JP2019081728A - GroEL-containing liquid, method for producing the same and method for using the same - Google Patents

Abstract

To provide means for controlling GroEL's collective and discrete states under simple and mild conditions and the shapes of collective states.SOLUTION: By contacting unpolymerized GroEL with ATP in an ATP-containing solution containing ATP at a concentration of 5 mM or more, a tube-like complex formed by polymerization of the plurality of GroELs is formed. Thereby, a GroEL-containing liquid containing a tube-like complex formed by polymerization of the plurality of GroELs, and ATP at a concentration of 5 mM or more can be obtained.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a GroEL-containing liquid, a method of producing the GroEL-containing liquid, and a method of using the GroEL-containing liquid.

  Chaperonin is one of the so-called molecular chaperones which aids in the correct folding of substrate proteins. The chaperonin family is a protein with a molecular weight of 50 kDa to 60 kDa, and has a common feature of taking a ring-like complex structure and assisting the folding of a substrate protein in an ATP-dependent manner. The chaperonin GroEL of E. coli has been shown to support folding of substrate proteins in an ATP and GroES-dependent manner.

  The wild-type chaperonin GroEL of E. coli consists of a heptamer of GroEL subunit which constitutes one ring, and this ring has a 14-mer structure in total in a state in which two rings overlap each other back to back. That is, wild-type chaperonin GroEL is surrounded by seven GroEL subunits, and has two cavities capable of containing a substrate protein. In addition, one GroEL subunit is composed of an equatorial domain including an ATP binding site, an apex domain including a binding site of a substrate protein and GroES, and an intermediate domain connecting the two domains.

  From these facts, GroEL, and further, a complex of GroEL and GroES is attracting attention as a drug delivery system (DDS) carrier candidate to replace conventionally proposed liposomes.

JP 2017-38610 A International Publication WO2016 / 185955

  There are many problems to use GroEL practically as DDS carrier, and one of them is to provide technology to control the form, control the release of aggregates, discretes and inclusions of GroELs, and aggregate GroEL. It can be mentioned. Here, in consideration of practical use as a DDS carrier, a technique involving a process with a high load on a living body at the time of morphology control of GroEL and a technique involving complicated chemical synthesis are not preferable.

  In consideration of the above-mentioned circumstances, the present invention aims to provide a means for controlling the shapes of GroEL assemblies and discrete states and aggregation states under simple and mild conditions.

As a result of intensive studies by the present inventors, the present invention has been completed.
The GroEL-containing solution of the present invention comprises a tube-like complex formed by polymerization of a plurality of GroELs, and ATP at a concentration of 5 mM or more, preferably 5 to 10 mM. GroEL may be a wild type of E. coli.
As a method for producing such a GroEL-containing liquid, a plurality of the GroELs are polymerized by bringing the unpolymerized GroEL into contact with the ATP in an ATP-containing liquid containing ATP at a concentration of 5 mM or more. A manufacturing method is provided, characterized in that a tube-like complex is formed.
According to the present invention, the concentration of GroES is set to GroEL by (I) lowering the ATP concentration of the above-mentioned GroEL-containing solution to 4 mM or less, or (II) mixing the above-mentioned GroEL-containing solution with GroES. By making it equal to or higher than the concentration, it is possible to depolymerize the above-mentioned tube-like complex into individual non-polymerized GroEL.

  According to the present invention, by adjusting the concentration of ATP in the GroEL-containing solution, a plurality of GroELs can be arranged and polymerized in the form of a tube, and it can be depolymerized. This can be interpreted as providing a means of morphological control, such as integrating GroEL in one-dimensional extension and dispersing it. The GroEL-containing solution of the present invention can be interpreted as a state in which the tube-like GroEL is stored, and the ability to store such a tube-like GroEL results in the local accumulation of drug capsules in the field of DDS. There may be advantages such as control of uptake concentration by tube length, and placement around cells as drug nanotapes that also have multiple types of drugs. Generally, since the concentration of ATP in vivo is less than 4 mM, for example, the GroEL-containing solution of the present invention containing a tube-like complex is used as a DDS carrier, and GroEL as a tube-like complex in vitro In handling and in vivo, applications such as using GroEL discrete from the complex are considered. At that time, if the inclusion is put in GroEL, it is usually considered that the inclusion is released by the discreteness of GroEL.

  Furthermore, according to the present invention, by adding GroES to the GroEL-containing solution under predetermined conditions, it is possible to control the tube-like complex to be in a discrete state, and as in the case of control by the above-mentioned ATP concentration. It can be used as one means of form control of GroEL as a DDS carrier.

It is a TEM image and DLS measurement result of GroEL ^WT . It is a TEM image and DLS measurement result of GroEL ^D52,398A . It is a TEM image and DLS measurement result of GroEL ^WT , GroEL ^D398A , GroEL ^D52 , ^398A . It is a TEM image and DLS measurement result of GroEL ^WT , GroEL ^D52 , ^398A . It is a TEM image and DLS measurement result of GroEL ^WT . It is a TEM image and DLS measurement result of GroEL ^WT . It is a TEM image and DLS measurement result of GroEL ^WT and GroEL ^D52,398A . It is a TEM image and DLS measurement result of GroEL ^WT . It is a TEM image and DLS measurement result of GroEL ^D52,398A . It is a TEM image and DLS measurement result of GroEL ^WT . It is a TEM image and DLS measurement result of GroEL ^D52,398A . It is a TEM image of GroEL ^WT . It is a TEM image of GroEL ^WT . It is a TEM image of GroEL ^WT .

Hereinafter, the present invention will be described in detail.
The inclusion solution of the present invention includes a tube-shaped complex. The tube-like complex is formed by polymerization of a plurality of GroELs. GroEL forms a 7-mer ring structure, and when the ring structures are connected back to back, it becomes a 14-mer double ring structure. The double ring structure is a three-dimensional structure having a cavity of about 4 to 8 nm in diameter (about 5 nm in E. coli wild type). In the present invention, the above 7-mer and 14-mer are referred to as "non-polymerized GroEL".

  In the present invention, GroEL may be wild type or mutant. Wild-type GroEL is derived from E. coli, and the hydrolysis time of ATP is as short as about 8 seconds. Therefore, it is not always preferable to adopt wild-type GroEL as it is as a carrier material for a drug delivery system.

  In the present invention, GroEL may be mutant. As a variant type, there is a variant protein in which the ATP hydrolysis activity (including an ATP alternative compound) is reduced relative to wild type GroEL. There is no particular limitation on such mutant GroEL, and preferred examples include those in which all of the GroEL heptamers constituting the ring structure exhibit the ATP hydrolysis activity-reduced type. The wild type or mutant GroEL is preferably one that can bind to ATP.

GroEL ^D398A can be mentioned as a specific example of the mutant GroEL having a reduced ATP hydrolysis activity relative to wild type GroEL (hereinafter also referred to as GroEL ^WT ). Specifically, GroEL ^D398A refers to a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing. The GroEL ^D398A is a GroEL mutant in which aspartate (D) at position 398 in the amino acid sequence of wild-type GroEL is substituted with alanine (A). While the reaction cycle time including ATP hydrolysis in wild-type GroEL is about 8 seconds, this variant gives a dissociation half-life of 30 to 60 minutes in the chaperonin complex (Rye et al., Cell, Vol. 97 , 1999).

  As the mutant GroEL, it consists of an amino acid sequence in which one or more amino acids other than alanine at position 398 in the amino acid sequence shown in SEQ ID NO: 1 is substituted, deleted and / or added, and chaperonin activity is The one which it has can be used similarly. GroEL-like proteins derived from bacteria other than E. coli and variants thereof that satisfy the conditions are also included here.

  Here, as a range relating to mutations other than alanine other than the 398th in the amino acid sequence of SEQ ID NO: 1, suitably 70% or more, preferably 70% or more in sequence homology to the amino acid sequence described in SEQ ID NO: 1 Is preferably one having a region showing sequence homology of 80% or more, more preferably 90% or more, still more preferably 95% or more. The number of amino acid substitution, deletion or addition mutation sites at positions other than the 398th alanine is preferably 100 or less, more preferably 50 or less, still more preferably 25 or less, particularly preferably Is preferably 10 or less.

GroEL ^{D52, 398A} is preferably mentioned as another specific example of the mutant GroEL. GroEL ^D52 , ^398A refers to a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2. The GroEL ^D52 , ^398A is a GroEL ^mutant in which the 52nd aspartic acid (D) is substituted with alanine (A) in GroEL ^D398A , and has a property in which the hydrolysis activity of ATP is significantly reduced.

In GroEL ^D52 , ^398A , the dissociation half life in the chaperonin complex shows an extremely long time of about 6 days. This is a dramatically high value of about 150 to 300 times that of GroEL ^D398A (Japanese Patent No. 5540367, Koike-Takeshita et al., J. Biol. Chem. 2014). Here, the finding that the mutation substitution for 52nd alanine in GroEL synergistically reduces the ATP hydrolysis activity is a finding that the present inventors have found for the first time (International Publication WO 2016/185955).

  As the mutant GroEL, an amino acid in which one or more amino acids other than alanine at position 52 and 398 in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing is substituted, deleted, and / or added. A sequence consisting of a sequence and having chaperonin activity can be used as well. GroEL-like proteins derived from bacteria other than E. coli and variants thereof that satisfy the conditions are also included here.

  Here, as a range related to mutations other than alanine other than the 52nd and 398th amino acids in the amino acid sequence of SEQ ID NO: 2, the sequence homology to the amino acid sequence described in SEQ ID NO: 2 is preferably 70. It is preferable to have a region showing a sequence homology of% or more, preferably 80% or more, more preferably 90% or more, more preferably 95% or more. The number of amino acid substitution, deletion or addition mutation sites at positions other than 52nd and 398th alanine is preferably 100 or less, more preferably 50 or less, and still more preferably 25 or less. Particularly preferably 10 or less is preferable.

  The method for preparing mutant GroEL is not particularly limited, and for example, it can be prepared by introducing a mutation into wild-type GroEL. As a method for introducing a mutation, a commonly used method can be used without particular limitation. For example, genetic engineering techniques such as a method using PCR and a site-directed mutagenesis kit (for example, Stratagene etc.) can be used. In addition, as mutations to be introduced, mutations that have less influence on chaperonin activity or ATP hydrolysis activity, neutral neutral mutations, mutations that add additional functions, and the like may be allowed and included.

  A plurality of non-polymerized GroEL as described above is polymerized to form a tube-like complex. At this time, polymerization is performed such that the cavities of GroEL are connected in series. FIG. 2 shows TEM images and DLS measurement results in the examples described later. The tubular complex is observed in the TEM image of FIG. The tubular complexes are particularly clearly depicted in the 4 minutes, 10 minutes and 30 minutes TEM images. As described above, a tube-shaped complex is one in which approximately 5 to 10 spherical bodies are linearly connected.

  Such a tube-like complex can be evaluated as one in which GroEL is integrated so as to extend in one dimension. The number of GroEL 14-mers polymerized in one tube is not particularly limited as long as it is 2 or more.

  It is necessary to obtain a tube-like complex by, for example, local accumulation of drug capsules, control of uptake concentration by tube length, optical tweezers (traps of fine particles of around 0.2 to 20 μm), etc. in DDS technology. It has the significance of being able to handle one tube and place it around cells as a drug nanotape having a combination of multiple drugs.

  According to the new findings of the present inventors, by controlling the concentration of ATP in the GroEL-containing solution, GroEL is polymerized to obtain a tube-like complex, or depolymerized from the complex to obtain GroEL. It can be polymerized. If the substance is contained in the cavity of GroEL, the inclusion is usually diffused / released by depolymerization.

In the present invention, a liquid containing GroEL is referred to as a GroEL-containing liquid. GroEL may not be polymerized in the GroEL-containing solution, or may be polymerized to form a tube-shaped complex. The liquid can be a solution, a suspension, or any other liquid like liquid without particular limitation. As a medium in the liquid, a buffer used in biochemistry can be used without particular limitation. Non-limiting examples of such buffers include N- (2-hydroxyethyl) piperazine-N'-2-ethanesulfonic acid (HEPES) and the like, including KCl, MgCl _{2 and the} like.

According to the present invention, a non-polymerized GroEL is brought into contact with ATP to form a tube-shaped complex in which the GroELs are polymerized if the concentration of ATP is 5 mM or more. Can. As demonstrated in the following examples, in the case of wild-type GroEL ^WT , a tube-like complex is most difficult to form, and as described above, tube-like in mutant GroEL in which ATP hydrolysis activity is reduced. Tends to form a complex of And even in the case of wild-type GroEL ^WT, a tube-like complex is formed if the concentration of ATP reaches 5 mM, and therefore, any GroEL liquid containing ATP at a concentration of 5 mM or more It was found that a tube-like complex was formed. There is no particular limitation on the upper limit of the concentration of ATP, and in consideration of actual ease of handling, for example, 10 mM and the like are preferably mentioned.

  Although the mechanism of the interaction with ATP when GroELs polymerize to form a tube-like complex is not strictly known, it is presumed that the mechanism is as follows. It is known that binding of ATP to GroEL causes a structural change, exposing H and I helices composed of hydrophobic amino acid residues, which are sites to which denatured polypeptides and GroES bind (Protein Science, Yugo Goto, Kunihiro Kabashima, Katsuyuki Tanizawa, ed. It is considered that GroELs in this state are linked to each other by hydrophobic interaction.

  There is no particular limitation on the method of contacting unpolymerized GroEL with ATP. The ATP or ATP-containing solution may be added to the GroEL-containing solution to adjust the concentration of ATP to 5 mM or more, or the ATP-containing solution may be adjusted to 5 mM or more. You may add it.

  The reaction temperature at the time of polymerizing GroELs to obtain a tube-like complex may be about room temperature, for example, a temperature range of 5 to 60 ° C. can be mentioned. The reaction time is about 30 seconds to 30 minutes, and after that, if the concentration of ATP is maintained at 5 mM or more, the GroEL-containing solution containing the tube-like complex is stably maintained.

  According to the present invention, it is possible to form a tube-like complex without particularly modifying GroEL. The formation of such a tube-like complex formed by polymerization of GroELs not specially modified with each other has not been conventionally obtained, and the GroEL-containing solution containing such complex is also disclosed. It is one form of the invention. As described above, the liquid containing the tube-like complex is excellent in storage and transportation, and easily administered to a living body.

  According to further new findings of the present inventors, with regard to a tube-shaped complex once formed in the GroEL-containing solution, the tube-shaped complex can be obtained by reducing the ATP concentration of the solution to less than 4 mM. Depolymerize into individual non-polymerized GroEL. Thus, the formation and dissociation of the tube-like complex can be controlled by raising or lowering the ATP concentration in the contained solution.

  As demonstrated in the following examples, GroEL, whether wild type or mutant, causes depolymerization if the ATP concentration is less than 4 mM. Taking into consideration the influence of ATP in the above-mentioned GroEL polymerization, it is also possible to remove ATP if the ATP concentration is less than 4 mM, regardless of whether it is a wild type with high ATP hydrolysis activity or a mutant with low activity. As polymerization occurred, it was found that the tube-like complex was depolymerized if the ATP concentration was less than 4 mM for any GroEL. The lower limit value of the ATP concentration for the above-mentioned depolymerization is not particularly limited, and may be zero.

  In humans, the concentration of ATP is usually about 3 mM, that is, less than 4 mM. Therefore, the in vivo administration of the GroEL-containing solution of the present invention containing a tube-like complex depolymerizes into individual GroELs in vivo. In this way, it is possible to store the tube-shaped complex while forming in vitro and convert it into individual GroEL by administering it in vivo, as described in the field of DDS. In addition, control of uptake concentration by tube length, handling of each tube with optical tweezers (trap of fine particles of around 0.2 to 20 μm), etc. is possible, and it is 1 as drug nanotape which also has multiple types of drugs. One advantage is that it can be placed around one cell to enable efficient administration of selective drugs to selective cells. In addition, the tube-like complex can be conveniently separated according to the length by centrifugation, and accumulation before administration is easy. To be able to enjoy these advantages can be interpreted as the meaning of the GroEL-containing liquid of the present invention itself including a tube-like complex.

  According to the invention, for tube-shaped complexes once formed in the GroEL-containing liquid, depolymerizing the tube-like complexes into individual non-polymerized GroELs also by mixing with GroES Can. In this case, it is finally necessary to make the concentrations of GroES and GroEL the same or to make GroES have a higher concentration. Here, the concentrations of GroEL and GroES are molar concentrations.

  The main body of GroES (region excluding intracellular organelle localization peptide) is a subunit capable of binding to GroEL and capable of forming the top of chaperonin complex, and the complex has molecular chaperone activity. It is an area that functions as a cofactor to exert. Such subunits usually constitute one molecule of 7-mers and function as a cofactor for the GroEL ring structure.

  As the GroES main body, any protein can be used as long as it is a GroES-like protein that exerts the above-mentioned function. For example, GroES derived from E. coli, GroES homologous proteins derived from bacteria other than E. coli, proteins having a three-dimensional structure and similar functions of GroES derived from phage, and further, mutant proteins derived from these proteins can be suitably used. .

  An example of GroES is a wild-type GroES consisting of the amino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing. In addition, a protein comprising the amino acid sequence set forth in SEQ ID NO: 3 and having the above-mentioned function in formation of a chaperonin complex can also be treated as GroES.

  In addition, the amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence described in SEQ ID NO: 3 comprises the amino acid sequence, and the above-mentioned function in formation of chaperonin complex The proteins possessed can also be treated as GroES, and include GroES-like proteins derived from bacteria other than E. coli, or variants thereof, which satisfy the conditions. Here, as a range relating to the mutation of the amino acid sequence of SEQ ID NO: 3, preferably 70% or more, preferably 80% or more, more preferably 90% or more in sequence homology to the amino acid sequence of SEQ ID NO: 3. It is preferable to have a region showing% or more, more preferably 95% or more of sequence homology. Further, the number of mutation sites in which the amino acid of SEQ ID NO: 3 has been substituted, deleted or added is preferably 20 or less, more preferably 10 or less, still more preferably 5 or less. Moreover, it comprises an amino acid sequence having a mutation with respect to SEQ ID NO: 3, and one having the above function in formation of a chaperonin complex can also be used as GroES.

  Hereinafter, the present invention will be more specifically described by way of examples. However, the present invention is not limited to the embodiments described in these examples.

Examples 1-3:
In this example, 5 μM GroEL was treated in the presence of 5 mM ATP.
After adding ATP to a final concentration of 5 mM in HKM buffer (100 mM KCl, 20 mM HEPES / KOH containing 5 mM MgCl ₂ , pH 7.4), wild-type GroEL (GroEL ^WT ) in Example 1 In Example 2, the GroEL mutant (GroEL ^D398A ) was ^mixed , and in Example 3, the GroEL mutant (GroEL ^{D52, 398A} ) was mixed to a final concentration of 5 μM and allowed to stand at room temperature. The time when the ATP and GroEL were in contact was taken as the reaction start (t = 0), and after 30 seconds from the start of the reaction, it was subjected to transmission electron microscope (TEM) observation and further subjected to dynamic light scattering (DLS) measurement.
In ^addition, the amino acid sequence of amino acid sequence and ^{GroEL D52,398A} of ^{GroEL D398A,} respectively, SEQ ID NO: 1 and SEQ ID NO: 2.

Comparative Examples 1 and 2:
In this example, 5 μM GroEL was treated in the presence of 5 mM ADP.
After adding ADP to a final concentration of 5 mM in HKM buffer (100 mM KCl, 20 mM HEPES / KOH containing 5 mM MgCl ₂ , pH 7.4), in Comparative Example 1, wild-type GroEL (GroEL ^WT ) In Comparative Example 2, GroEL mutants (GroEL ^{D52, 398A} ) were mixed to a final concentration of 5 μM, respectively, and allowed to stand at room temperature. The time when ADP and GroEL were in contact was taken as the reaction start (t = 0), and it was subjected to DLS measurement and TEM observation 4 minutes after the reaction start.

Examples 4 and 5:
In this example, 0.1 μM GroEL was treated in the presence of 5 mM ATP.
After adding ATP to a final concentration of 5 mM in HKM buffer (100 mM KCl, 20 mM HEPES / KOH containing 5 mM MgCl ₂ , pH 7.4), wild-type GroEL (GroEL ^WT ) in Example 4 In Example 5, GroEL mutants (GroEL ^{D52, 398A} ) were mixed to a final concentration of 0.1 μM and allowed to stand at room temperature. The time when the ATP and GroEL were in contact was taken as the reaction start (t = 0), and it was subjected to DLS measurement and TEM observation 4 minutes after the reaction start.

Comparative Examples 3 and 4:
In this example, 5 μM GroEL was treated in the presence of 4 mM ATP.
After adding ATP to a final concentration of 4 mM in HKM buffer (20 mM HEPES / KOH containing 100 mM KCl, 5 mM MgCl ₂ , pH 7.4), in Comparative Example 3, wild-type GroEL (GroEL ^WT ) In Comparative Example 4, GroEL mutants (GroEL ^{D52, 398A} ) were mixed to a final concentration of 5 μM and allowed to stand at room temperature. The time when the ATP and GroEL were in contact was taken as the reaction start (t = 0), and it was subjected to DLS measurement and TEM observation 4 minutes after the reaction start.

Examples 6, 7
In this example, the same processing and observation as in Comparative Examples 3 and 4 were performed except that the concentration of ATP was changed from 4 mM to 6 mM. In Example 6, wild-type GroEL (GroEL ^WT ) was used, and in Example 7, the GroEL mutant (GroEL ^{D52, 398A} ) was used.

Comparative example 5, 6:
In this example, 5 μM GroEL was treated in the presence of 3 mM ATP and 0.3 mM ADP (physiological concentration).
After adding ATP to a final concentration of 3 mM and ADP to a final concentration of 0.3 mM in HKM buffer (100 mM KCl, 20 mM HEPES / KOH containing 5 mM MgCl ₂ , pH 7.4), Comparative Example 5 Then, wild type GroEL (GroEL ^WT ) was mixed, and in Comparative Example 6, GroEL mutant (GroEL ^{D52, 398A} ) was mixed so as to have a final concentration of 5 μM and allowed to stand at room temperature. The time when the ATP and GroEL were in contact was taken as the reaction start (t = 0), and it was subjected to DLS measurement and TEM observation 4 minutes after the reaction start.

Examples 8 and 9:
In this example, 5 μM GroEL (tube state) formed in the presence of 5 mM ATP was transferred to 3 mM ATP and 0.3 mM ADP (physiological concentration) conditions.
In order to reduce the concentration of ATP in the sample, add ADP to a final concentration of 0.75 mM in HKM buffer (concentration 100 mM KCl, concentration 5 mM MgCl ₂ containing 20 mM HEPES / KOH, pH 7.4) After addition to the above, a sample solution A was prepared by mixing wild-type GroEL (GroEL ^WT ) in Example 8 and GroEL mutant (GroEL ^{D52, 398A} ) in Example 9 to a final concentration of 5 μM.

After adding ATP to a final concentration of 5 mM in HKM buffer (concentration 100 mM KCl, concentration 20 mM HEPES / KOH containing 5 mM MgCl ₂ , pH 7.4), wild-type GroEL (Example 8) Sample solution B was prepared by mixing GroEL ^WT ) and GroEL mutant (GroEL ^{D52, 398A} ) in Example 9 at final concentration of 5 μM and ^leaving them to stand at room temperature.

After preparing the sample solution B, immediately mix the two so that the volume ratio of sample solution A: sample solution B = 2: 3, and ATP: ADP = 10: 1, final concentration 3 mM ATP, final concentration 0 A sample solution C was prepared which had a final concentration of 5 μM of GroEL ^WT (Example 8) or GroEL ^{D52, 398A} (Example 9) in the presence of 3 mM ADP. When the sample solution C was prepared, the reaction was initiated (t = 0), and the sample solution C was subjected to TEM observation from 30 seconds after the initiation of the reaction, and was provided for DLS measurement from 1 minute after the initiation of the reaction.

Examples 10 and 11:
In this example, GroES was added to the GroEL tube formed in the presence of 5 mM ATP.
In Example 10, GroEL ^WT was used. After adding ATP to a final concentration of 5 mM in HKM buffer solution (concentration 100 mM KCl, concentration 20 mM HEPES / KOH containing concentration 5 mM MgCl ₂ , pH 7.4), GroEL ^WT becomes final concentration 5 μM After mixing to form a tube, GroES was added to a final concentration of 10 μM. The time point after GroES addition was regarded as the reaction start (t = 0), and was used for TEM observation from 30 seconds after the reaction start, and was used for DLS measurement from 1 minute after the reaction start. The amino acid sequence of GroES used here is as SEQ ID NO: 3.

In Example 11, GroEL ^D52 , ^398A was used. After adding ATP to a final concentration of 5 mM in HKM buffer solution (concentration 100 mM KCl, concentration 20 mM HEPES / KOH containing concentration 5 mM MgCl ₂ , pH 7.4), GroEL ^D52 , ^398A at final concentration 5 μM The mixture was mixed to form a tube. After 4 minutes at room temperature, GroES was added to a final concentration of 10 μM. The time point after GroES addition was regarded as the reaction start (t = 0), and was used for TEM observation from 30 seconds after the reaction start, and was used for DLS measurement from 1 minute after the reaction start.

  The GroEL-containing solution in Example 1 and the GroEL-containing solution obtained in the same manner as in Example 1 except that ATP was not present were subjected to a centrifugation operation, and then subjected to TEM observation.

  The various samples prepared as described above were subjected to the following measurement.

Size characterization of GroEL:
Each sample after 1 minute, 2 minutes, 4 minutes, 6 minutes, 10 minutes, 30 minutes and 60 minutes of sample preparation was used as Zetasizer Nano ZSP (Malvern) with ZEN 1010 cell as a measurement container. It was measured by dynamic light scattering intensity (DLS). GroEL size distribution was drawn by Zetasizer Software Ver 7.11 attached to the device.

TEM observation:
30 seconds after sample preparation, 2 minutes after, 4 minutes after, 6 minutes after, 6 minutes after, 6 minutes after, 10 minutes after, 30 minutes after, 60 minutes after the sample preparation with GroM in the sample with HKM buffer containing 5 mM ATP The GroEL mutant was diluted to 0.1 μM and immediately used for TEM observation.
Preparation of the TEM grid was performed as follows. Glow discharge was performed using an ion coater IB-2 (Eiko Engineering) under conditions of 3 mA for 30 seconds to hydrophilize a collodion-supported 200 mesh copper grid (EM Japan). Within 1 hour after the grid hydrophilization treatment, 2 μL of the diluted sample solution is placed on the grid for 20 seconds, suctioned with filter paper, 6 μL of ultrapure water, immediately suctioned with filter paper, and then 0.5% phosphotungstic acid (pH 4.). 0) 6 μL was loaded, and after 20 seconds, it was blotted with filter paper. The prepared sample grid was dried overnight with a dry keeper (Samplertech).

  Image acquisition application Digital Micrograph Ver. A sample grid was observed and photographed at an acceleration voltage of 100 kV using a transmission electron microscope JEM2100 (Nippon Electron) equipped with a cooled CCD camera ORIUS SC200 (Gatan) to which 2.111404.0 was attached.

Results and discussion:
GroEL tube formation and maintenance time:

About Examples 1-3:
5 μM GroEL ^{WT in the} presence of a final concentration of 5 mM ATP (Example 1), according to DLS measurement, an average sample size peaking at about 39 nm from 1 minute to 10 minutes after the reaction start, 30 minutes after the reaction start Shows an average sample size with a peak of about 34 nm, and an average sample size with a peak of about 27 nm even at 60 minutes after the start of the reaction, which is larger than the average sample size of about 20 nm shown in the absence of ATP. Indicated. As a result of TEM observation, it was confirmed that the GroEL ^WT tube exists at least for 4 minutes after the start of the reaction. FIG. 1 shows TEM images and DLS measurement results.

In 5 μM GroEL ^{D 52} , ^398A in the presence of 5 mM ATP (Example 3), DLS measurement shows an average sample size peaking at about 51 nm from 1 minute to 60 minutes after sample preparation, in the absence of ATP It was shown to be more than twice as large as the average sample size of about 20 nm shown. As a result of TEM observation, five or less GroEL ^{D 52, 398A} were connected in the direction of the long side between 30 seconds to 2 minutes after sample preparation, indicating an early stage of tube formation. After 4 minutes of sample preparation, it was shown that a tube state in which five or more GroEL ^{D 52, 398A} were connected was formed and maintained. FIG. 2 shows TEM images and DLS measurement results.

In comparison of 4 minutes after starting the reaction of 5 μM GroEL ^WT , GroEL ^D398A and GroEL ^{D 52} , ^{398A in} the presence of final concentration of 5 mM ATP (Examples 1 to 3), the average sample size of each sample is GroEL by DLS measurement ^WT at about ^33nm, about ^49nm in ^{GroEL D398A,} was 54nm in ^{GroEL D52,398A.} As a result of TEM observation, it is found that GroEL ^WT and GroEL ^D398A form about 4 to 5 GroEL tubes, and GroEL ^D52 and ^398A include GroEL tubes having about 10 connected lengths, and the ATP hydrolysis activity of GroEL It was shown that the number of connected GroELs increases as the FIG. 3 shows TEM images and DLS measurement results of GroEL ^WT , GroEL ^D398A and GroEL ^D52 , ^398A tubes.

About Examples 4 and 5:
0.1 μM GroEL ^{WT in the} presence of a final concentration of 5 mM ATP (Example 4) is smaller than the average sample size of 33 nm in the presence of 5 mM ATP by DLS measurement, but it is shown in the absence of ATP It showed a peak of about 28 nm slightly larger than the average sample size of 20 nm. 0.1 μM GroEL ^{D52, 398A in the} presence of a final concentration of 5 mM ATP (Example 5) shows by DLS measurement smaller than an average sample size of 54 nm in the presence of 5 mM ATP but in the absence of ATP Showed a peak of about 37 nm larger than the average sample size of about 20 nm. As a result of TEM observation, the GroEL tube was formed under the condition of 0.1 μM. FIG. 4 shows TEM images and DLS measurement results of GroEL ^WT and GroEL ^D52 , ^398A tubes.

From these results, in the presence of 5 mM ATP, GroEL ^WT , GroEL ^D398A , and GroEL ^{D 52} , ^398A can form GroEL tubes in the range of 0.1 ^μM to 5 μM, and a GroEL mutant having a slower ATP hydrolysis activity can be obtained. It was confirmed that the chain length and formation maintenance time of the tube were long.

Effect of Nucleotide Concentration on GroEL Tube Formation:

Regarding Examples 1 and 6 and Comparative Examples 1 and 3:
As a result of DLS of GroEL ^WT in which the final concentration of ATP is in the range of 4 to 6 mM (Comparative Example 3, Example 1, Example 6), the average sample size is about 20 nm, final concentration at 4 mM of final concentration ATP The peak showed about 33 nm at 5 mM ATP and about 52 nm at a final concentration of 6 mM ATP. Since GroEL in the absence of ATP has an average sample size of 20 nm, it was suggested that GroEL ^WT did not form a tube in the presence of 4 mM ATP at a final concentration, which was also consistent with the result of TEM observation. At a final concentration of 5 mM or more of ATP, tube formation of GroEL ^WT was shown by DLS and TEM, indicating that the GroEL ^WT tube may become longer depending on the ATP concentration. In addition, GroS ^WT tubes were formed in the presence of 5 mM ATP (Example 1), whereas in the presence of 5 mM ADP (Comparative Example 1), the GroEL ^WT tube was not formed, as shown by DLS and TEM. . FIG. 5 shows TEM images of GroEL ^WT and DLS measurement results.

Regarding Examples 3 and 7 and Comparative Examples 2 and 4:
Results of DLS of GroEL ^D52 , ^398A in which final concentration of ATP is in the range of 4 to 6 mM (Comparative Example 4, Example 2, Example 7), the average sample size of GroEL ^D52 , ^398A is the final concentration of 4 mM of ATP The peak showed about 50 nm at about 20 nm, about 53 nm at a final concentration of 5 mM ATP, and at a final concentration of 6 mM ATP. The average sample size of 20 nm of GroEL in the absence of ATP suggested that GroEL ^D52 , ^398A did not form a tube in the presence of 4 mM ATP. As a result of TEM observation, a tube of GroEL ^D52 , ^398A was not formed at a final concentration of 4 mM of ATP, and tube formation of GroEL ^D52 , ^398A was confirmed at an final concentration of 5 mM or more of ATP. While GroEL ^{D52, 398A} tubes were formed in the presence of 5 mM ATP (Example 3), DLS showed that GroEL ^{D 52} , ^398A tubes were not formed in the presence of 5 mM ADP (Comparative Example 2). . FIG. 6 shows TEM images of GroEL ^WT and DLS measurement results.

About Comparative Examples 5 and 6:
When 3 mM, which is an intracellular nucleotide concentration, is a final concentration of ATP, the DLS measurement results of 5 μM GroEL ^WT (Comparative Example 5) and 5 μM GroEL ^{D52, 398A} (Comparative Example 6) are average samples in the absence of ATP It showed an about 20 nm peak that is the same as the size, and it was found that any GroEL was an ATP concentration that did not form a tube. FIG. 7 shows TEM images and DLS measurement results of GroEL ^WT and GroEL ^D52 , ^398A .

About Example 8:
From the results of DLS measurement, when the protein concentration of 5 μM GroEL ^WT formed in the presence of 5 mM ATP is changed to the presence of 3 mM ATP / 0.3 mM ADP (physiological concentration) without changing the protein concentration, It was found that the average sample size peaking at about 40 nm in the presence of 5 mM ATP was reduced to an average sample size peaking at about 20 nm after transitioning in the presence of 3 mM ATP / 0.3 mM ADP . As a result of TEM observation, the GroEL ^WT tube was confirmed at 30 seconds after transition in the presence of 3 mM ATP, but at 10 minutes, the tube was degraded. FIG. 8 shows a TEM image of GroEL ^WT and DLS measurement results. About DLS, the thing of measurement time 2 minutes is illustrated.

About Example 9:
Results of DLS measurement when transferred to the presence of 3 mM ATP / 0.3 mM ADP (physiological concentration) without changing the protein concentration of 5 μM GroEL ^D52 , ^398A forming GroEL tubes in the presence of 5 mM ATP From the above, it was found that the average sample size peaking at about 50 nm in the presence of 5 mM ATP was reduced to an average sample size peaking at about 20 nm after transfer in the presence of 3 mM ATP. As a result of TEM observation, the GroEL ^{D52, 398A} tube was confirmed at 30 seconds after transition to the presence of 3 mM ATP / 0.3 mM ADP, but the number of ^GroELs connected became about half compared to before the decrease in ATP concentration. The tube was disassembled at 10 minutes. The GroEL ^{D52, 398A} tube also ^showed that when the surrounding nucleotide concentration decreased from 5 mM to 3 mM, the tube was degraded and dispersed for each molecule by 10 minutes after the concentration change. FIG. 9 shows TEM images and DLS measurement results of GroEL ^D52 , ^398A . About DLS, the thing of measurement time 2 minutes is illustrated.

From these results, it was found that GroEL tube formation requires ATP at a final concentration of 5 mM or more. Further, it was suggested that the GroEL tube was not formed below the final concentration of 4 mM ATP, and the GroEL tube did not occur spontaneously at the final concentration of 3 mM ATP which is the intracellular nucleotide concentration. Moreover, it turned out that ADP can not form GroEL tube. It can be said that it is possible to determine the nucleotide concentration that causes the formation and degradation of the GroEL tube based on ATP at a final concentration of 5 mM.

The influence of GroES on GroEL tube formation:

About Examples 10 and 11:
For 10 μM GroES added to 5 μM GroEL ^WT (tubes) formed in the presence of 5 mM ATP (Example 10), the measured DLS results show an average sample size of about 40 nm before GroES addition What decreased was reduced to about 20 nm one minute after the addition of GroES, implying that the GroEL ^WT tube was rapidly degraded and returned to GroEL ^WT . As a result of TEM observation, GroEL ^WT showed both GroEL ^WT tube and 1 molecule GroEL ^WT at 30 seconds after GroES addition, but 2 minutes after addition, GroEL ^WT tube had completely disappeared . FIG. 10 shows TEM images of GroEL ^WT and DLS measurement results.

For 10 μM GroES added to a 5 μM GroEL ^{D52, 398A} tube formed in the presence of 5 mM ATP (Example 11), the measured DLS results show that the average sample size is about 71 nm before GroES addition The amount decreased to about 61 nm at 1 minute after the addition of GroES and to about 41 nm at 10 minutes. As a result of TEM observation, in GroEL ^D52 , ^398A , at 30 seconds after GroES addition, both GroEL ^D52 , ^398A tube and 1 molecule GroEL ^D52 , ^398A were observed, but at 10 minutes after addition, GroEL ^{D 52, 398A} tube Was almost gone. FIG. 11 shows TEM images and DLS measurement results of GroEL ^D52 , ^398A .

These results indicated that GroES has the ability to disassemble GroEL tubes. GroEL ^D52 , ^398A, in which ATP hydrolysis activity is reduced, maintains the tube formed in the presence of 5 mM of ATP even after 60 minutes, but by adding GroES, the tube is forcibly forced in about 10 minutes. Since it was degraded, it was found to be the second means of degrading the tube without changes in nucleotide concentration.

  The result of TEM observation of the GroEL-containing solution obtained in Example 1 and the GroEL-containing solution obtained in the same manner as in Example 1 except that ATP is not present is as follows. is there. FIG. 12 is a TEM image in a state of being allowed to stand on ice before centrifugation. In the state of standing on ice, no significant difference was found between the upper side and the lower side. FIG. 13 is a TEM image after a 3000 × g centrifugation operation. In this state, a tube with a large molecular weight appeared slightly more downward than above. FIG. 13 is a TEM image after operation at 19000 × g centrifugation. In this condition, many tubes were clearly seen below. This phenomenon was not seen in ATP (-). From the above, it was found that GroEL can be accumulated by centrifugation when the tube-like complex is formed.

Claims (6)

  A GroEL-containing solution comprising a tube-like complex formed by polymerization of a plurality of GroELs, and ATP at a concentration of 5 mM or more.   The GroEL-containing solution according to claim 1, wherein the concentration of ATP is 5 to 10 mM.   The GroEL-containing solution according to claim 1 or 2, wherein GroEL is a wild type of E. coli. A method for producing the GroEL-containing liquid according to any one of claims 1 to 3, wherein
A non-polymerized GroEL is brought into contact with the ATP in an ATP-containing solution containing ATP at a concentration of 5 mM or more to form a tube-like complex formed by polymerization of the plurality of GroELs. A method for producing a GroEL-containing liquid.   A method of depolymerizing the tube-like complex to individual non-polymerized GroEL by lowering the concentration of ATP of the GroEL-containing solution according to any one of claims 1 to 3 to 4 mM or less.   4. The GroEL-containing solution according to any one of claims 1 to 3 is mixed with GroES to make the concentration of GroES equal to or higher than the concentration of GroEL, thereby individually polymerizing the tube-like composite. Method of depolymerizing to GroEL not.

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