of ways in which refolded polypeptides can be analysed. SDS-PAGE and
analytical h.p.l.c. or f.p.l.c. can be used to examine the state of
aggregation of the final product. SDS-PAGE analysis in the presence and
absence of 2-ME may reveal incorrect, intermolecular disulphide bonds. If
there are contaminating proteins still remaining, interpretation of a
stained gel may be difficult. This problem can be overcome by Western
blotting. The best measure of the efficiency of refolding to yield active
protein is to assay biological activity and this is feasible for many
enzymes.

Hope this helps.
Nikolai.





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