AU646759B2

AU646759B2 - Method of stabilizing proteins during optical tests

Info

Publication number: AU646759B2
Application number: AU23498/92A
Authority: AU
Inventors: Johannes Buchner; Barbel Holl-Neugebauer; Rainer Rudolph
Original assignee: Boehringer Mannheim GmbH
Current assignee: Roche Diagnostics GmbH
Priority date: 1991-07-22
Filing date: 1992-07-22
Publication date: 1994-03-03
Anticipated expiration: 2012-07-22
Also published as: AU2349892A; ZA925458B; IL102561A0; WO1993002211A1; JPH06500705A; DE4124286A1; CA2092098A1; KR930702537A; EP0549778A1

Description

OPI DATE 23/02/93 APPLN. ID 23498/92 IIIH 111111Ifl"I AOJP DATE 29/04/93 PCT NUMBER PCT/EP92/01676 II 11111 IIuih.

AU9223498

E

(51) Internationale Patentk'2ssifikation 5 (U1) Internationale Ver6ffentlichungsnummer: WMO 93/02211 C12Q 1/34, 1/00 Al (43) Internationales Ver~iffentlichungsdatum: 4. Februar 1993 (04.02.93) (21) Internationales Aktcnzeichen: (22) Internationales Anmneldedatum: Prioritlitsdaten: P 4124 286.6 22. Ju] PCT/EP92/0 1676 22. Juli 1992 (22.07.92) (74)Anwiilte: WEICKMANN, H. usw. Kopern;KUsstrage 9, D-8000 Mflnchen 80 (DE).

(81) Bestinimungsstaaten: AU, CA, JP, KR, U europiiischers Patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IT, LU, MC, NL, SE).

Ver6ffentlicht Mit internationalem Recherclienbericht.

I i 1991 (22.07.9 1) (71) Anmelder (fdr alle Bestirnrungssiaten ausser US): BOEH- RINGER MANNH-EIM GMBH [DE/DE]; Sandhofer Strage 112 132, D-6800 Mannheim -Waldhof (DE).

(72) Erfinder; mid Erfinder/Anmelder (nurfiir US) RUDOLPH, Rainer [DE/ DE]; Fiirbergasse 17, D-8120 Weiheim HOLL- NEUGEBAUER, Bfirbel [DE/DE]; Geistb0helstrage 17, D-8 120 Weilheim BUCH-NER, Johannes [DE/ DE]; Arndtstrage HI, D-8400 Regensburg (DE).

(54) Title: METHOD OF STABILIZING PROTEINS DURING OPTICAL TESTS (54) Bezeichnung: VERFAHREN ZUR STABILISIERUNG VON PROTEINEN BEI OPTISCHEN TESTS (57) Abstract The invention rns a method of holding the sensitivity constant in optical tests, using protein -containing solutions, in wvhich the results can be affected by the low stability of proteins present as components of the test solution. The method is characterized in that one or more proteins of the "chaperonin 60" class, plus an optical-test reagent containing one or more "chaperonin proteins, are added to the test reagent and/or the test solution.

(57) Zusammenfassung Die Erfindung betrlfft emn Verfahren zur Konstanthaltung der Empf indlichkeit bei optischen Tests in proteinhaltigen Li~sungen, bei demn Stbrungen durch eine geringe Stabilitit von in der TestU~sung, vorhandenen Protein kompon enten auftreten k6nnen, welches daidurch gekennzeichnet ist, dali man dem Testreagenz oder/und der Testkisung en oider mehrere Proteine aus der Substanzklasse der "Chaperonin 60"-Proteine zusetzt, sowie emn Reagenz fOr einen optischen Test, das emn oder mehrere "Chaperonin enthilt, 00 000OCO00O00000 0 C9 0 0 0 0 0 0 0 0 0 0 0 20 71ME (iun) ZE1(.in) 30 Process for the stabilization of proteins in optical tests D e s cr i p t i on The present invention concerns a process for maintaining a constant sensitivity in optical tests in solutions containing protein in which interferences causel by a low stability of protein components present in the test solution can occur.

The GroE complex occurring in prokaryotic organisms 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 recently described "chaperone" protein family which usually 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. 6L (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), 2 Nature 333, 330-334) and various bacterial species (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 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 RNA polymerase from heat inactivation and can ATP-dependently renature an RNA polymerase which has already been inactivated by heat (Skowyra et al., Cell 62 (1990), 939-944). However, for this purpose a very large excess of DnaK protein is necessary in relation tu 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 proteir components are present in the test solution. Thus for example in investigations on the stability of a-glucosidase from baker's yeast it was found that this protein is very temperature sensitive. Above 40 0 C the protein unfolds and this is followed by aggregation. The precipitates which form in this process which can also occur at 37 0

C

at a lower rate impede the use of this protein in 3 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 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 process for increasing the stability of labile protein components in optical tests, in particular enzymatic tests, in which the disadvantages of 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 containing protein in whicn 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 proteins are added to the test reagent or/and the test solution.

It was surprisingly f und that the aggregation of a-glucosidase from baker's yeast can be completely suppressed when thermally stressed by the presence of GroEL protein from E. coli. Further addition of Mg-A't., 4 GroES protein and potassium ions to a solution containing a-glucosidase and GroEL protein causes precipitation at higher temperatures and reactivation of the a-glucosidase at lower temperatures.

The isolation of GroEL protein is described in an article by Georgopoulos, Mol. Gen. Genet. (1986), 202.

The purification 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 of the "chaperonin 60" 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 binding protein" from chloroplasts or/and analogous cytosolic proteins which are ubiquitouc 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 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 14 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 5 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 the 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 equimolar amount of the chaperone has to be added and it is even better to add an excess.

The process according 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 prevents 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 advantage of the process according to the present invention is that by preventing aggregation, measurement 6 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 a-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 preferably 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 ctass of "chaperonin 60" proteins, in particular the GroEL protein from E. coli, the hsp 60 protein from mitochondria, the "Rubisco subunit binain- 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 "chaperonin 60" protein in a dissolved form or/and as a lyophilisate. The "chaperonin 60" protein is preferably the GroEL protein from E. coli.

7 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 a-glucosidase at 46.3 0

C

in the absence or in the presence of a molar excess of GroEL protein, Fig. 2: shows the aggregation of a-glucosidase in the presence of different amounts of GroEL protein, Fig. 3: shows the dissociation of the a-glucosidase- GroEL complex caused by the addition of ATP, MgC1 2 K+ and GroES protein and Fig. 4 shows the reactivation of a-glucosidase at room temperature after thermal denaturation in the presence of GroEL protein by the addition of ATP, MgC12, K+ and GroES protein.

Examp 1 es Materials: a-glucosidase PI was expressed in yeast (strain ABYSMAL81, transforme.l 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.

8 GroEL and GroES were purified from an -ver-expressing E. coli strain (Fayet et al., Mol. Gen. Genet. 202 (1986), 435-445) by means of molecular sieve and ionexchange chromatography (Buchner et al., Biochem. (1991), 1587-1591). Adenosine triphosphate (ATP) and p-nitrophenyl-a-D-glucopyranoside (pNPG) were from Boehringer Mannheim GmbH.

Example 1 Suppression of the aggregation of a-glucosidase PI during thermal stress by GroEL A cuvette with 0.1 mol/l Tris buffer, pH 7.6 is thermostatted at ca. 46.5 0 C in the cuvette holder of a fluorescence spectrophotometer. The temperature in the cuvette is monitored with a thermosensor. a-glucosidase PI is added (final concentration 10 /g/ml 0.146 Amol/l); the aggregation of the enzyme is monitored by measurement of light scattering in a Hitachi fluorescence spectrophotometer F 4000 with the following instimnent settings: 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 a-glucosidase is added.

a-glucosidase is an enzyme which is very sensitive to temperature. At a temperature of 46 0 C and in the absence jI 9 of GroEL protein a very pronounced aggregation (light scattering) can already be seen within 10 minutes (Fig. 1).

However, if a-glucosidase is thermally stressed at 46 0

C

in the presence of a 1.5-fold molar excess of GroEL protein 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 a-glucosidase GroEL is varied (a-glucosidase GroEL 1 0.25 1 1 1 1 1.5 or 1 2 at an a-glucosidase concentration of 10 Ag/ml 0.146 Mmol/l) show that even small amounts of GroEL slow the formation of aggregates; an excess of GroEL completely suppresses the aggregation (Fig. 2).

Example 2 Dissociation of the a-glucosidase-GroEL complex by the addition of ATP, GroES and K+ a-glucosidase (10 g/ml, 0.146 mol/l) is incubated at 46 0 C with a 1.5-fold excess of GroEL (0.219 pmol/l in relation to the 14mer) in 0.1 mol/l Tris, 10 mmol/l KC1, pH 7.6 as described above. 2 mmol/l ATP, 10 mmol/l MgC12 as well as 0.146 Amol/l GroES are added after minutes.

When ATP/MgC1 2 and GroES are added at the same time the binding between GroEL and the a-glucosidase is broken and the liberated enzyme molecules aggregate (Fig. 2 mmol/l ATP and 10 mmol/l MgC12 (without GroES) also lead to a dissociation of the a-glucosidase- 10 GroEL complex; however, the light scattering shows an increase 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 liberated a-glucosidase.

Example 3 Reactivation of thermally denatured a-glucosidase PI a-glucosidase (10 Mg/ml) ii incubated for 60 minutes at 47 0 C 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 25 0 C, 2 mmol/l ATP, 10 mmol/l MgC12, mmol/l KC1 as well as 0.146 pmol/1 GroES are added In the control there are no additions. The reactivation of the a-glucosidase is monitored by means of an activity test using p-nitrophenol-a-Dglucopyranoside as an a-glucosidase substrate (Fig. 4).

Claims

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, wherein 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, wh 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" pLotein 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, wherein 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.

Process as claimed in one of the previous claims, wherein the optical test includes an enzymatic reaction. 12

6. Process as claimed in claim wherein the protein component to be stabilized is a-glucosidase PI.

7. Process as claimed in one of the previous claims, w h e r e i 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 protein.

8. Process as claimed in claim 7, wherein the GroEL protein from E. coli is used.

9. Process as claimed in one of the previous claims, wherein in addition a "chaperonin 10" protein, in particular the GroES protein from E. coli, is added for the renaturation of denatured protein components.

Reagent for an optical test, when used in the method of any one of claims 1 to 9 wherein said contains one or several proteins from the substance of class "chaperonin proteins.

11. Reagent as claimed in claim where in the "chaperonin 60" protein is present in a dissolved or/and lyophilized form. 13

12. Reegent as claimed in claim 9 or wh 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, whe r e i n the "chaperonin 60" protein is the GroEL protein from E. coli.

14. Processes for maintaining a constant sensitivity in optical tests according to the present invention substantially as hereinbefore described with reference to any one of the Examples. Dated this TWENTY-NINTH day of NOVEMBER 1993 Boehringer Mannheim GmbH Jy DAVIES COLLISON CAVE Patent Attor ys for the Applicant(s) A b st r a ct 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 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.