JPH10262693A - Regeneration of protein by using modified molecular chaperone - Google Patents

Abstract

PROBLEM TO BE SOLVED: To effectively regenerate a protein used for a drug, etc., by using a modified chaperone readily prepared and capable of being repeatedly used and by reacting the modified molecular chaperone with the protein to be regenerated. SOLUTION: A DNA fragment coding for GroEL, which is a molecular chaperone derived from Escherichia coil, is amplified by using a primer by a PCR and prepared, and the molecular chaperone is produced by using the DNA fragment. An (His)6 tag is added to the C-terminal of the molecular chaperone to provide a modified molecular chaperone. The modified chaperone is brought into contact with a protein to be regenerated such as modified enolase in 4M guanidine hydrochloride to react the modified chaperone with the protein to be regenerated in the method for regenerating the protein. The protein such as an enzyme utilized in a wide use such as a drug is effectively regenerated by using the modified chaperone capable of being readily prepared and repeatedly used.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for regenerating a protein. More specifically, the present invention relates to a method for regenerating a recombinant protein expressed as a denatured protein or an inactive protein.

[0002]

2. Description of the Related Art At present, proteins such as enzymes are used in a wide range of applications including pharmaceuticals, but proteins produced in trace amounts in nature are generally expensive. Further, proteins are easily denatured and deactivated in the purification step or the reaction step due to physical or chemical factors such as heat or chemical substances. When a protein is produced using a gene recombination technique, a heterologous protein highly expressed in a bacterial cell in particular is often obtained as an insoluble and inactive protein called an inclusion body. Methods for solubilizing and activating these denatured or inactive proteins by various means have been attempted.

[0003] Recently, it has been revealed that a heat shock protein called a so-called molecular chaperone is constitutively expressed in cells (Hlodan et al., Edited by Pain, Mechanism O.).
f protein folding, Oxford University Press p194-22
8, (1994)). This molecular chaperone is responsible for the formation and maintenance of protein higher-order structures in cells,
It has been clarified that it is involved in the physiological functions common to many species including cell cycle regulation, individual development / differentiation, and the immune system (Hyundai Kagaku, August 1996, pp. 14-20, Protein Nucleic acid enzyme 41, 849-859 (1996), 860-867 (199
6)).

[0004] There are several families of heat shock proteins, the Hsp60 family being particularly called chaperonin. Among the chaperonins, E. coli GroE is the best studied to date. GroE is a 14-mer of GroEL (molecular weight 57,000) and a 7-mer of GroES (molecular weight 10,000).
Consisting of two types of subunits, the chaperonin GroE, under normal cellular conditions, acts on newly synthesized proteins, depending on ATP, to fold into its native structure. Also, between 42 ° C and 46 ° C,
GroE rises to about 10% of bacterial proteins, and the majority of proteins are bound to chaperonin (GroE) (Mar
tin et al., Science 258: 995-998 (1992)).

[0005] In vitro, GroE binds to heat-denatured proteins and prevents aggregation at high temperatures, after which the temperature decreases.
It has been shown to catalyze folding in the presence of ATP and GroES (Holl-Neugebauer et al., Biochemistry 30: 116).
09-11614 (1991), Martin et al., Nature 352: 36-42 (199
2)).

The enolase and DNase denatured with guanidine hydrochloride are regenerated using the molecular chaperone GroE or GroEL. GroE or Gr with this immobilized
The present inventors have developed what is done using oEL (Fukuda et al., The 61st Annual Meeting of the Chemical Society of Japan, Abstracts of Lectures)
(1996)). In this method, GroE can be reused by immobilizing GroE on formyl cellulofine, but a method for preventing a decrease in reproduction capability is required. Therefore, there is a demand for an efficient protein purification method that can reuse these molecular chaperones.

[0007]

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above problems, and it has been found that a molecular chaperone can be easily prepared and repeatedly used by modifying the molecular chaperone. The present invention has been completed.

[0008]

According to the present invention, there is provided a method for regenerating a protein, comprising the step of reacting a modified molecular chaperone with a protein to be regenerated.

[0009] In a preferred embodiment, the modified molecular chaperone is reused.

In a preferred embodiment, the modified molecular chaperone is a (His) ₆ -tagged molecular chaperone.

In a preferred embodiment, the (His) ₆
Tags are added to the C-terminus of the molecular chaperone.

In a preferred embodiment, the reaction is carried out in a state where the modified molecular chaperone is bound to a Ni ⁺ chelate resin.

[0013] In a preferred embodiment, the molecular chaperone is GroEL.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The molecular chaperones used in the present invention are GroEL, Hsp6 of the HSP60 (chaperonin) family.
0, Cpn60, DnaK of Hsp70 family, Hsp70, Bip, Hsp90
HtpG, Hsp90, Grop94 of the family, T of the TRiC family
F55, TRiC (TCP1), Hsp28, Hsp45 and the like. Preferably, the chaperonin family GroEL, GroE
S, GroE (consisting of GroEL 14-mer and GroES 7-mer)
Can be used.

The origin of these molecular chaperones does not matter. Furthermore, a thermostable molecular chaperone derived from a thermophilic bacterium, a hyperthermophilic primordial bacterium and the like can also be used.

E. coli GroE (GroEL and GroES) is produced by genetic recombination. For example, λ649 (NATIONAL INATITUTE OF GENETICS (S
HIZUOKA, JAPAN) is cut with EcoRI and HindIII, and a 8.5 kbp fragment is recovered. This fragment contains GroEL
And GroES genes are included. From this fragment, a fragment containing a gene encoding a target molecular chaperone is cut out and inserted into an expression vector. After culturing the host carrying the obtained vector under appropriate conditions, by recovering the molecular chaperone from the culture,
Molecular chaperones can be produced.

The modification of the molecular chaperone is performed by adding an amino acid sequence capable of affinity purification to the molecular chaperone. The site to be added may be at the N-terminus, C-terminus or inside as long as the regeneration activity of the molecular chaperone is maintained. Examples of added are affinity purified possible amino acid sequences, but such as glutathione S-transferase and (His) ₆ tag and the like, (His) ₆
Tags are particularly preferred. Small molecules that do not adversely affect the formation of higher-order structures of molecular chaperones are preferable (for example, GroEL
Forms a 14-mer). The (His) ₆ tag is an amino acid sequence consisting of six histidines and has a property of specifically binding to Ni ⁺ .

The addition of an affinity-purifiable amino acid sequence to a molecular chaperone is performed, for example, by ligating a DNA fragment encoding the molecular chaperone in frame to an expression vector in which the amino acid sequence to be added has already been cloned. Is done. As an example of an expression vector in which the (His) ₆ tag has been cloned, pET21a-d (+)
(Novagen), pQE-30 (Qiagen) and the like, which are commercially available. If modification of the DNA sequence is required to ligate in-frame, DNA encoding the molecular chaperone using methods well known in the art, such as site-directed mutagenesis, PCR or addition of adapters
Process the pieces.

The modified molecular chaperone is purified by affinity purification using a carrier to which a substance to which the amino acid sequence to be added specifically binds is bound. For example, a molecular chaperone to which a (His) ₆ tag is added is (His) ₆
Utilizing the specific binding between the tag and Ni ⁺ , for example, purification is performed using a Ni ⁺ chelating column.

The protein to be regenerated used in the present invention may be a protein that has undergone physical or chemical denaturation such as heat denaturation, denaturing agent treatment, or a chemical treatment, or an inactive body such as a recombinantly expressed inclusion body. A protein or the like can be used, and the origin of the protein does not matter. Preferable examples include glucose-6-phosphate dehydrogenase, lactate dehydrogenase, α-amylase, DNase, and enolase.

The molar ratio of the molecular chaperone used in the reaction to the protein to be regenerated is about 2 or more, preferably 3 or more. If the molar ratio is too small, the reaction does not proceed sufficiently.

The reaction conditions between the molecular chaperone and the protein to be regenerated depend on the protein to be regenerated, but in consideration of the pH stability, thermal stability, etc. of the protein, the reaction pH, temperature, etc. Conditions apply. The regeneration reaction is carried out by incubating a reaction solution containing the molecular chaperone and the protein to be regenerated, but it is also possible to carry out the reaction using a molecular chaperone previously bound to an affinity column. When chaperonin is used, the reaction can be promoted by adding ATP. Regeneration is performed in a regeneration buffer (eg, 10 mM MgC
l _2, 20 mM KCl and 50mM acetate pH5.0 containing 2mM dithiothreitol, phosphoric acid pH 6.0-7.0, or 50 mM Tris-HCl pH 7.
The protein to be regenerated is added to 8-10), ATP is added to a final concentration of 2 mM, and GroE21 mer or GroEL14 mer is added at a predetermined molar ratio.

The completion of the reaction can be determined by measuring the enzyme activity and the like of the regenerated protein. After the reaction,
The regenerated protein can be recovered and purified by a general protein separation and purification method well known to those skilled in the art. Alternatively, the molecular chaperone can be removed from the reaction solution by the affinity purification, and the regenerated protein can be recovered from the remaining reaction solution. The affinity-purified molecular chaperone can be used repeatedly. If the regeneration reaction is performed using a molecular chaperone pre-bound to the affinity column, the regenerated protein is recovered as eluate from the column.

[0024]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

(Example 1) Addition of His ₆ tag to GroEL D coding for GroEL which is a molecular chaperone derived from Escherichia coli
NA fragments were prepared by PCR. PCR was performed using phage DNA cloned from a DNA fragment encoding GroEL (λ649 (NATIONAL
Using INATITUTE OFGENETICS (SHIZUOKA, JAPAN)) as a template, a sense primer (5'-GGGGAATTCCCATGGGGATGGCA-3 ') containing a nucleotide sequence encoding the N-terminus of GroEL and an EcoRI site, GroEL
An antisense primer containing a nucleotide sequence complementary to the sequence encoding the C-terminus of HindII and a HindII site (5′-T
TAGGATCCTTAAAGCTTCATCATGCCGCCCATGCC-3 ') and Taq
Using polymerase enzyme (Cetus, USA), 94
℃ 1 minute, 98 ℃ 20 seconds and 68 ℃ 3 minutes under the condition of 30 cycles
PCR was performed. Obtained product and pCR II vector (Invit
rogen) at an insert to vector ratio of 2.5: 1 (weight ratio) and ligated overnight at 16 ° C. The ligation mixture was then used to transform Escherichia coli DH5α competent cells, and kanamycin and ampicillin resistant colonies were selected. Next, plasmid DNA was extracted from some of the resulting colonies, clones that produced a fragment of the expected length (108 bp) by restriction digestion were selected, and the nucleotide sequence of the insert was changed to Thermo Sequenase.
fluorescent labelled primer cycle sequencing kit
(RPN2436) and a fluorescent DNA sequencer (DQS-1000, manufactured by Shimadzu Corporation) to determine a clone having a correct sequence.

Next, the PCR amplified fragment was converted to an expression vector pET-21a-d (+) (No.
vagen). The insert was excised from the resulting clone by digestion with EcoRI and HindIII and ligated to pET-21a-d (+) digested with the same restriction enzymes as described above. Using the resulting ligation mixture, Escherichi
E. coli HMS174 competent cells were transformed and ampicillin-resistant colonies were selected. A clone expected from the obtained colonies was selected in the same manner as above, and His
P encoding GroEL with tag added (GroEL- (His) ₆ )
TM6 was obtained.

(Example 2) Preparation of GroEL- (His) ₆ Escherichia coli H containing pTM6 obtained in Example 1
The MS174 strain was transformed into an LB medium (yeast extract 5 g / l, tryptone 10 g / l, NaCl 5 g / l) containing ampicillin (100 μg / ml).
And cultured with shaking at 37 ° C. overnight. The culture was then diluted 10-fold with the same medium, and diluted until the OD ₆₀₀ was 0.6-1.0.
The cells were cultured with shaking at 7 ° C. At this point, isopropyl β-D-
Thiogalactopyranoside was added to a final concentration of 1 mM and shaken for an additional 4 hours. In FIG. 1, lane 1 is the result of SDS-PAGE analysis of the extract extracted from the cells before the addition of IPTG, and lane 2 is the result of the IPTG addition.
Was found to induce the production of GroEL- (His) ₆ . Also, as seen in lane 3, GroEL- (Hi
s) ₆ was also included in the insoluble fraction.

Cells were collected from the resulting culture by centrifugation, and then sonicated to obtain an extract. 40
A chelate cellofine column (manufactured by Chisso Corporation) was charged with Ni ⁺ ions using 0 mM NiSO ₄ . Binding extract
Buffer solution (40 mM imidazole, 4 M NaCl, 100 mM Tris-HCl
(pH 7.8)).
Then, elution was performed with 4 M imidazole to obtain a fraction containing GroEL- (His) ₆ . SDS-PAGE confirmed that this fraction showed a single band with the expected molecular weight (60 kd) (FIG. 1, lane 4).

Example 3 GroEL- (H) obtained in Example 2
is) ₆ examined the ability to regenerate denatured proteins.
Enolase or DN denatured in 4M guanidine hydrochloride
The ase was added to a final concentration of 0.17 μM in a regeneration buffer (50 mM Tris-HCl pH 7.8 containing 10 mM MgCl ₂ , 20 mM KCl and 2 mM dithiothreitol) to give GroEL- (His) ₆ or a control. Reactions were performed at various temperatures for 30 minutes with or without the addition of GroEL at equimolar concentrations. After completion of the reaction, each enzyme activity was measured.

The activity of DNase I was determined by adding 10 μl of the enzyme solution to 40 μl.
l of assay buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3
mM MgCl ₂ ) and 50 μl of bovine thymus DNA solution (2.5 mg / ml assay buffer) were mixed and incubated at 37 ° C. for 10 minutes. Stop the reaction by adding 25 μl of cold 1 M perchloric acid, 12,000 r
After centrifugation at pm for 10 minutes and dilution with water by 20 times, the absorbance at 260 nm was measured.

The enzyme activity of the enolase was determined by adding 40 μl of the enzyme solution to 0.96 ml of the substrate solution (50 mM Tris-HCl, pH 7.8, 1 mM MgC
l ₂ , 1mM 2-PGA (phosphoglyceric acid)
The increase in absorbance at was measured as a function of time.

The results for enolase are shown in FIG.
The result for e is shown in FIG. In the figure, the ordinate indicates the relative activity when the native activity of each enzyme is defined as 100%, and the abscissa indicates the reaction. In the figure, (●) indicates regeneration in the absence of chaperonin, (△) indicates regeneration in the presence of GroEL, and (□) indicates regeneration in the presence of GroEL- (His) ₆ . As is clear from these results, it was shown that all of the denatured proteins were equally remarkably increased in activity using GroEL and GroEL- (His) ₆ and were regenerated. This indicates that the His ₆ tag does not affect the GroEL regeneration activity.

[0033]

According to the protein regeneration system using the modified molecular chaperone that can be affinity-purified, the molecular chaperone can be easily recovered and reused many times.

[Brief description of the drawings]

FIG. 1 is an electrophoretic photograph showing expression of GroEL- (His) ₆ in E. coli and affinity purification of the expression product.

FIG. 2. GroEL of enolase at various temperatures.
It is a graph which shows reproduction | regeneration in the presence or absence (●) of (△) or GroEL- (His) ₆ (□).

[FIG. 3] DNase at various temperatures in the presence or absence of GroEL (G) or GroEL- (His) ₆ (□) (●)
6 is a graph showing the reproduction in.

Claims (6)

[Claims] 1. A method for regenerating a protein, comprising reacting a modified molecular chaperone with a protein to be regenerated. 2. The method of claim 1, wherein said modified molecular chaperone is reused. 3. The method according to claim 1, wherein the modified molecular chaperone is a (His) ₆ -tagged molecular chaperone. 4. The method of claim 3, wherein said (His) ₆ tag is added to the C-terminus of a molecular chaperone. 5. The method according to claim 3, wherein the reaction is performed in a state where the modified molecular chaperone is bound to a Ni ⁺ chelate resin. 6. The method according to claim 1, wherein the molecular chaperone is GroEL.