Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
2~912~9~
Process for the stabilization of proteins in optical
tests
D e s c r i p t i o n
The present invention concerns a process for maintaining
a constant sensitivity in optical tests in solutions
containing protein in which interfexences caused by a
low stability of protein components present in the test
solution can occur.
The GroE complex occurring in prokaryotic oxganisms
which consists of the proteins GroEL and GroES is
involved in vitro as well as in vivo in the
reconstitution and association of proteins (Goloubinoff
et al., Nature 337 (1989), 44 47; Goloubinoff et al.,
Nature 342 (1989), 884-889 and Viitanen et al.,
Biochemistry 29 (1990), 5665-5671). GroEL is a member of
the "chaperonin 60" group of proteins, whereas GroES is
assigned to the "chaperonin 10" group. Both protein
groups are classified as belonging to the recent:Ly
described "chaperone" protein family which usual:Ly
depend on ATP and are actively involved in the folding,
association and translocation processes in the cell
(Landry and Gierasch, TIBS 16 (1991), 159-163, Ellis and
van der Vies, Ann. Rev. Biochem. 60 (1991), 321-347).
According to these articles the GroEL complex consists
of 14 subunits and promotes the translocation and
folding of proteins. Further heat-shock proteins which
are related to GroEL are known from mitochondria
(McMullin and Hallberg (1988), Molec. Cell. Biol. 8,
371-380), chloroplasts (Hemmingsen et al., (1988),
9 8
Nature 333, 330-334) and various bacterial species
fVodkin and Williams (1988), J. Bact. 170, 1227-1234;
Mehra et al., (1986), Proc. Natl. Acad. Sci. USA 83,
7013-7017 and Torres-Ruiz and McFadden (1989), Arch.
Biochem. Biophys. 261, 196-204).
The interaction of chaperones with newly synthesized or
denatured proteins in order to increase the yield of
active protein during renaturation in vitro or in
coexpression in vivo is already known and has been
described in several publications. For example in the in
vitro renaturation of citrate synthase it was possible
to show by light scattering measurements that the GroE
complex or the GroEL protein can prevent the development
of aggregation processes which occur during renaturation
(Buchner et al., Biochemistry 30 (1991), 1586-1591). In
addition it is known that the DnaK protein from E. coli,
another chaperone, protects RN~ polymerase from heat
inactivation and can ATP-dependently renature an ~NA
polymerase which has already been inactivated by heat
(Skowyra et al., Cell 62 (1990), ~39-944). However, for
this purpose a very large excess of Dna~ protein is
necessary in relation to the RNA polymerase.
The accuracy of optical tests, in particular of
enzymatic tests, often depends on their sensitivity.
This sensitivity is often reduced during long periods of
storage when unstable or labile protein components are
present in the test solution. Thus for example in
investigations on the stability of ~-glucosidase from
baker's yeast it was found that this protein is very
temperature sensitive. ~bove ~0C the protein unfolds
and this i.s followed hy aggregation. The precipitates
which form in this process which can also occur at 37C
at a lower rate impede the use of this protein in
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photometric test systems e.g. in the determination of
~-amylase.
Methods are already known for maintaining the
sensitivity of optical tests constant over a certain
time period by increasing the stability of labile
protein components. Thus for example one can add bovine
serum albumin or certain detergents at low
concentrations to the test solution. A disadvantage of
known methods is, however, that the stabilization is
often inadequate and that interf~ring interactions of
the stabilizer with other components of the test system
can occur.
The object of the present invention was therefore to
provide a process for increasing the stability of labile
protein components in optical tests, in particular
enzymati.c tests, in which the disadvantages o the state
of the art are at least partially eliminated.
The object according to the present invention is
achieved by a process for maintaining a constant
sensitivity in optical tests in solutions containin~
protein in which interferences caused by a low stability
of the protein components present in the test solution
can occur which is characterized in that one or several
proteins from the substance class of "chaperonin ~0"
proteins are added to the test reagent or/and the test
solution.
It was surprisingly found that the aggregation of
~-glucosidase from baker's yeast can be completely
suppressed when thermally stressed by the presence of
GroEL protein from E. coli. Further addition of ~g-ATP,
GroES protein and potassium ions to a solution
containing ~-glucosidase and GroEL protein causes
precipitation at higher temperatures and reactivation of
the ~-glucosidase at lower temperatures.
The isolation of GroEL protein is described in an
article by Georgopoulos, Mol. Gen. Genet. (19~6), 202.
The purificati.on of GroEL protein is described in an
article by Buchner et al. (Biochemistry 30 (1991), 1586-
1591).
In addition to GroEL protein from E. coli, other members
or the "chaperonin ~0" family of proteins are suitable
for the process according to the present invention e.g.
proteins from other bacterial species which are
homologous to GroEL or "cpn 60" proteins from eukaryotes
such as the hsp 60 protein which occurs in mitochondria,
the "Rubisco subunit bindin~ protein" from chloroplasts
or/and analogous cytosolic proteins which are ubiquitous
in eukaryotic organisms. Lists of "cpn 60" proteins may
be found for example in Hallberg (1990), Semin. Cell.
Biol. 1, 37-45 and Hemmingsen (1990), Semin. Cell.
Biol. 1, 47-54. The GroEL protein from E. coli is
particularly preferably used for the process according
to the present invention.
The molar ratio between the added "chaperonin 60"
protein and the protein component to be stabilized is
preferably 0.0001 : 1 to 20 : 1 in the process according
to the present invention. In this context this molar
ratio relates to the "chaperonin 60" complex which has
1~ subunits. The "chaperonin 60" protein is particularly
preferably added in a molar ratio of 0.001 : 1 to 10 : 1
in relation to the protein components to be stabilized
2 ~ 3 Q~ ~
and most preferably in a molar ratio of 0.1 : 1 to 5 : 1
in relation to the protein components to be stabilized.
In cases in which only a small portion of th~ protein
component to be stabilized has a tendency to aggregate
it is sufficient to add the "chaperonin 60" protein in a
molar deficit since in this case only a relative small
amount of protein is subject to unfolding. This is,
however, different in those cases in which due to
external circumstances, in particular due to thermal
stress it is probable that the major portion of the
protein components to be stabilized are liable to unfold
and thus finally to aggregate. In this case at least an
e~uimolar amount of the chaperone has to be added and it
is even better tv add an excess.
The process accordiny to the present invention can be
applied to every optical test in which interferences can
be caused by a low stability of protein components
present in the test solution. Such test procedures
usually include an enzymatic reaction. An "optical test"
within the sense of the present invention is a
determination in which an optical quantity or the change
in an optical quantity e.g. absorbance, transmission,
scattered light etc. is measured.
The addition of one or several "chaperonin 60" proteins
according to the present invention increases the
stability of the protein components in a test solution
and this p~events the occurrence of turbidity in the
solution. This leads to a considerable improvement in
the maintenance of a constant sensitivity which is shown
by the low blank values for the test. A further
advantaye of the process according to the present
invention is that by preventing aggregation, measurement
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errors caused by carry-over of protein aggregates are
prevented.
A preferred example of a protein component which is to
be stabilized by "chaperonin 60" proteins, e.g. by
GroEL, is ~-glucosidase PI from baker's yeast. The
process according to the present invention is, however,
not limited to this protein.
In the process according to the present invention a
"chaperonin 10" or GroES protein is not usually added
since the "chaperonin 60" protein already by itself
causes a stabilization of labile protein components by
preventing the formation of aggregates. The addition of
"chaperonin 10" proteins, i.e. of proteins which
together with "chaperonin 60" proteins and Mg-ATP can
form a complex, is only necessary when it is intended to
reactivate denatured protein components. In this case
one prefera~ly uses the GroES protein from E. coli as
the "chaperonin 10" protein.
The present invention also concerns a reagent for an
optical test which can be solid or liquid and contains
one or several proteins from the substance class of
"chaperonin 60" proteins, in particular the GroEL
protein from E. coli, the hsp 60 protein from
mitochondria, the "Rubisco subunit binding protein" from
chloroplasts or/and an analogous protein from the
cytosol of prokaryotic or eukaryotic cells. A reagent
according to the present invention can for example
contain the "chaperonirl 60" protein in a dissolved form
or/and as a lyophilisate. The "chaperonin 60" protein is
preferably the GroEL protein from E. coli.
2 ~
Finally the present invention also concerns the use of
"chaperonin 60" proteins, in particular GroEL proteins,
to stabilize protein components for an optical test.
It is intended to further elucidate the invention by the
following examples in conjunction with the figures.
Fig. 1: shows the aggregation of ~-glucosidase at 46.3C
in the absence or in the presence of a 1.5-fold
molar excess of GroEL protein,
Fig. 2: shows the aggregation of ~-glucosidase in the
presence of different amounts of GroEL protein,
Fig. 3: shows the dissociation of the ~~glucosidase-
GroEL complex caused by the addition of ATP,
MgC12, K+ and GroES protein and
Fig. 4 shows the reactivation of ~ glucosidase at room
temperature after thermal denaturation in the
presence of GroEL protein by the addition of
ATP, MgC12, K~ and GroES protein.
E x a m P 1 e s
Materials:
~-glucosidase PI was expressed in yeast (strain
ABYSMAL~1, transformed with the plasmid YEp/5c6b3)
(Kopetzki et al. (1989), EP 0 323 838, Kopetzki et al.,
Yeast 5 (1989), 11-24) and purified with the usual
methods of ion-exchange and hydrophobic interaction
chromatography.
., .
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GroEL and GroES were purified from an over-expressing
E. coli strain (Fayet et al., Mol. Gen. Genet. 202
(1986), 435-445) by means of molecular sieve and ion-
exchange chromatography (Buchner et al., ~iochem. 30
(1991), 1587-1591). Adenoslne triphosphate (ATP) and
p-nitrophenyl-~-D-glucopyranoside (pNPG) were from
Boehringer Mannheim Gmb~I.
Example
Suppression of the aggregation of ~-glucosidase PI
during thermal stress by GroEL
A cuvette with 0.1 mol/l Tris buffer, pH 7.6 i5
thermostatted at ca. 46.5C in the cuvette holder of a
fluorescence spectrophotometer. The temperature in the
cuvette is monitored with a thermosensor. ~-glucosidase
PI is added (final concentration 10 ~gtml = 0.146
~mol/l~; the aggregation of the enzyme is monitored by
measurement of light scattering in a Hitachi
fluorescence spectrophotometer F 4000 with the following
instrument settinys:
Time scan
Excitation wavelength: 360 nm
Emission wavelength: 360 nm
Slit width excitation: 5 nm
Slit width emission: 5 nm
The fluorimeter has a cuvette holder with a magnetic
stirrer which can the thermostatted. In the experiments
to suppress aggregation, GroEL is firstly mixed with the
buffer and subsequently ~-glucosidase is added.
~-glucosidase is an enzyme which is very sensitive to
temperature. At a temperature of 46C and in the absence
2 ~ 9 8
of GroEL protein (o) a very pronounced aggregation
(light scattering) can already be seen within 10 minutes
(Fig. l).
However, if ~-glucosidase is thermally stressed at 46C
in the presence of a 1.5-fold molar excess of GroEL
protein (o) in relation to the GroEL complex with 14
subunits then the formation of aggregates can be
completely suppressed (Fig. 1).
Experiments in which the ratio of ~-glucosidase : GroEL
is varied (~-glucosidase : GroEL = 1 : 0.25 (a)J 1 o 0 5
( J), 1: 1 (0), 1: 1.5 (~) or 1 : 2 (0) at an
~-glucosidase concentration of 10 ~g~ml = 0.146 ~mol/l)
show that even small amounts of GroEL 510w the formation
of aggregates; an excess of GroEL completely suppresses
the aggregation (Fig. 2).
Exam~le 2
Dissociation of the ~-glucosidase-GroEL complex by the
addition of ATP, GroES and K+
~-glucosidase (10 ~g/ml, 0.146 ~mol/l) is incubated at
46C with a 1.5-fold excess of GroEL (0.219 ~mol/l in
relation to the 14mer) in 0.1 mol/l Tris, 10 mmol/l KCl,
pH 7.6 as described above. 2 mmol/l ATP, 10 mmol/l MgCl2
as well as 0.146 ~mol/l GroES are added after 20
minutes.
When ATP/MgCl2 and GroES (o) are added at the same time
the binding between GroEL and the a-glucosidase is
broken and the liberated enzyme molecules aggregate
(Fig. 3). 2 mmol/l ATP and 10 mmol/l MgCl2 ~without
GroES) also lead to a dissociation of the ~-glucosidase-
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GroEL complex; however, khe light scattering shows anincrease which is smaller than in the previous
experiment; the subsequent addition of GroES (~) leads
to the rapid and complete dissociation of the complex or
to aggregation of the libera~ed ~-glucosidase.
Example 3
Raactivation of thermally denatured ~-glucosidase PI
~-glucosidase (10 ~g/ml) is incubated for 60 minutes at
47C in 0.1 mol/l Tris buffer, pH 7.6 in the presence of
a 1.5-fold excess of GroEL. After the protein mixture
has been cooled to 25C, 2 mmol/l ATP, 10 mmol/l MgCl2,
10 mmol/l KCl as well as 0.146 ~mol/l GroES are added
(a). In the control (~) there are no additions. The
reactivation of the ~-glucosidase is monitored by means
of an activity test using p-nitrophenol ~-D-
glucopyranoside as an ~-glucosidase substrate (Fig. 4).
