JP2010246522A - Fusion protein and gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for more reliably maintaining activity of target protein and acquiring a large amount of target protein in high efficiency. <P>SOLUTION: The fusion protein includes two or more chaperonin subunits connected to each other in series, forms a ring-like structure in which the chaperonin subunits are arranged in a ring-like state and stably maintains an open ring type form. Preferably the fusion protein has a constitution that the target protein is further connected to an N-terminal or C-terminal and the target protein is stored in the inside of the ring-like structure. The gene encodes the fusion protein. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a fusion protein and a gene, and more particularly to a fusion protein of a chaperonin and a target protein, and a gene encoding the fusion protein. The present invention makes it possible to produce a target protein maintaining its activity with high efficiency.

  Analysis of whole genome sequences of various species including humans, mice and yeast has been completed, and the flow of bio research is shifting from gene analysis to comprehensive protein research. As a result, the discovery of proteins involved in diseases and the elucidation of protein-protein interactions in vivo are expected to lead to the development of new drugs. In recent years, two-hybrid methods for detecting protein-protein interactions and protein chips for detecting protein expression in vivo have been developed and widely used as research tools for that purpose. That is, until now, DNA chips have been mainly used as research tools for screening genes related to diseases, but it cannot always be said that there is a correlation between gene translation and protein expression. Therefore, instead of this, research tools that use proteins, such as protein chips, are attracting particular attention.

  What should be taken into account when handling proteins is to prevent denaturation and maintain activity. However, when a target protein is to be expressed / produced by genetic recombination technology, a protein having a function / activity maintained is not always obtained, and trial and error are often repeated. As a technique for solving such a problem, a method using chaperonin is known (Patent Document 1).

  Chaperonin is a kind of molecular chaperone and is a complex protein composed of subunits (chaperonin subunits) having a molecular weight of about 60,000. A typical chaperonin is a cylinder having a total molecular weight of about 800,000 to 1,000,000 having a two-layer structure in which two layers of a ring-shaped structure composed of 7 to 9 chaperonin subunits are non-covalently associated via a ring surface. A huge complex is formed. Chaperonins contain other proteins inside and can fold correctly.

  In the invention described in Patent Document 1, an artificial protein (chaperonin linked body) formed by linking two or more chaperonin subunits in series is prepared, and a fusion protein in which a target protein is linked to this end is used. A chaperonin conjugate can also form a ring-like structure like a natural chaperonin, and can be folded correctly by storing the target protein therein. According to the method of Patent Document 1, (a) degradation of a target protein by a host-derived protease, which frequently occurs during forced expression of the target protein by a gene recombination technique using a host vector system, and (b) the target protein is originally Problems such as difficulty in protein production due to host toxicity and (c) formation of inclusion bodies due to poor structure formation of the target protein can be solved.

International Publication No. 02/052029 Pamphlet

  The invention described in Patent Document 1 provides a method capable of maintaining the activity of the target protein and expressing it with high efficiency. However, depending on the type of the target protein, it is still not possible to obtain a target protein that maintains its activity even when chaperonin is allowed to act. Therefore, the present invention aims to further develop the method of Patent Document 1, to provide a method for more reliably maintaining the activity of the target protein and obtaining a large amount of the target protein with higher efficiency. .

  The present inventors examined the cause that the target protein that maintained the activity could not be obtained even when the chaperonin was acted on, and focused on the storage stage of the target protein in the ring-shaped structure. As a result of studying measures for solving this problem, the present inventors have found that the problem can be solved by using a mutant chaperonin that can stably maintain an open ring type form, and completed the present invention. The gist of the present invention is as follows.

  The invention according to claim 1 is a fusion protein comprising two or more chaperonin subunits connected in series, and capable of forming a ring-shaped structure in which the chaperonin subunits are arranged in a ring shape. It is a fusion protein characterized by being able to stably maintain a ring-shaped form.

  The present invention relates to a fusion protein in which two or more chaperonin subunits are connected in series (that is, a chaperonin conjugate), and is characterized in that it can stably maintain an open ring type form. . Since the fusion protein of the present invention maintains an open ring type form, the ring-shaped structure has a larger cavity than the natural type chaperonin. Therefore, even a target protein that cannot be stored in the cavity of the natural chaperonin when folded into the correct structure can be stored securely.

  Here, the “open ring type” refers to a form in which the top portion of the ring is open in a direction perpendicular to the ring surface in the chaperonin ring-shaped structure. The open ring type morphology is observed in a very short time (several seconds to several tens of seconds) during the process of storing the target protein by the natural chaperonin. The chaperonin employed in the present invention can stably maintain an open ring type form, and does not temporarily take an open ring type form like a natural type chaperonin, and is open ring even in a normal state. Take the form of a mold. An open ring type chaperonin can be produced, for example, by substituting a part of amino acid residues of a natural type chaperonin.

  The chaperonin subunit is preferably a group 1 type chaperonin subunit (claim 2).

  The group 1 type chaperonin is preferably an E. coli-derived chaperonin (Claim 3).

  The structure which forms the ring-shaped structure which consists of a single ring is preferable (Claim 4).

  The invention according to claim 5 is characterized in that the target protein is further linked to the N-terminal or C-terminal, and the target protein can be stored inside the ring-shaped structure. The fusion protein described in 1.

  The fusion protein of the present invention is obtained by linking a target protein to the end of a chaperonin conjugate. According to the fusion protein of the present invention, the target protein can be reliably stored inside the ring-shaped structure.

  The invention according to claim 6 is a gene encoding the fusion protein according to any one of claims 1 to 5.

  The present invention relates to a gene and encodes the above-described fusion protein of the present invention. By transcribing and translating the gene of the present invention in a host, the fusion protein of the present invention can be produced with high efficiency.

  Since the fusion protein of the present invention maintains an open ring type form, the ring-shaped structure has a larger cavity than the natural type chaperonin. Therefore, even a target protein that cannot be stored in the cavity of the natural chaperonin when folded into the correct structure can be stored securely.

  According to the gene of the present invention, the fusion protein of the present invention can be produced with high efficiency.

It is explanatory drawing explaining the process in which colon_bacillus | E._coli GroEL correctly folds | denatures a denatured protein, (a) is a normal state (closed type), (b) is the state which trapped the target protein in the middle of folding (closed type), (c) is The state in which the target protein is stored inside (open ring type), (d) shows the state in which the target protein has been folded (open ring type), and (e) shows the state in which the target protein has been released (closed type). It is a graph which shows the measurement result of the arrest activity performed in the Example.

  The fusion protein of the present invention is a fusion protein comprising two or more chaperonin subunits connected in series, and capable of forming a ring-shaped structure in which the chaperonin subunits are arranged in a ring shape. It is characterized in that the form of can be stably maintained. First, the “open-ring type” chaperonin will be described in detail using Escherichia coli GroEL as an example. The series of mechanisms shown in FIG. 1 is described in, for example, Nature, 388, 792-798 (1997).

  The process of correctly folding the denatured protein by Escherichia coli GroEL (chaperonin) is as shown in FIGS. 1 (a) to 1 (e). First, on the top of one of the two-layered ring structure (FIG. 1 (a)). The target protein during folding is trapped (FIG. 1 (b)). Next, the target protein is stored in the cavity in the ring with the binding of GroES, which is a cofactor (co-chaperonin), and ATP (FIG. 1 (c)). At this time, it is known that when the target protein is stored in the cavity, the top domains of the seven GroEL subunits forming the ring change the angle in the vertical direction. This form is called “open ring type”. It is called. Next, ATP is converted to ADP and the target protein is correctly folded in the cavity (FIG. 1 (d)), and then the target protein that is correctly folded is released from the cavity (FIG. 1 (e)). That is, regarding the form of GroEL, the “open ring type” form is adopted at the stage shown in FIGS. 1C to 1D, and at the stage shown in FIGS. 1A to 1B and FIG. 1E. Takes the form of a “closed type” similar to the normal state. Here, the stage of the open ring type shown in FIGS. 1C to 1D is a temporary step of several seconds to several tens of seconds, and the open ring type state is stably maintained for a long time. There is no. Specifically, it is said that GroEL takes an open ring type form between 8 seconds and 15 seconds. Note that the open-ring chaperonin has a larger cavity volume than the closed type because the top domain changes the angle in the vertical direction.

  As described above, in the natural chaperonin, taking the open ring type form is temporary, and the open ring type form is not stably maintained. However, on the other hand, a mutant chaperonin that can stably maintain an open ring type form is known. The fusion protein of the present invention utilizes this mutant chaperonin. A mutant chaperonin capable of stably maintaining an open ring type form can be obtained, for example, by modifying a part of amino acids constituting the chaperonin subunit. For example, in the case of Escherichia coli GroEL (SEQ ID NOs: 1 and 2), the distance between the 384th alanine of one adjacent subunit and the 509th serine of the other subunit is relatively distant from the closed chaperonin. The open ring type is closer. Therefore, by substituting these two amino acids with cysteine and bridging between them by a disulfide bond, the two amino acids cannot return to their original positions, and can be kept in close proximity at all times. As a result, the chaperonin ring is stably maintained in an open type.

  When an open ring chaperonin utilizing a disulfide bond between cysteines is employed, it is desirable to place the fusion protein (chaperonin conjugate) in a mild oxidative environment. For example, after obtaining a cysteine-substituted chaperonin subunit in the presence of a reducing agent such as DTT, the reducing agent is completely removed by dialysis or the like in an appropriate buffer, followed by formation of disulfide bonds by natural oxidation, It is preferable to perform a treatment for forming a disulfide bond in the presence of oxidized glutathione or diamide at a concentration.

  Next, chaperonin groups will be described. In general, chaperonins are roughly classified into group 1 type and group 2 type. Chaperonins present in bacteria and eukaryotic organelles are classified as group 1 type, all of which form a ring structure in which seven chaperonin subunits having a molecular weight of 60 kDa are linked in a ring, and two ring structures overlap. A 14-mer homo-oligomer having a two-layer structure. These cofactors are cyclic heptamers of a protein with a molecular weight of about 10 kDa called co-chaperonin. A specific example of the group 1 chaperonin is GroEL, which is a chaperonin derived from E. coli. On the other hand, group 2 type chaperonins are found in eukaryotic cytoplasm and archaea, and have a bilayer structure in which ring-shaped structures composed of 8 to 9 chaperonin subunits are usually overlapped. Homo or hetero oligomers are formed. The chaperonin employed in the fusion protein of the present invention may be either group 1 type or group 2 type, but group 1 type chaperonin is preferred, and the above-mentioned Escherichia coli GroEL is particularly preferred from the viewpoint of ease of handling. Here, the base sequence and amino acid sequence of GroEL are shown in SEQ ID NO: 1, and only the amino acid sequence is shown in SEQ ID NO: 2.

  A general chaperonin is composed of a two-layered ring-shaped structure, but there is also a “single-ring” chaperonin composed of a single-layered ring-shaped structure. In the fusion protein of the present invention, it is preferable to employ a chaperonin that forms a ring-shaped structure consisting of a single ring. That is, by forming the chaperonin ring into a single layer, the target protein and the external environment stored in the chaperonin ring can be accessed from the external environment through the two openings of the chaperonin ring. As a result, when the target protein needs to act with, for example, a substrate or a low molecular ligand, the accessibility to the target protein is improved.

  Single-ring chaperonins can be obtained, for example, by modifying a part of amino acids constituting chaperonin subunits. For example, in the case of Escherichia coli GroEL (SEQ ID NOs: 1 and 2), four charged amino acids (R452, E461, S463, V464) in the equator domain strongly contribute to the bond between the rings by crystal structure analysis. I know it. Therefore, by replacing the 452nd amino acid residue (arginine) with glutamic acid and the 461st (glutamic acid), 463 (serine) and 464th (valine) amino acid residues with alanine, the interaction between rings is reduced, It is known to constitute a single ring (Weissman et al., Cell, 1995, 83, 577-587). In addition, chaperonin 60 derived from eukaryotic mitochondria is usually purified as a single ring and thus can be obtained without special genetic manipulation (Viitanen et al., J. Biol. Chem., 1992, 267, 695-698). Here, an example of an open ring type single ring mutant (open type single ring mutant) of Escherichia coli GroEL is shown in SEQ ID NOs: 3 and 4. The single-ring chaperonin is also a means for producing a mutant chaperonin that can basically fold the target protein by the mechanism shown in FIGS. 1 (a) to 1 (e) and can stably maintain an open-ring form. The above-described means can be applied as it is.

  In a preferred embodiment, the target protein is further linked to the N-terminus or C-terminus of the fusion protein (chaperonin conjugate), and the target protein can be stored inside the ring-shaped structure. According to the fusion protein of this embodiment, the target protein can be reliably stored inside the ring-shaped structure.

  The target protein employed in the present invention is not particularly limited, but the molecular weight is preferably a molecular weight that can be stored in the ring structure of chaperonin, for example, 100,000 or less, more preferably 60,000 or less. There are no particular limitations on the type of protein, and for example, all proteins derived from animals such as humans, mice and nematodes, pathogenic bacteria, infectious viruses and the like can be employed. For example, multiple transmembrane proteins including G protein-coupled receptors, channel-type receptors, etc .; single transmembrane proteins including phosphorylase, phosphatase, cell adhesion factor, and the like. In addition, not only a transmembrane protein but also a membrane surface protein present in the vicinity of the surface layer of a biological membrane. It is difficult to produce by the conventional gene recombination technology such as virus-derived protease, nuclear hormone receptor protein, reverse transcriptase, etc. A protein or the like can be preferably employed.

  The gene of the present invention encodes the fusion protein of the present invention. By transcribing and translating the gene of the present invention in a host, the fusion protein of the present invention can be produced with high efficiency. For example, the gene of the present invention may be incorporated into an expression vector, the recombinant vector is introduced into an appropriate host to produce a transformant, and the gene is expressed in the transformant. The host used at this time is not particularly limited, but microorganisms such as bacteria are preferable from the viewpoint of low culture cost, short culture days, and easy culture operation, and Escherichia coli is particularly easy to handle. More preferred.

  Since chaperonin is expressed in the soluble fraction of the host organism's cytoplasm or body fluid, even if the target protein stored in the chaperonin ring is a membrane-bound protein, it migrates to the membrane and changes the membrane structure of the host organism. There is no destruction and no toxicity to the host organism is manifested. Regardless of the type of target protein, it can be purified as a fusion protein under the same purification conditions as long as it is stored in the chaperonin ring. In addition, the method for purifying the fusion protein of the present invention is not particularly limited, and for example, it can be appropriately purified using a method such as salting out, hydrophobic chromatography, ion exchange chromatography, gel filtration, affinity chromatography and the like. it can. Further, by adding a histidine sequence of about 6 residues to the N-terminal or C-terminal of the fusion protein, it is possible to purify the fusion protein also by chelate chromatography using a nickel chelate carrier.

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

(1) Expression vector construction of open ring chaperonin A DNA (SEQ ID NO: 3) containing a gene encoding an open single ring mutant (OSREL; SEQ ID NO: 4) of Escherichia coli GroEL (SEQ ID NO: 1, 2) was artificially synthesized. . The synthetic DNA was provided with a SpeI site at the 5 ′ end and an XbaI site at the 3 ′ end. The synthetic DNA was incorporated on the pUC57 plasmid to obtain pUC57-OSREL. pUC57-OSREL was treated with restriction enzymes SpeI / XbaI, and a DNA fragment containing the OSREL gene was recovered (DNA fragment A). This DNA fragment A was introduced into a pTrc99A vector (Amersham Pharmacia) previously treated with XbaI. The sequencer confirmed that the OSREL gene was introduced in the correct orientation with respect to the promoter. This vector was named pTrc99A (OSREL) 1.

  pUC57-OSREL was treated with restriction enzymes SpeI / XbaI, and DNA fragment A containing the OSREL gene was obtained again. This DNA fragment A was introduced into pTrc99A (OSREL) 1, which had been treated with XbaI in advance. Thereby, the vector pTrc99A (OSREL) 2 in which one more OSREL gene was inserted downstream of the OSREL gene was constructed. The same procedure was repeated to construct a vector containing a fusion gene ((OSREL) 7 gene) in which seven OSREL genes were linked downstream of the promoter. At this time, it was confirmed at any time by a DNA sequencer that each OSREL gene was introduced in the correct orientation. As a result, a vector pTrc99A (OSREL) 7 expressing a chaperonin conjugate formed by connecting seven OSRELs (subunits) in series was constructed.

  Furthermore, a multicloning site for introducing a gene encoding the target protein was introduced downstream of the (OSREL) 7 gene of pTrc99A (OSREL) 7, and a gene sequence for expressing the FLAG peptide was provided on the 3 ′ side. . This expression vector was named Trc99A (OSREL) 7-F.

(2) Production of fusion protein Trc99A (OSREL) 7-F constructed in (1) above was introduced into E. coli BLR (DE3) strain, and 1 colony of the transformant was transformed into 2 × YT liquid medium (bactotryptone 16 g, Yeast extract (10 g, NaCl 5 g / L) was inoculated and cultured with stirring at 25 ° C. for 48 hours in the presence of ampicillin (100 μg / mL). After completion of the culture, the cells were collected.

  The collected cells were suspended in a PBS buffer containing 1 mM DTT, and the cells were disrupted by ultrasonic treatment. The cell disruption solution was centrifuged and the supernatant was collected. The supernatant was subjected to an immunoprecipitation reaction using anti-FLAG antibody-immobilized beads (manufactured by Sigma). After washing the beads, the fraction eluted with the FLAG peptide was applied to 5 mL of HiTrap Butyl FF column (Amersham Pharmacia) equilibrated with 10% ammonium sulfate / PBS buffer (containing 1 mM DTT). Next, elution was performed with PBS buffer (containing 1 mM DTT), and the fraction containing the fusion protein was collected. This fraction was subjected to gel filtration using TSKgel G4000SWXL pre-equilibrated with PBS buffer (containing 1 mM DTT), and the fraction containing (OSREL) 7 was collected. The obtained fraction was concentrated and dialyzed against PBS buffer solution to remove DTT, thereby subjecting it to natural oxidation treatment. As a result, a chaperonin conjugate (OSREL) 7 in which seven open single ring variants of GroEL subunits were linked in series was obtained.

  When the molecular weight of the obtained (OSREL) 7 was evaluated by native electrophoresis using a 2-15% acrylamide gradient gel, a band was detected at the same position as the single ring type chaperonin. From this, it was confirmed that the obtained (OSREL) 7 formed a single ring.

(3) Evaluation of interaction of open ring chaperonin with denatured protein Closed chaperonin has a function of capturing denatured protein (arrest activity), whereas open chaperonin is known to have very low arrest activity. ing. The arrest activity of (OSREL) 7 obtained in (2) above was evaluated by the following procedure using guanidine-modified citrate synthase (CS). As a control, a closed-type single-ring GroEL seven-fold conjugate was used.

CS (C-9454) purchased from Sigma-Aldrich was completely unfolded by acting in 6M guanidine hydrochloride for 60 minutes. TMK buffer (100 mM Tris-HCl pH 7.4, 10 mM MgCl ₂ , 10 mM KCl) containing 1.5 μM (OSREL) 7 in 300 μL of the unfolded modified CS as described above at a final concentration of 0.5 μM (TMK buffer) Spontaneous aggregation reaction of the denatured CS was started. CS aggregation was evaluated by light scattering intensity of 320 nm. The results are shown in FIG. In the figure, A (solid line) is added (OSREL) 7 (open type single ring), B (broken line) is added control GroEL 7-fold linked body (closed type single ring), C (thin broken line) is chaperonin linked body The case where it is not added is shown. As a result, in the case of the closed type single ring chaperonin, the aggregation of the denatured CS was efficiently suppressed, while in the case of using the open type single ring chaperonin (OSREL) 7, the aggregation inhibition effect of the denatured CS was low. . This indicated the nature of the open chaperonin. From this, it was shown that the chaperonin conjugate (OSREL) 7 prepared in the above (2) stably maintains an open ring type form.

(1) Construction of expression vector of open ring type chaperonin linked with target protein In this example, human endothelin receptor type A (hETAR) was adopted as the target protein.
PCR was performed using a clone containing the hETAR gene of Accession Number NM_001957 obtained from OriGene as a template, and using the oligo DNAs of SEQ ID NO: 5 and SEQ ID NO: 6 as a primer set, and BglII was added to the 5 ′ end of the hETAR gene (SEQ ID NO: 7). A DNA fragment having an XhoI site introduced on the 3 ′ side of the site was obtained. This DNA fragment was introduced into the pT7blueT vector. After confirming the nucleotide sequence of the introduced DNA fragment, the hETAR gene was excised from the same vector with BglII / XhoI. The excised hETAR gene was introduced into the BglII / XhoI site included in the multicloning site of Trc99A (OSREL) 7-F constructed in Example 1. As a result, an expression vector Trc99A (OSREL) 7-ETAR-F that can express a fusion protein in which hETAR (target protein) is linked to the C-terminus of (OSREL) 7 was constructed.

(2) Production of fusion protein The constructed expression vector Trc99A (OSREL) 7-ETAR-F was introduced into the E. coli BLR (DE3) strain in the same manner as in (2) of Example 1, and after culturing, the cell pellet was obtained. It was collected. Further, the collected cells were crushed in the same manner as in Example 1 (2), and subjected to immunoprecipitation using anti-FLAG antibody-immobilized beads, hydrophobic chromatography using HiTrap Butyl FF column, and gel filtration using TSKgel G4000SWXL. As a result, a fusion protein in which hETAR was linked to the C-terminus of (OSREL) 7 was obtained. The purity of the obtained fusion protein was 90% or more, and the recovery rate was about 1 mg / 1 L culture.

Claims (6)

  A fusion protein in which two or more chaperonin subunits are linked in series and can form a ring-shaped structure in which the chaperonin subunits are arranged in a ring shape, stably maintaining an open ring-type form A fusion protein characterized in that it is possible.   The fusion protein according to claim 1, wherein the chaperonin subunit is a subunit of a group 1 type chaperonin.   The fusion protein according to claim 2, wherein the group 1 type chaperonin is E. coli-derived chaperonin.   The fusion protein according to any one of claims 1 to 3, which forms a ring-shaped structure composed of a single ring.   The fusion protein according to any one of claims 1 to 4, wherein the target protein is further linked to the N-terminal or C-terminal, and the target protein can be stored inside the ring-shaped structure.   A gene encoding the fusion protein according to any one of claims 1 to 5.

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