EP0549778A1

EP0549778A1 - Method of stabilizing proteins during optical tests

Info

Publication number: EP0549778A1
Application number: EP92916184A
Authority: EP
Inventors: Rainer Rudolph; Bärbel HÖLL-NEUGEBAUER; Johannes Buchner
Original assignee: Roche Diagnostics GmbH; Boehringer Mannheim GmbH
Current assignee: Roche Diagnostics GmbH
Priority date: 1991-07-22
Filing date: 1992-07-22
Publication date: 1993-07-07
Also published as: ZA925458B; IL102561A0; AU646759B2; KR930702537A; JPH06500705A; DE4124286A1; AU2349892A; WO1993002211A1; CA2092098A1

Abstract

L'invention concerne un procédé destiné à assurer un maintien constant de la sensibilité lors d'essais optiques effectués dans des solutions protéiniques, au cours desquels des perturbations peuvent se produire en raison d'une faible stabilité des composants protéiniques présents dans la solution d'essai, caractérisé en ce qu'on ajoute au réactif d'essai ou/et à la solution d'essai, une ou plusieurs protéines appartenant à la classe de substances des protéines du type "Chaperonine 60", ainsi qu'un réactif pour essai optique renfermant une ou plusieurs protéines "Chaperonine 60".The invention relates to a method for ensuring constant maintenance of sensitivity in optical tests performed in protein solutions, in which disturbances may occur due to poor stability of the protein components present in the solution of. test, characterized in that one or more proteins belonging to the class of substances of proteins of the type "Chaperonin 60" are added to the test reagent and / and to the test solution, as well as a test reagent optic containing one or more "Chaperonin 60" proteins.

Description

Process for stabilizing proteins in optical tests

Description

The present invention relates to a method for keeping the sensitivity constant in optical tests in protein-containing solutions, in which disturbances can occur due to low stability of protein components present in the test solution.

The GroE complex occurring in prokaryotic organisms, consisting of the proteins GroEL and GroES, is involved both in vitro and 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., Bioche istry 29 (1990), 5665-5671). GroEL belongs to the group of "chaperonin 60" proteins, while GroES belongs to the "chaperonin 10" group. Both protein groups are assigned to the most recently described, mostly ATP-dependent protein family of "chaperones", which are actively involved in 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. supports the translocation and folding of proteins. Other heatshock proteins related to GroEL are from mitochondria (Mcullin and Hallberg (1988), Molec.Cell.Biol. 8, 371-380), chloroplasts (Hemmingsen et al. (1988), Nature 333, 330-334 ) and various types of bacteria (Vodkin 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 to increase the yield of active protein in renaturation in vitro or in co-expression in vivo is already known and has been described in several publications. Using the example of in vitro renaturation of citrate synthase, light scattering measurements have shown that the GroE complex or GroEL protein can prevent the formation of the aggregation processes that occur during renaturation (Buchner et al., Biochemistry 30 (1991), 1586-1591). Furthermore, it is known that the DnaK protein from E. coli, another chaperone, protects the RNA polymerase from inactivation by heat and that an RNA polymerase which has already been inactivated by heat can renaturate depending on ATP (Skowyra et al., Cell 62: 939-944 (1990)). For this purpose, however, a very large excess of DnaK protein over the RNA polymerase is required.

The accuracy of optical tests, especially enzymatic tests, often depends on their sensitivity. This sensitivity is often reduced with a longer storage period if unstable or unstable protein components are present in the test solutions. For example, in studies of the stability of α-glucosidase from baker's yeast, it was found that this protein is very temperature sensitive. The protein unfolds above 40 ° C, followed by aggregation. The precipitates formed in this way, which can also occur at a slower rate at 37 ° C., hinder the use of this protein in photometric test systems, e.g. in the determination of α-amylase.

There are already known ways of constantly increasing the sensitivity in optical tests over a certain period of time hold by increasing the stability of unstable protein components. For example, bovine serum albumin or certain detergents can be added to the test solution in a low concentration. A disadvantage of known methods, however, is that the stabilization is often insufficient and that interfering interactions of the stabilizer with other components of the test system can occur.

The object of the present invention was therefore to provide a method for increasing the stability of unstable protein components in optical tests, in particular enzymatic tests, in which the disadvantages of the prior art are at least partially eliminated.

The object of the invention is achieved by a method for keeping the sensitivity constant in optical tests in protein-containing solutions, in which disturbances can occur due to a low stability of protein components present in the test solution, which is characterized in that the test reagent or / and the Test solution adds one or more proteins from the class of "Chaperonin 60" proteins.

It was surprisingly found that the aggregation of the α-glucosidase from baker's yeast under thermal stress can be completely suppressed by the presence of GroEL protein from E. coli. A further addition of g- _^ ATP, GroES protein and potassium ions to a solution containing α-glucosidase and GroEL protein causes precipitation at higher temperatures and reactivation of the α-glucosidase at low temperatures.

GroEL protein recovery is described in the article by Georgopoulos, Mol.Gen.Genet. (1986), 202. The Purification of GroEL protein is described in the article by Buchner et al. (Biochemistry 30 (1991), 1586-1591).

In addition to GroEL protein from E. coli, other members of the family of "chaperonin 60" proteins are suitable for the process according to the invention, e.g. proteins from other types of bacteria which are homologous to GroEL or "cpn 60" proteins from eukaryotes such as the hsp 60 protein which occurs in mitochondria, the "Rubisco subunit binding protein" from chloroplasts and / or analogous cytosolic proteins which are ubiquitous in eukaryotic - occur in organisms. Listings of "cpn 60" proteins can be found e.g. in Hallberg (1990), Semin.Cell Biol. 1, 37-45 and Hemmingsen (1990), Semin.Cell.Biol. 1, 47-54. GroEL protein from E. coli is particularly preferably used for the method according to the 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 invention. This molar ratio relates to the "chaperonin 60" complex having 14 subunits . The "chaperonin 60" protein is particularly preferred in a molar ratio of 0.001: 1 to 10: 1 in relation to the protein components to be stabilized and most preferably in a molar ratio of 0.1: 1 to 5: 1 in relation added to the protein components to be stabilized.

In cases in which only a small part of the protein component to be stabilized tends to aggregate, it is sufficient to add the "chaperonin 60" protein in molar deficit, since only a relatively small amount of protein is subject to aging. However, this is different in those cases in which, due to external circumstances, in particular due to thermal stress, it can be expected that the largest Part of the protein component to be stabilized will be subject to unfolding and thus ultimately to aggregation. In this case, the chaperone must be added at least in an equimolar amount, better still in excess.

The method according to the invention can be used in any optical test in which disturbances due to low stability of protein components present in the test solution can occur. Such test methods usually involve an enzymatic reaction. An "optical test" in the sense of the present invention is a determination in which an optical variable or the change in an optical variable, e.g. Absorption, transmission, stray light etc. is measured.

The stability of the protein components in a test solution is increased by the addition of one or more "chaperonin 60" proteins according to the invention, so that the occurrence of turbidity in the solution is prevented. This leads to a significant improvement in keeping the sensitivity constant, which is reflected in the low blank values of the test. Another advantage of the method according to the invention is that the prevention of aggregation prevents measurement errors which occur due to the carryover of protein aggregates.

A preferred example of a with "Chaperonin 60" proteins, e.g. Protein component to be stabilized with GroEL is the α-glucosidase PI from baker's yeast. However, the method according to the invention is not restricted to this protein.

In the process according to the invention, no "chaperonin 10" or GroES protein is usually added, since the "chaperonin 60" protein alone stabilizes the labile protein component by preventing aggregate formation. The addition of "chaperonin 10" proteins, ie of proteins which form a complex together with "chaperonin 60" proteins and Mg-ATP is only required if reactivation of denatured protein components is to take place. In this case, the GroES protein from E. coli is preferably used as the "chaperone 10" protein.

The present invention also relates to a reagent for an optical test, which can be solid or liquid, and one or more proteins from the substance class of the "chaperonin 60" proteins, in particular the GroEL protein from E. coli , the hsp 60 protein from mitochondria, the "Rubisco Subunit binding protein" from chloroplasts and / or an analog protein from the cytosol of prokaryotic or eukaryotic cells. A reagent according to the invention can contain, for example, the "chaperonin 60" protein in dissolved form and / or as a lyophilisate. Preferably the "chaperonin 60" protein is the GroEL protein from E. coli.

Finally, the present invention also relates to the use of "chaperonin 60" proteins, in particular GroEL protein, for stabilizing protein components for an optical test.

The invention is further illustrated by the following examples in conjunction with the drawing. It shows:

Fig. 1: the aggregation of α-glucosidase at 46.3 ° C in

Absence or in the presence of a 1.5-fold molar excess of GroEL protein,

Fig. 2 the aggregation of α-glucosidase in the presence of different amounts of GroEL protein, Fig. 3 shows the dissociation of the α-glucosidase-GroEL complex by adding ATP, MgCl ₂ , K ⁺ and GroES protein, and

Fig. 4 the reactivation of α-glucosidase at room temperature after thermal denaturation in the presence of GroEL protein by adding ATP, MgCl ₂ , K ⁺ and GroES protein.

Examples

Materials: α-glucosidase PI was expressed in yeast (strain ABYSMAL81, 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.

GroEL and GroES were purified from an overexpressing 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., Biochem. 30 (1991 ), 1587-1591). Adenosine triphosphate (ATP) and p-nitrophenyl-α-D-glucopyranoside (pNPG) were from Boehringer Mannheim GmbH.

example 1

Suppression of the aggregation of α-glucosidase PI under thermal stress by GroEL

A cuvette with 0.1 mol / 1 Tris buffer, pH 7.6 is heated to approx. 46.5 ° C. in the cuvette holder of the fluorescence spectrophotometer for 15 minutes. The temperature in the cuvette is checked with a thermal sensor. ^{'Α-glucosidase} PI is zuge¬ give (final concentration 10 ug / ml = 0.146 mol / 1); the aggregation of the enzyme is measured by light scattering in one Hitachi F 4000 fluorescence spectrophotometer tracked with the following device settings:

Time scan

Excitation wavelength: 360 n

Emission wavelength: 360 nm

Gap width excitation: 5 nm

Gap width emission: 5 nm The fluorimeter has a temperature-controlled cuvette holder with magnetic stirrer. In the experiments to suppress aggregation, GroEL is first mixed with the buffer and then the α-glucosidase is added.

α-Glucosidase is a very temperature sensitive enzyme. At a temperature of 46 ° C and in the absence of GroEL protein (O) very strong aggregation (light scattering) can be seen within 10 minutes (Fig. 1).

However, if α-glucosidase is thermally stressed in the presence of a 1.5-fold molar excess of GroEL protein (#) based on the GroEL complex with 14 subunits at 46 ° C., the aggregate formation can be completely suppressed (Fig. I. ¬

Experiments in which the ratio of α-glucosidase: GroEL is varied (α-glucosidase: GroEL = 1: 0.25 (Q), 1: 0.5 (1), 1: 1 _(O). 1 5 _(. #) or 1: 2 “>) at an α-glucosidase concentration of 10 μg / ml = 0.146 μmol / 1), show that even small amounts of GroEL slow the formation of aggregates; an excess of GroEL completely suppresses aggregation (Fig. 2). Example 2

Dissociation of the α-glucosidase-GroEL complex by adding ATP, GroES and K ⁺

α-glucosidase (10 μg / ml, 0.146 μmol / 1) is mixed with a 1.5-fold excess of GroEL (0.219 μmol / 1 based on 14-mer) in 0.1 mol / 1 Tris, 10 mmol / 1 KCl, pH 7.6 incubated at 46 ° C as described above. After 20 minutes, 2 mmol / 1 ATP, 10 mmol / 1 MgCl ₂ and 0.146 μmol / 1 GroES are added.

If ATP / MgCl ₂ and GroES (o) are added at the same time, the bond between GroEL and the α-glucosidase is released, and the enzyme molecules released aggregate (Fig. 3). 2 mmol / 1 ATP and 10 mmol / 1 MgCl ₂ (without GroES) also lead to a dissociation of the α-glucosidase-GroEL complex; however, the light scatter shows a flatter rise than in the previous experiment; the subsequent addition of GroES (φ) leads to rapid complete dissociation of the complex or aggregation of the released α-glucosidase.

Example 3

Reactivation of thermally denatured α-glucosidase PI

α-Glucosidase (10 μg / ml) is incubated in the presence of 1.5-fold GroEL excess for 60 minutes at 47 ° C. in 0.1 mol / 1 Tris buffer, pH 7.6. After the protein mixture has cooled to 25 ° C., 2 mmol / 1 ATP, 10 mmol / 1 MgCl ₂ , 10 mmol / 1 KCl and 0.146 μmol / 1 GroES are added (D) no additives. The reactivation of the α-glucosidase is followed using an activity test with p-nitrophenol-α-D-glucopyranoside as the α-glucosidase substrate (Fig. 4).

Claims

Patent claims

1. A method for keeping the sensitivity constant in optical tests in protein-containing solutions, in which disturbances can occur due to low stability of protein components present in the test solution, characterized in that one or more proteins from the substance class are added to the test reagent and / or the test solution the "chaperonin 60" protein.

2. The method of claim 1, d a d u r c h g e k e n n z e i c h n e t that the "chaperonin 60" protein is added in a molar ratio of 0.0001: 1 to 20: 1 with respect to the protein components to be stabilized.

3. The method of claim 2, d a d u r c h g e k e n n z e i c h n e t that the "chaperonin 60" protein is added in a molar ratio of 0.001: 1 to 10: 1 with respect to the protein components to be stabilized.

4. The method of claim 3, d a d u r c h g e k e n n z e i c h n e t that the "chaperonin 60" protein is added in a molar ratio of 0.1: 1 to 5: 1 with respect to the protein components to be stabilized.

5. The method according to any one of the preceding claims, characterized in that the optical test includes an enzymatic reaction.

6. The method of claim 5, d a d u r c h g e k e n n z e i c h n e t that the protein component to be stabilized is α-Glucosi¬ dase PI.

7. The method according to any one of the preceding claims, characterized in that the GroEL protein from E. coli, the hsp60 protein from mitochondria, the "Rubisco subunit binding protein" from chloroplasts and / or as a "chaperonin 60" protein analog protein from the cytosol of prokaryotic or eukaryotic cells is used.

8. The method of claim 7, d a d u r c h g e k e n n z e i c h n e t that the GroEL protein from E. coli is used.

9. The method according to any one of the preceding claims, that the addition of a "chaperonin 10" protein, in particular the GroES protein from E. coli, for the renaturation of denatured protein components is added.

10. Reagent for an optical test, so that it contains one or more proteins from the class of "chaperonin 60" proteins.

11. Reagent according to claim 10, characterized in that the "chaperonin 60" protein is present in dissolved or / and lyophilized form.

12. Reagent according to claim 9 or 10, characterized in that the "chaperonin 60" protein, the GroEL protein from E. coli, the hsp60 protein from mitochondria, the "Rubisco subunit binding protein" from chloroplasts and / or an analog Protein from the cytosol of prokaryotic or eukaryotic cells.

13. Reagent according to claim 12, so that the "chaperonin 60" protein is the GroEL protein from E. coli.

14. Use of "Chaperonin 60" proteins for stabilizing protein components for an optical test.

15. Use according to claim 14, that the use of GroEL from E. coli as a "chaperonin 60" protein is used.