LT5053B - Chaperons DnaK, DnaJ and GrpE from Meiothermus ruber having increased thermal stability and system and process for refolding proteins in vitro - Google Patents

Abstract

The present invention claims protein refolding system which includes thermophilic chaperons cloned from mezophilic microorganism Meiothermus rubber. The chaperons mentioned above are characterized by increased thermal stability and refolding activity compared to chaperons known previously. The refolding system claimed (the refolding buffer) consists of denaturated protein, neorganic salts, the mixture of three purified thermophilic chaperons (DnaK, DnaJ and GrpE) and adenosintriphosphate. The invention belongs to biotechnology and may be useful in restoring in vitro the active structure of native and recombinant proteins in the process of their preparation.

Description

The present invention relates to the field of protein biotechnology. Its essence is the use of new chaperones to restore the active protein structure (by refolding denatured proteins) in vitro.

Molecular chaperones are proteins that restore the tertiary structure (conformation) of denatured or 10 non-formed proteins, which enable the latter to acquire their characteristic biochemical or physiological activity. If the activity of denatured enzymes is restored, this case of refolding is called reactivation. The thermophilic chaperones DnaK, DnaJ and GrpE are recombinant proteins cloned from Meiothermus ruber (M. ruber) and expressed in E. coli (hereafter thermophilic chaperone proteins are designated as Λ / r u. DnaK, Mrw.DnaJ, Λ / rn.GrpE, and their genes, respectively, Mru.dnaK, Mru.dnaJ, Mru.grpE). All of these proteins are purified from E. coli and utilized in an in vitro system for the refolding of denatured proteins.

There are several patents known for the use of chaperone proteins or genes for the production of active conformation proteins, including: EP0885967 (with equivalents JP11009274,

US6159708), EP1016724 (with equivalents JP 2000189163, US6197547), EP0998485,

WO9905163, EP1149909, W00071723.

The authors of EP0998485, WO9905163 propose the use of chaperonines Hsp60 (GroEL) or fragments thereof together with foldases for protein refolding, which may be peptidylprolylisomerases or thiol / disulfide oxidoreductase (often referred to as protein disulfide25 isomerases). The authors of EP1149909, W00071723 use the in vitro non-thermophilic chaperones DnaK, DnaJ and GrpE from various sources (E. coli, yeast, plants and mammals) together with another group of chaperones ClpB and ClpS. The latter authors and the authors of EP0885967, EP1016724 propose variants of co-expression of chaperones DnaK, DnaJ, GrpE, GroES and GroEL with the target protein of interest in order to obtain them in active conformation in vivo. However, none of the analogues in question used thermophilic chaperones for protein refolding. Furthermore, as will be shown below, the unique thermophilic chaperones of the present invention outperform analogs from other sources in their activity.

It was an object of the present invention to provide an in vitro protein refolding system using recombinant thermophilic chaperones. To this end, thermophilic chaperone Mru. The dnaK, Mru.dnaJ, and Mru.grp E genes were cloned from the mesophilic microorganism M. ruber, transferred to E. coli, and expressed. The novel recombinant chaperones thus obtained were purified and used in the construction of protein refolding systems in vitro. Chaperone systems have been found which efficiently restore the structure of denatured proteins and can be used to restore protein activity in a variety of applications in biotechnological processes.

v

The DnaK, DnaJ and GrpE chaperone proteins of the present invention were first identified, cloned, and expressed, and exhibited high activity in protein refolding. The complete sequence of the M.ruber dnaK (hsp70) operon was disclosed and deposited on behalf of the authors in the GenBank Database, AF507046 (http://www.nebi.nlm.nih.gov/).

Chaperones A / rw.DnaK, A / r «.DnaJ and Mru. GrpE are thermophilic and differ from those previously known for their origin, high temperature resistance, and higher refold activity. The new thermophilic chaperones from M. ruber have the characteristics shown in Table 1:

Table 1.

Chaperones Chaperones class An amino acid chicks number Molecular mass, kDa Isoelectric dot, PI Mru.DnaK Hsp70 630 66.7 5.17 Mru.DnaJ Hsp40 293 32.1 9.22 Mru.GrpE Hsp22 176 19.5 4.86

Their amino acid sequences are shown below (Figs. 1-3).

Recombinant plasmids pUC19TR1, pUC19-TR2 and pUC19-TR3 were used to generate novel chaperone proteins; these plasmids are stored in the Recombinant Protein Research Laboratory of the Institute of Biotechnology (Vilnius) under the numbers BI-TR1, BI-TR2, BI-TR3, respectively.

The recombinant plasmid pUC19-TRl (Accession number BI-TR1) has the following properties:

- it encodes part of the M.ruber dnaK operon;

- has a total length of 5500 base pairs;

- it consists of the following elements:

EcoRl-HindlII 2.7 ths. a pore size pUC19 plasmid fragment containing a gene conferring ampicillin resistance and a pMB1 replicon (ori region); 2.8 thousand EcoR1-HindIII fragment. the size of the base pore encoding part of the Mru.dnaK gene, the entire Mru.grpE and part of the Mru.dnaJ gene.

The plasmid pUC19-TR2 (deposit number BI-TR2) has a total length of 3500 bp, encodes a portion of the M.ruber dnaK operon, and consists of the following elements:

EcoRl-HindlII 2.7 ths. a pore-size plasmid fragment of base pore containing the gene-specific resistance to ampicillin and the replicon (orI region) to pMB1; EcoRl-HindlII 0.8 thousand. a pore-size fragment encoding the 5'-terminal Mru. part of the dnaK gene and its promoter.

Plasmid pUC19-TR3 (deposit number BI-TR3) has a total length of 5500 bp, encodes part of the M.ruber dnaK operon, and consists of the following elements:

Ecl \ 3611 2,7 ths. a pore size base of a pUC19 plasmid containing gene-specific ampicillin resistance and a replicon (orI region) of pMB1;

Ecl \ 3611 2.8 thousand. a pore-size fragment encoding part of the Mru.grpE gene and the entire Mru.dnaJ gene.

The in vitro protein active structure refolding system comprises a lost active structure protein buffer containing an ionic salt salt disulfide protein bridge reducing agent adenosine triphosphate and cloned thermophilic Mru.DnaK, Mru.OnaS and Mru.GrpE chaperones. The system is in the form of an aqueous solution and the amount of components is determined from the conditions:

Protein whose structure requires 3-5 μΜ

Bumper (optimum TRIS-HC1)

Salts required to maintain ionic strength Agent for reducing disulfide protein bridges (optimally dithiothreitol)

Thermophilic chaperone proteins:

Mru. DnaK,

Afru.DnaJ

A / ru.GrpE

Adenosine triphosphate pH 7.5 - 8.5 0.38-0.42 M

2-5 μΜ

- 5 μΜ 3 - 5 μΜ

0.3 - 0.5 μΜ 9-11 mM.

An in vitro method of restoring the active structure of a protein using chaperones according to the invention is characterized in that the denatured protein is diluted with a buffer such as TRISHC1, pH 7.5 + 0.1, containing ionic salts such as Na ₂ SO 4, KC 1, Mg ( CH3COO) 2, add dithiothreitol to 2-5 μΜ for reduction of disulfide protein bridges, then add a mixture of thermophilic chaperones Mrw.DnaK, Mru.OnaJ and Mru.GrpE, optimally at 10: 10: 1, after incubation at 37 ° C. adenosine triphosphate to a concentration of 10 + 1 mM and retains for at least 5 min.

The in vitro protein refolding system of the present invention using recombinant thermophilic chaperones is illustrated in Figs. 11, together with the methodologies used.

Figure 1 shows the amino acid sequence of A / ru.DnaK thermophilic chaperones of the invention;

Fig. 2 shows the amino acid sequence of Λ / ru.GrpE thermophilic chaperones of the invention;

Figure 3 shows the amino acid sequence of A / rw.DnaJ thermophilic chaperones of the invention;

FIG. 4 shows the nucleotide sequence of the M. ruber operon encoding the genes Mru.dnaK, Mru.dnaJ, Mru.grpE. Mru.dnaK gene 634 to 2499 nucleotides, Mru.grpE gene 2578 to 3108 nucleotides, Mru.dnaJ gene 3182 to 4063 nucleotides.

FIG. 5 replicates the Southem blot image of Meiothermus ruber chromosomal DNA using a [ ³² P] -labeled fragment of the foaA 'gene. Selected fragments for use in cloning.

Chromosomal DNA performed:

\ -EcoRV

2- EcoRV + Eco47III 3 - Eco471II 4-NsbI 5 - Bspl20I

- Bspl20I + PaeI

- Pael & -Alw44I

- Alw44I + EheI \ 0-EheI

M - DNA marker (3.0; 3.5; 4.0; 5.0; 6.0; 8.0; 10.0 kb)

FIG. Scheme of plasmids pUC19-TR1, pUC19-TR2 and pUC19-TR3 with M. ruber DNA fragments. FIG. 7 illustrates purification steps of Mru.DnaK protein by fractional analysis on SDS-PAA gels (12%, reducing conditions), where

A Chromatography on a DEAE-Sepharose column:

- molecular weight standards for protein (from above: 116, 66.2, 45, 35, 25, J8.4 kDa),

- biomass extract supernatant prior to application to DEAE-Sepharose,

- non-adsorbed fraction,

4-10 are fractions from a DEAE-Sepharose column.

. 5 B Chromatography on Phenyl-Sepharose columns:

- molecular weight standards for the protein, (top: 116, 66.2, 45, 35, 25 kDa),

- pooled fraction after DEAE-Sepharose column,

3-10 are fractions from the Phenyl-Sepharose column.

C Gel filtration on Sephacryl S-300 columns:

1 - molecular weight standards for the protein, (from above: 116, 66.2, 45, 35, 25, 18.4, 14.4 kDa),

- pooled fraction after selection of Phenyl-Sepharose column,

3-10 are fractions from a Sephacrylic column.

FIG. 8 depicts the purification steps of Λ / ru.DnaJ protein by fractional analysis on 15 SDS-PAA gels (12%, reducing conditions), where

A Chromatography on an LRY2KT-Sepharose column:

- molecular weight standards for the protein, (top: 116, 66.2, 45, 35, 25, 18.4, 14.4 kDa).

- biomass extract supernatant prior to application to LRY2KT-Sepharose,

- non-adsorbed fraction,

4-10 are fractions from the LRY2KT -Sepharose column;

B Chromatography on a CM-Sepharose column:

- molecular weight standards for the protein, (top: 116, 66.2, 45, 35, 25 kDa).

- pooled fraction fraction after LRY2KT-Sepharose column;

- non-adsorbed fraction,

4-9 are fractions from the CM-Sepharose column.

FIG. 9 depicts the purification steps of A / ru.GrpE protein, fractional analysis by electrophoresis on SDS-PAA gels (15%, reducing conditions), where A Chromatography using DEAE-Sepharose column:

1 - molecular weight standards of protein (from above: 116, 66.2, 45, 35, 25, 18.4 kDa),

- biomass extract supernatant prior to application to DEAE-Sepharose,

- Unsorbed fraction;

4-10 are fractions from a DEAE-Sepharose column.

B Chromatography on an Octyl-Sepharose column:

- molecular weight standards for protein (from above: J16, 66.2, 45, 35, 25, 18.4, 14.4 kDa),

- pooled fraction after DEAE-Sepharose column,

3-10 are fractions from the Octyl-Sepharose column.

C Gel filtration using a Sephacryl S-100 column:

- molecular weight standards for protein (from above: 116, 66.2, 45, 35, 25, 18.4, 14.4 kDa),

- pooled fractions after octyl-Sepharose column,

3-8 are fractions from a Sephacryl S-100 column.

FIG. 10 shows the thermal stability of recombinant A / rzz.DnaK (Hsp70) and Mru.DnaJ (Hsp40).

FIG. 11 shows reactivation (restoration of activity) of MDH using E. coli

DnaK / J / GrpE and M. ruber DnaK / J / GrpE chaperone systems according to the invention, wherein α- MDH + M.ruber chaperones; b - MDH + E.coli chaperones; c - spontaneous reactivation of MDH;

Details of the cloning of novel chaperone genes, purification of gene expression in E.coli, purification of the resulting chaperones, and determination of their thermal stability are described in the examples below. These examples, as well as protein-refolding systems with thermophilic chaperones Mru.DnaK, Mr «.DnaJ and Mru. An example of the preparation and use of GrpE for the recovery of malate dehydrogenase activity does not limit the scope of the invention as defined in the claims.

example.

Cloning of thermophilic chaperone genes

The bacterial strain Meiothermus ruber, which was isolated from Kamchatka (RU) geysers, was selected for cloning thermophilic chaperones. The strain was kindly provided by MBI Fermentas (Vilnius). M ruber is a Gram-negative bacterium that does not form spores and has a growth temperature optimum of 60-65 ° C. The chromosomal DNA isolated from the M. ruber strain was digested with several restriction enzymes, electrophoretically fractionated on 1% agarose, and blotted with the [ ³² P] -labeled fragment of the M. ruber dnaK gene obtained by polymerase chain reaction (PCR) amplification of the conserved dnaK gene 5'- a portion with degenerate primers selected for analysis of the E. coli and Thermus thermophilus dnaK gene sequences:

'-ATTGACCTGGGIACIACIAAC-3' '-C (G / T) GCGGTIGG (C / T) TCGTTGAT-3' wherein I is an inosine nucleotide.

The autoradiogram of the Southern blot is shown in Figs. 5.

M. ruber chromosomal DNA fragments (2.8 and 0.8kb) obtained by EheI restriction enzyme were selected for cloning. These fragments were isolated from preparative gel electrophoresis and ligated to the target of plasmid pUC19 Ecll36II and transformed into E. coli. The resulting clones were re-hybridized with the above fragment of the M. ruber dnaK gene and two colonies containing plasmids with 2.8 and 0.8 kb fragments were selected. These plasmids (pUC19-TR1 and pUC19-TR2) were sequenced. Analysis of the obtained data revealed that their fragments comprise most of the M. ruber operon with dnaK, grpE and dnaJ genes (Fig. 6).

After detecting that the Mru.dnaJ gene was deficient in plasmid pUC19-TR1, another clone containing the plasmid pUC19-TR3 with the Ecll36-Cfr42I fragment (2.8 kb) was identified by hybridization as described above. Sequencing data confirmed that plasmid pUC19TR3 contains the remainder of the dnaJ gene. The complete sequence of the M. ruber dnaK-grpE-dnaJ operon is presented in Figs. 4.

example.

Expression of the thermophilic chaperone Mru.dnaK, Mru.dnaJxrMru.grpE genes in E. coli The thermophilic chaperone genes Mru.dnaK, Mru.dnaJ and Mru.grpE were transferred from pUC19-TR1 and pUC19-TR3 into the expression plasmid pET21b (+) (Novagen , published in US4952496). This was done by PCR using Ndel targets at the 5'-end of each gene and BamHI targets at the 3'-end. Thus modified fragments of the respective genes were ligated with pET21b (+) plasmid cut through Ndel-BamHI targets. Selected plasmids pET21-Mru-dnaK, pET21-Mru-dnaJ and pET21-Mru-grpE were further transformed into the E. coli BL (DE3) expression strain. Colonies expressing high levels (10-20% of total cellular protein) of recombinant thermophilic chaperone after induction with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) were selected. Such strains are suitable for further purification of thermophilic chaperones, providing sufficient amounts for in vitro protein refolding.

example

Purification of thermophilic chaperones 3 / r «.DnaK, Λ / rn.DnaJ andMru.GrpE.

The purification of the recombinant chaperones Mru.DnaK and Mru.DnaJ by analytical amounts was performed as follows. E. coli BL (DE3) pET21-Mru.dnaK and E. coli BL (DE3) pET21 -Mru.dnaJ strains were grown to OD6oonm ⁼ 0.6-1.0 and induced for 2-3 h. with the addition of lmM IPTG. The resulting biomass is centrifuged, resuspended in 10mM TRIS-HCl, pH 7.8, 1mM EDTA buffer and sonicated. After centrifugation of the lysate, supernatant is diluted 10 times with A / ru.DnaJ in 20mM CFbCOONa, pH 5.6 buffer.

The solution was applied to an Imi SP-Sepharose Fast Flow (Amersham Pharmacia Biotech) column (AKTA explorer chromatography system) and a 0-1M NaCl gradient in 20mM CHiCOONa pH 5.6 buffer was run. In the case of Λ / ru.DnaK, the supernatant was diluted 10 together with 50 mM TRIS-HCl, pH 8.5 buffer. The solution was applied to an Imi Q-Sepharose Fast Flow column and a 0-1M NaCl gradient in 50mM TRIS-HCl, pH 8.5 buffer was run.

After analysis of the obtained fractions on SDS-PAA gel, those containing the purer T / ry.DnaK or A / ru.DnaJ proteins are selected.

Recombinant A / ru.DnaK, Λ / ru. Preparative purification of DnaJ and Λ / ru.GrpE is accomplished by an individual method for each of the proteins, nailed to its physicochemical properties such as isoelectric point (pi), hydrophobicity, histidine amino acid number and position.

Λ / ru.DnaK (pi 5.17) was purified by three chromatographic steps: DEAE-Sepharose, Phenyl-Sepharose and Sephacryl S-300 columns (Fig. 7).

A / ru.DnaJ (pi 9.22) was purified by two chromatographic steps: on LRY2KTSepharose (sorbent with biomimetic dye - light-resistant yellow containing copper ions) and on CM-Sepharose (Fig. 8).

Λ / ru.GrpE (pi 4.86) was purified through three chromatographic steps: DEAESepharose, Octyl-Sepharose and Sephacryl S-100 (Fig. (Fig.9). 9). The applied purification methodology gave satisfactory results - more than 85% purity for M. ruber chaperones Λ / ru.DnaK, A / ru.DnaJ, A / ru.GrpE.

example

Determination of thermal stability of chaperones Λ / ru.DnaK and A / ru.DnaJ

The thermal resistance of chaperones Λ / ru.DnaK and A / ru.DnaJ was tested in ultrasonic clarified extracts of induced biomasses. E. coli cells expressing A / ru.DnaJ (Fig. 10, lanes 2–7) and A / ru.DnaK (Fig. 10, lanes 8, 9) were centrifuged and resuspended in 10 mM TRIS-HCl, 1 mM EDTA in pH 7.4 buffers; 7.8; 8.2 (25 ° C). Cells were sonicated and the soluble lysate fraction was incubated at 37 ° C for 10 min, transferred to 65 ° C for 10 min. and centrifuged. The soluble fraction (S) and the precipitate (P) were analyzed by electrophoresis

12% in SDS-PAA gel. FIG. Lane 10 - protein molecular weight standards (top: 116.66.2.45, 35.25, 18.4.14.4 kDa).

It has been observed that the thermal stability of recombinant Λ / ru.DnaJ increases with increasing pH of the buffer (Fig. 10). At pH 7.4 Mru.OnaJ settles completely and at pH 7.8 it becomes semi-soluble. The protein A / rw.DnaJ remains thermally resistant at pH 8.2 (Fig. 10, lanes 6 and 7). The thermal resistance of recombinant A / rw.DnaK is pH independent. This protein was soluble in the pH range 7.4 to 8.2 (Fig. 10, lanes 8 and 9).

example

Preparation and use of a protein active structure reflux system with thermophilic chaperones Mr «.DnaK, Mrrr.DnaJ and Mrr / .GrpE to restore malate dehydrogenase activity.

Chaperone activity was determined as follows. 3.8 μΜ of mitochondrial malate dehydrogenase (MDH) was denatured in buffer with 3 M guanidine hydrochloride for 1 h. At 37 ° C and then diluted 30-fold (to 0.127 μΜ) in folding buffer (25 mM TRIS-HCl, pH 7.5, 0.25 M Na ₂ SO ₄ , 150 mM KCl, 20 mM Mg (CH ₃ COO) ₂ , 3 mM DTT ) with the chaperones 3.5μΜ A / rw.DnaK, 3.5μΜ Afrir.DnaJ and 0.35μΜ A / rw.GrpE or with the same amounts of E. coli recombinant chaperones DnaK, DnaJ and GrpE. The mixture was incubated for 5 min. At 37 ° C and then 10 mM adenosine triphosphate (ATF) was added. Recovery (reactivation) of mitochondrial malate dehydrogenase activity was measured for 10 min. interval 2 or. on the track. MDH activity was measured according to known procedures (Sanchez SA, Hazlett TL, Brunet JU, Jameson DM, 1998, Aggregation statuses of mitochondrial malate dehydrogenase. Protein Sci 7: 2184-2189). The control sample with denatured MDH was incubated without chaperones, a sample of spontaneous reactivation of MDH.

From FIG. From Figure 11, it is evident that in the sample with M. ruber chaperones, MDH reflux occurs about 10-fold more intensively than spontaneous MDH reactivation and about 3-fold more intensively than with an analogous system with E. coli chaperones. Thus, it can be argued that the protein active structure refolding system using the recombinant chaperonic M. ruber proteins of the invention is superior to the analogous proteins of E. coli.

Claims (10)

DEFINITION OF INVENTION 1. The protein active structure refolding protein Mrw.DnaK is a cloned thermophilic chaperone having the amino acid sequence set forth in Figs. 1. 2. Protein Active Structure Refolding MruGrpE is a cloned thermophilic chaperone having the amino acid sequence shown in Figs. 2. 3. Protein Active Structure Refolding Protein Mrw.DnaJ - Cloned 10 is a thermophilic chaperone having the amino acid sequence set forth in FIGS. 3. Protein active structure refolding chaperones according to claims 1-3, characterized by increased thermal stability at 62-65 ° C, pH 8.2-8, 5 interval. 5. The M. ruber operon encoding the Mru.dnaK, Mru.dnaJ, Mru.grpE genes having the nucleotide sequence shown in Figs. 4. 6. The recombinant plasmid pUC19-TR1, accession number BI-TR1, has a total length of 5500 base pairs encoding a portion of the Mru dnaK operon and consisting of the following elements: 20 EcoRI-Hindffl 2, 7 thousand a pore-size plasmid fragment of base pUC19 containing a gene conferring ampicillin resistance and a replicon (ori region) of pMB1; 2.8K of the EcoRI-HindIII fragment. the size of the base pore encoding part of the Mru.dnaK gene, the entire Mru.grpE and part of the Mru.dnaJ gene. 7. Recombinant plasmid pUC19-TR2, accession number BI-TR2, total length 3500 base pairs encoding part of the Mru dnaK operon and consisting of the following elements: EcoRI-HindlII 2,7k. a pore size pUC19 plasmid fragment containing a gene conferring ampicillin resistance and a pMB1 replicon (ori region); EcoRI-HindlII 0.8 thousand. a pore size fragment encoding the 5'-terminus 30 of the Mru.dnaK gene and its promoter. 8. The recombinant plasmid pUC19-TR3, accession number BI-TR3, has a total length of 5500 base pairs encoding a portion of the Mru dnaK operon and consisting of the following elements: £ c / 136II 2,7k. a pore-size plasmid fragment of pUC19 containing a gene conferring ampicillin resistance and a replicon (ori region) of pMB1; Ecl \ 3611 2.8 thousand. a pore-size fragment encoding part of the Mru.grpE gene and the entire Mru. dnaJ gene. 9. In vitro protein active structure refolding system with loss of active protein, buffer, ionic salts, disulfide protein bridge reducing agent, adenosine triphosphate and chaperones, characterized in that it is an aqueous solution used for cloning by chaperones. /rM.DnaK, A / r «.DnaJ and A / rw.GrpE chaperones according to claims 1-4 and the amount of system components are determined from the following conditions: Protein whose structure requires 3-5 μΜ Buffer, optimally TRIS-HC1 pH 7.5 -8.5 Salts required to maintain an ionic strength of 0.38 - 0.42 M Agent for reducing disulfide protein bridges, 2-5 μΜ Thermophilic chaperone proteins: Afru.DnaK 3-5 μΜ Mrw.DnaJ 3-5 μΜ A / rt / .GrpE Adenosine triphosphate 0.3 - 0.5 μΜ, 9–11 mM. 10. In vitro method of restoring the active structure of proteins using chaperones in a refolding system, characterized in that the denatured protein is diluted with a buffer such as TRIS-HCl pH 7.5 + 0.1 containing ionic salts such as Na ₂ SO ₄ , KCI, Mg (CH ₃ COO) 2, dithiothreitol is added to reduce disulfide protein bridges to a concentration of 2-5 μΜ, followed by addition of thermophilic A / ru.DnaK, Λ / rn.DnaJ and Λ / rn.GrpE chaperones according to claims 1-4. the adenosine triphosphate to a concentration of 10 + 1 mM after incubation at 37 ° C, optimally in a molar ratio of 10: 10: 1, and maintained for at least 5 min.