CA2092098A1 - Process for the stabilization of proteins in optical tests - Google Patents
Process for the stabilization of proteins in optical testsInfo
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- CA2092098A1 CA2092098A1 CA002092098A CA2092098A CA2092098A1 CA 2092098 A1 CA2092098 A1 CA 2092098A1 CA 002092098 A CA002092098 A CA 002092098A CA 2092098 A CA2092098 A CA 2092098A CA 2092098 A1 CA2092098 A1 CA 2092098A1
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- protein
- chaperonin
- groel
- proteins
- coli
- Prior art date
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
- C07K1/1133—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by redox-reactions involving cystein/cystin side chains
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- Life Sciences & Earth Sciences (AREA)
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- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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- Analytical Chemistry (AREA)
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- Chemical Kinetics & Catalysis (AREA)
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- Genetics & Genomics (AREA)
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- General Physics & Mathematics (AREA)
- Pathology (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
A b s t r a c t The invention concerns a process for maintaining a constant sensitivity in optical tests in solutions containing protein in which interferences caused by a low stability of protein components present in the test solution can occur which is characterized in that one or several proteins from the substance class of "chaperonin 60" proteins are added to the test reagent or/and the test solution, as well as a reagent for an optical test which contains one or several "chaperonin 60" proteins.
Description
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), 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 - 2~2~8 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 20~2~
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.
., .
2~2~98 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-2~9~8 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).
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), 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 - 2~2~8 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 20~2~
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.
., .
2~2~98 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-2~9~8 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).
Claims (15)
1. Process for maintaining a constant sensitivity in optical tests in solutions containing protein in which interferences caused by a low stability of protein components present in the test solution can occur, w h e r e i n one or several proteins from the substance class of "chaperonin 60" proteins are added to the test reagent or/and the test solution.
2. Process as claimed in claim 1, w h e r e i n the "chaperonin 60" protein is added at a molar ratio of 0.0001 : 1 to 20 : 1 in relation to the protein components to be stabilized.
3. Process as claimed in claim 2, w h e r e i n the "chaperonin 60" protein is added at a molar ratio of 0.001 : 1 to 10 : 1 in relation to the protein components to be stabilized.
4. Process as claimed in claim 3, w h e r e i n the "chaperonin 60" protein is added at a molar ratio of 0.1 : 1 to 5 : 1 in relation to the protein components to be stabilized.
5. Process as claimed in one of the previous claims, w h e r e i n the optical test includes an enzymatic reaction.
6. Process as claimed in claim 5, w h e r e i n the protein component to be stabilized is .alpha.-glucosidase PI.
7. Process as claimed in one of the previous claims, w h e r e i n 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 is used as the "chaperonin 60"
protein.
protein.
8. Process as claimed in claim 7, w h e r e i n the GroEL protein from E. coli is used.
9. Process as claimed in one of the previous claims, w h e r e i n in addition a "chaperonin 10" protein, in particular the GroEL protein from E. coli, is added for the renaturation of denatured protein components.
10. Reagent for an optical test, w h e r e i n it contains one or several proteins from the substance class of "chaperonin 60" proteins.
11. Reagent as claimed in claim 10, w h e r e i n the "chaperonin 60" protein is present in a dissolved or/and lyophilized form.
12. Reagent as claimed in claim 9 or 10, w h e r e i n the "chaperonin 60" protein is 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.
13. Reagent as claimed in claim 12, w h e r e i n the "chaperonin 60" protein is the GroEL protein from E. coli.
14. Use of "chaperonin 60" proteins to stabilize protein components for an optical test.
15. Use as claimed in claim 14, w h e r e i n GroEL from E. coli is used as the "chaperonin 60"
protein.
protein.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4124286A DE4124286A1 (en) | 1991-07-22 | 1991-07-22 | METHOD FOR STABILIZING PROTEINS IN OPTICAL TESTS |
| DEP4124286.6 | 1991-07-22 | ||
| PCT/EP1992/001676 WO1993002211A1 (en) | 1991-07-22 | 1992-07-22 | Method of stabilizing proteins during optical tests |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2092098A1 true CA2092098A1 (en) | 1993-01-23 |
Family
ID=6436753
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002092098A Abandoned CA2092098A1 (en) | 1991-07-22 | 1992-07-22 | Process for the stabilization of proteins in optical tests |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP0549778A1 (en) |
| JP (1) | JPH06500705A (en) |
| KR (1) | KR930702537A (en) |
| AU (1) | AU646759B2 (en) |
| CA (1) | CA2092098A1 (en) |
| DE (1) | DE4124286A1 (en) |
| IL (1) | IL102561A0 (en) |
| WO (1) | WO1993002211A1 (en) |
| ZA (1) | ZA925458B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5646249A (en) * | 1994-02-28 | 1997-07-08 | The United States Of America As Represented By The Department Of Health And Human Services | Isolation and characterization of a novel chaperone protein |
| US6013488A (en) | 1996-07-25 | 2000-01-11 | The Institute Of Physical And Chemical Research | Method for reverse transcription |
| CN104459127B (en) * | 2013-12-16 | 2018-03-23 | 顾晋元 | Bio-carrier and its application in the detection |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE1598157C3 (en) * | 1966-12-12 | 1973-11-22 | Boehringer Mannheim Gmbh, 6800 Mannheim | Stabilizing agent for enzymatic test reagents |
| US4956274A (en) * | 1987-04-06 | 1990-09-11 | Microgenics Corporation | Reagent stabilization in enzyme-donor and acceptor assay |
-
1991
- 1991-07-22 DE DE4124286A patent/DE4124286A1/en not_active Withdrawn
-
1992
- 1992-07-20 IL IL102561A patent/IL102561A0/en unknown
- 1992-07-21 ZA ZA925458A patent/ZA925458B/en unknown
- 1992-07-22 AU AU23498/92A patent/AU646759B2/en not_active Ceased
- 1992-07-22 WO PCT/EP1992/001676 patent/WO1993002211A1/en not_active Application Discontinuation
- 1992-07-22 KR KR1019930700801A patent/KR930702537A/en not_active Application Discontinuation
- 1992-07-22 EP EP92916184A patent/EP0549778A1/en not_active Withdrawn
- 1992-07-22 CA CA002092098A patent/CA2092098A1/en not_active Abandoned
- 1992-07-22 JP JP5502605A patent/JPH06500705A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| ZA925458B (en) | 1994-03-09 |
| JPH06500705A (en) | 1994-01-27 |
| EP0549778A1 (en) | 1993-07-07 |
| AU2349892A (en) | 1993-02-23 |
| AU646759B2 (en) | 1994-03-03 |
| IL102561A0 (en) | 1993-01-14 |
| WO1993002211A1 (en) | 1993-02-04 |
| DE4124286A1 (en) | 1993-01-28 |
| KR930702537A (en) | 1993-09-09 |
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| FZDE | Discontinued |