JPH11225763A - Purification of protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently and economically producing and purifying a protein. SOLUTION: A cellulose carrier is treated with a solution containing a protein having a cellulose bond domain of a cellulolytic enzyme such as xylanase to adsorb the protein on the carrier. The protein is treated with a saccharide to elute the protein from the carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing and purifying a recombinant protein using a genetic engineering technique. More specifically, the present invention relates to a method for easily purifying a target protein from a transformant of a host cell using a solid phase carrier. And a method for purifying with good purity. In addition, this method can be applied to a vector for producing a recombinant protein and a reagent for purifying a fusion protein.

[0002]

2. Description of the Related Art With the advent of recombinant DNA technology, it has become possible to express foreign genes as heterologous proteins by introducing them into cells such as animals, plants, yeasts and bacteria. At present, it is possible to produce various useful recombinant proteins such as hormones and enzymes using this technology.

Hitherto, in producing such a recombinant protein, Escherichia coli, which is a gram-negative bacterium, has been widely used as a host. Generally, when producing a recombinant protein using this Escherichia coli as a host, after disrupting the cells, a large amount of proteins and nucleic acids originally retained by the cells,
It is necessary to separate and purify the target protein from contaminants such as polysaccharides. The same applies to the case where cells other than Escherichia coli are used as hosts when producing recombinant proteins. Therefore, in the production of a recombinant protein using a cell such as Escherichia coli as a host, efficient separation and purification of the target recombinant protein is an important issue for efficient production activities. .

[0004] One of the effective means for achieving such a problem is a method using a protein fusion technique and an affinity chromatography technique. In this method, a target protein is produced as a fusion protein with a protein (affinity tag) having an affinity for a specific ligand, and further, the fusion protein is efficiently subjected to affinity chromatography using the ligand as a carrier. This is a method for purification.

[0005] In addition, between the target protein and the affinity tag is recognized by a specific protease,
By inserting the amino acid sequence to be cleaved in advance, it is possible to cleave the fusion protein and recover only the target protein. For example, as an affinity tag, maltose binding protein (molecular weight 43
K dalton), glutathione S-transferase (molecular weight 26K dalton), protein A (molecular weight 52
K Dalton) and Factor Xa, thrombin, collagenase and the like as specific proteases for cleavage of the fusion protein are typical examples (Sassenfe).
ld, HM, TIBTECH 8: 88-93, 1990).

[0006] However, many of the methods developed so far have the following problems. 1. When purifying the fusion protein by affinity chromatography, the elution conditions from the carrier may be low pH (pH <3) that may cause inactivation of the target protein, or the target protein such as guanidine, ethylene glycol, and reducing agent may be used. Often it is necessary to add a substance that can also be an inhibitor. 2. Many carriers are very expensive or use a ligand that is relatively inactive and has low stability. 3. Very expensive specific proteases are often used for cleavage with an affinity tag.

On the other hand, most cellulolytic enzymes are:
A domain having a cellulose-degrading activity and a domain binding to cellulose (cellulose binding domain; hereinafter, CB
D). These CBDs are classified into nine families (family) based on their amino acid sequence homology.
I to IX) (Tomme, P. et al., Adv. Mi
crobiol. Physiol., 37, 1-81 (1995)).

Since cellulose to which CBD binds is very inexpensive as compared with other carriers, many attempts have been made to use these CBDs as affinity tags. For example, Greenwood et al. (FEBS Letter, 2
24: (1) 127-131 (1989)) is a CBD of Cellulomonas fimi endoglucanase.
(Family II) has been successfully fused with alkaline phosphatase. However, this CBD of Cellulomonas fimi has a low affinity for cellulose, and more than 30% of the fusion protein contains buffer (50 mM T
ris-HCl (pH7.5), 0.5M NaCl). Furthermore, Cellulomonas Fimi's CBD destroys cellulose fibers (Din et al., Bio Technology,
9: 1096-1098 (1991)).

[0009] Pseudomonas fluorescens
Cellulosa (Pseudomonas fluorescens subsp. Cellulo
sa) CBD of cellulase and xylanase obtained from
(Family X) binds very strongly to cellulose. Therefore, these enzymes are eluted from cellulose by boiling in 10% SDS, which is a condition under which many proteins are denatured (Poole, DM et al., Biochem. J., 279: 787-79
2 (1991)). Thus, it is clear that these CBDs are not optimal as affinity tags for fusion protein purification.

On the other hand, among the cellulolytic enzymes produced by Clostridium stercorarium, the gene for xylanase A has already been cloned and its nucleotide sequence has been elucidated (Sakka, K. et al. , Biosci. Biotech. Biochem.,
57, 237-277 (1993)). Furthermore, from the amino acid sequence deduced from this nucleotide sequence, the xylanase A is composed of about 130 amino acids in the vicinity of the C-terminal,
Two CBDs belonging to Family VI (CBD I and CBD II)
Which contain arginine and are linked by a linker of about 10 amino acids rich in serine and proline (Sakka, K. et al.
al., Biosci. Biotech. Biochem., 57, 237-277 (199
3)).

Recently, attempts have been made to fuse these two CBDs with endoglucanase IV from Ruminococcus albus,
BD has been shown to increase binding to insoluble cellulose without destroying the cellulose structure (Karit
a, S. et al., J. Ferment. Bioeng., 81, 553-556 (19
96)). However, there has been no attempt to purify a protein by affinity chromatography using a single CBD belonging to Family VI as an affinity tag, such as the CBD of xylanase A derived from Clostridium stercolarium.

[0012]

It is an object of the present invention to provide a method for efficiently and economically producing and purifying proteins.

[0013]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, the use of CBD of xylanase A derived from Clostridium stercolarium as an affinity tag enables efficient and economical use. The present inventors have found that the recombinant protein can be purified by the present invention, and reached the present invention.

[0014] That is, the present invention provides a method in which a solution containing a protein having a cellulose-binding domain of a cellulolytic enzyme is allowed to act on a cellulose carrier, whereby the protein is adsorbed on the carrier, and then a saccharide is allowed to act.
A protein purification method characterized by eluting the protein from the carrier.

The present invention is a fusion protein comprising (a) a protein to be purified and (b) a polypeptide chain containing a cellulose binding domain of a cellulolytic enzyme.

Further, the present invention comprises inserting a gene encoding a protein and a gene encoding a polypeptide chain containing a cellulose-binding domain of a cellulolytic enzyme into a vector, transforming a host cell with the vector, and transforming the host cell. Culturing, expressing a fusion protein of a polypeptide chain containing a protein and a cellulose-binding domain of a cellulolytic enzyme in the host cell, and collecting the fusion protein from the host cell to produce a fusion protein. Is the way.

Further, the present invention provides the above fusion protein,
Further, the present invention provides a method for producing a protein, comprising separating a polypeptide chain containing a cellulose binding domain from the fusion protein by treating with a specific protease, and recovering only the protein.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to a gene encoding a protein of interest and a cellulolytic enzyme such as xylanase A derived from Clostridium stercolarium.
And a step of synthesizing a fusion protein gene composed of a CBD-encoding gene, and a step of purifying the target fusion protein from a mixture containing the fusion protein synthesized from the gene.

Examples of the target protein include enzymes, antibodies, hormones, etc., which are industrially or academically useful. In general, the target protein is an in vitro (in vitro) synthesis system or an expression system using a host cell. If it can be synthesized,
Any protein can be used in the method of the present invention. For example, Ruminococcus albus
) Produced by Endoglucanase IV.

As cellulolytic enzymes, xylanase (EC 3.2.1.8), cellobiohydrolase (EC 3.2.1.
91), endoglucanase (EC 3.2.1.4) and the like, for example, Clostridium stercolarium (Clostridium
xylanase A produced by stercorarium).

The cellulose-binding domain is a polypeptide chain containing at least 40%, preferably at least 60%, of a homologous amino acid sequence with respect to a contiguous portion of the amino acid sequence shown in SEQ ID NO: 1 or 2, more preferably, a sequence chain. A polypeptide chain comprising the amino acid sequence shown in 1 or 2.

The protein having the cellulose binding domain of the cellulolytic enzyme is a fusion protein of the protein to be purified and the polypeptide chain containing the cellulose binding domain of the cellulolytic enzyme.

D encoding CBD used in the present invention
To prepare an NA fragment, first, a genomic DNA library is prepared from an organism having a cellulase gene suitable for carrying out the present invention. For example, such a cellulase gene can be obtained from a strain such as Clostridium stercolarium. Genomic DN from such strains
After preparing A, the fragment is partially digested with an appropriate restriction enzyme, for example, Sau 3AI, and the obtained fragment is digested with an appropriate vector,
For example, a genomic DNA library is obtained by integrating E. coli into pBR322 and transforming E. coli. A clone having a cellulase gene can be found from the obtained library by, for example, measuring cellulase activity of a randomly selected clone and isolating a clone exhibiting the activity. further,
Plasmid DNA is prepared from the clone having the cellulase activity selected in this way, and cut with an appropriate restriction enzyme, whereby a DNA fragment having the cellulase gene can be obtained.

Xylane, carboxymethylcellulose and the like are used as a substrate used for measuring cellulase activity. For example, xylan is suitable for measuring xylanase activity.

Further, DNA having a cellulase gene
In order to obtain a CBD-encoding DNA fragment from the fragment, for example, a DNA fragment having a cellulase gene was cut to an appropriate length using a restriction enzyme or exonuclease, or was cut from the end, so that the DNA fragment was first deleted. Obtain a cellulase gene fragment. Subsequently, after inserting the defective cellulase gene fragment into an appropriate vector again, the cells were introduced into a host cell, for example, E. coli, and the cellulose binding ability of the defective protein produced from the transformant was measured.
By using this as an index, the DN that codes the CBD
It is possible to prepare the A fragment.

Avicell (trade name, FMC) is used as the cellulose used for examining the cellulose binding ability.
Crystalline cellulose or non-crystalline cellulose is used. For example, non-crystalline cellulose is suitable for measuring the cellulose binding ability of CBD of xylanase A produced by Clostridium stercolarium.

By using the thus prepared DNA fragment containing the gene encoding CBD as a probe for hybridization, it is possible to isolate structurally similar CBD genes present in other organisms. It is. For example, if a DNA fragment containing the gene encoding the CBD of xylanase A produced by Clostridium stercholarium is used as a probe, DN containing the gene encoding the CBD belonging to the same family VI
The A fragment can be obtained from other organisms.

In the present invention, first, a fusion protein composed of a target protein and CBD of a cellulolytic enzyme is synthesized. For example, inserting the gene encoding the fusion protein into an expression vector suitable for recombinant protein production, introducing this into a host organism such as E. coli,
The cells may be grown under suitable conditions to express the fusion protein of interest.

Further, an expression vector having a multiple cloning site adjacent to a previously inserted gene encoding CBD may be used. In this case, the gene encoding the target protein is inserted into the expression vector using this multi-cloning site, and the base sequences of DNA comprising various combinations of promoters and restriction enzyme recognition sequences are prepared as gene cassettes. By doing so, it becomes easy to obtain an expression vector having a combination suitable for producing the target fusion protein.

When the fusion protein of interest is originally a secretory protein, a subsequence encoding an appropriate secretion signal is arranged upstream of the gene encoding the fusion protein, whereby the fusion protein can be obtained. Can be secreted into the periplasm.

On the other hand, as a method for purifying the fusion protein, a mixture containing the synthesized fusion protein is used as a starting material, and the purification is performed by affinity chromatography using a cellulose carrier. When a target fusion protein is synthesized in an expression system using host cells, a bacterial cell extract containing the fusion protein obtained by sonicating or mechanically disrupting the host cells is a starting material.

As the cellulose carrier for affinity chromatography, insoluble cellulose, for example, a commercially available cellulose carrier can be used. Xylanase A from Clostridium stercolarium
When using the CBD as an affinity tag,
Insoluble amorphous cellulose is preferred. Further, the adsorption capacity can be increased by pulverizing a commercially available cellulose carrier using a pulverizer such as a ball mill.
Further, a process for efficiently separating the carrier and the liquid, for example, a superparamagnetic metal compound may be combined with the cellulose carrier. In this case, it is possible to quickly and easily separate the carrier and the liquid by using a magnet.

In the purification step by affinity chromatography, first, a mixture containing the fusion protein of interest is prepared under buffer conditions suitable for adsorption to a cellulose carrier, and then contacted with the cellulose carrier to adsorb only the fusion protein. For example, when the CBD of Clostridium stercholalium-derived xylanase A is used as an affinity tag, a pH 7 potassium phosphate buffer is preferably used as the adsorption buffer.

The adsorption method may be either a batch method or a column method. In any method, the mixture containing the target fusion protein prepared in an appropriate buffer condition is brought into contact with the cellulose carrier, and if necessary, left for a certain period of time to allow only the fusion protein to be adsorbed to the cellulose carrier, thereby eliminating unnecessary The substance can be removed by washing the cellulose carrier with an adsorption buffer.

The target fusion protein adsorbed on the cellulose carrier is recovered by bringing an eluate containing a substance that prevents the binding between the cellulose carrier and the fusion protein into contact with the cellulose carrier. For example, when the CBD of Clostridium stercolarium-derived xylanase A is used as an affinity tag, it is preferable that saccharides such as cellobiose, maltose, glucose, and xylose are contained, and cellobiose is more preferable. In addition, these saccharides can be obtained relatively inexpensively. The concentration of the saccharide in the eluate used may be such that the binding between the CBD and the cellulose carrier is sufficiently prevented and the activity of the target protein is not affected, and more preferably 1 to 10%. A concentration is desirable.

According to the method of the present invention, a gene encoding a linker region containing a recognition site for a specific protease is located between the gene encoding the target protein and the gene encoding the CBD, Cleavage of the target protein and CBD by the treatment is possible. This is presumed to be because the linker region functions as a flexible hinge connecting the target protein and CBD in the expressed fusion protein, so that the recognition site of the specific protease is exposed on the surface of the fusion protein.
For example, after the eluted fusion protein is treated with a protease, if necessary, only the target protein can be separated using existing separation means such as various types of chromatography. Alternatively, the target protein may be recovered by bringing the protease digestion product of the fusion protein into contact with the cellulose carrier again, and adsorbing the peptide chain containing CBD used as the tag for purification onto the cellulose carrier. In addition, if a treatment with a protease is performed in a state where the fusion protein is bound to cellulose, only the target protein can be separated more easily.

As the specific protease, various known proteases can be used. For example,
Factor Xa, collagenase, enterokinase, thrombin and the like are preferred because they have very high specificity. Regarding the conditions for treating the fusion protein with a specific protease, the optimal reaction conditions for each protease may be considered.

However, it is necessary to select processing conditions such that the protein of interest does not contain amino acids that are recognized and cleaved by the protease, or that only the recognition site in the linker sequence is selectively cleaved. .
When a CBD of Clostridium stercholarium-derived xylanase A is used as an affinity tag, an expression vector is designed so that a linker sequence existing between the two CBDs is fused to a target protein together with one of the CBDs. Is also good. In this case, it is possible to specifically cleave the arginine residue present in the linker sequence at the carboxy terminus by allowing trypsin, which is a specific protease, to act under mild conditions.

[0039]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Unless otherwise specified, the gene manipulation methods used in the examples are described in Maniatis et al.'S literature (Molecu
lar Cloning, A Laboratory Manual; 2nd. Edition, Co
ld Spling Harbor Laboratry, 1989.).

Example 1 Xylanase A from Clostridium stercorarium
Preparation of DNA Fragment Containing DNA Sequence Encoding CBD In this example, a DNA fragment containing a DNA sequence encoding the CBD of xylanase A, one of the cellulolytic enzymes produced by Clostridium stercolarium, was prepared. In this xylanase A, two CBDs are present via a linker consisting of about 10 amino acid sequences (Sakka, K. et al., Biosci. Biotech.
Biochem., 57, 237-277 (1993)), and in this example, the linker sequence and the CBD located on the more carboxy-terminal side were used.
A DNA fragment (SEQ ID NO: 4) consisting of a DNA sequence encoding II was prepared (see FIG. 1).

In preparing the present DNA fragment, a polymerase chain reaction (PCR) using two types of primers each having an NspV or KpnI recognition sequence is used to prepare an NspV site on the 5 ′ side. Introduced the Kpn I site on the 3 'side.

First, genomic DNA is prepared from Clostridium stercoralium, partially cut with a restriction enzyme, Sau3AI, the obtained fragment is incorporated into a plasmid vector, and Escherichia coli is transformed to obtain a genomic DNA library. I got Subsequently, a plasmid pYK208 having a 6 kb DNA fragment containing a gene encoding xylanase A as an insert was obtained by randomly selecting clones contained in this library. Further, a 4.5 kb fragment obtained by digesting the 6 kb DNA fragment with EcoRV was subcloned into pBluescript II KS (+) (Stratagene) to obtain a plasmid pXYN containing a gene encoding xylanase A. Obtained. These methods are described in the literature (Sakka et.
al., Agric. Biol. Chem., 54 (2), 337-342 (1990) or Sakka et. al., Biosci. Biotech. Biochem., 57.
(2), 273-277 (1993)).

Next, two types of oligonucleotides (SEQ ID NOS: 5 and 6) having an NspV or KpnI recognition sequence were synthesized as PCR primers for amplifying DNA encoding a linker sequence and CBDII. The annealing site of each primer is shown in FIG.

Next, using the DNA of plasmid pXYN as a template,
PCR was performed using AmpliTaq polymerase (manufactured by PE Applied Biosystems) and two kinds of synthesized primers 1 and 2 (SEQ ID NOS: 5 and 6). Subsequently, the obtained amplification product was digested with Nsp V (manufactured by Toyobo) and KpnI (manufactured by Toyobo) to prepare a DNA fragment (SEQ ID NO: 4) containing a DNA sequence encoding CBD. .

Example 2 Luminococcus albus (Rum
Construction of Expression Vector (pCsCBD2) Encoding Endoglucanase IV and CBD from Inococcus albus) In this example, endoglucanase IV, one of the cellulolytic enzymes produced by Luminococcus albus, was used.
Expression vector into which the gene encoding (EGIV) is inserted
pRA11 (Karita, S. et al., J. Ferment. Bioeng., 7
6, 439-444 (1993)), the DNA fragment encoding CBD (SEQ ID NO: 4) prepared in Example 1 was inserted into EGI.
An expression vector pCsCBD2 for a fusion protein of V and CBD (EGIV-CBD fusion protein) was constructed.

A DNA fragment encoding CBD was inserted between the Nsp V site located near the stop codon at the 3 'end of the coding region of the EGIV gene and the Kpn I site located downstream of the EGIV gene, followed by open reading. A fusion gene was constructed so that the frame was not split between EGIV and CBD (see FIGS. 1 and 3).

First, genomic DNA was prepared from the Luminococcus alps, and then partially cut with PstI.
A genomic DNA library was obtained by incorporating the obtained fragment into a plasmid vector and transforming Escherichia coli. Subsequently, among the clones contained in this library, those having an endoglucanase activity are selected to contain the gene encoding EGVI 4.
Plasmid pRA102 with 4kb DNA fragment as insert
I got Furthermore, a plasmid pRA11 containing a gene encoding EGVI was obtained by subcloning a 2.3 kb fragment obtained by digesting the 4.4 kb DNA with BglII into pUC119 (Takara Shuzo). These methods are described in the literature (Karita et al., J. Ferment. Bioeng. 76 (6),
439-444 (1993)).

Next, the DNA of pRA11 was replaced with NspV and KpnI
This was separated by agarose gel electrophoresis to obtain a fragment containing the coding region of the EGIV gene. Next, this fragment and the DNA fragment obtained in Example 1 were ligated using a commercially available ligation kit (manufactured by Toyobo Co., Ltd.), and a part of the obtained product was used to transform Escherichia coli.
JM109 competent cells (manufactured by Toyobo Co., Ltd.) were transformed to obtain a recombinant Escherichia coli JM109 / pCsCBD2 strain having an expression vector for the EGIV-CBD fusion protein.

Example 3 Production and Purification of EGIV-CBD Fusion Protein by Culture of Recombinant Escherichia coli JM109 / pCsCBD2 In this example, production of EGIV-CBD fusion protein was carried out by culturing recombinant Escherichia coli JM109 / pCsCBD2. The fusion protein was further purified by using cellulose as a carrier for purification.

First, the recombinant E. coli JM109 / pCsCBD2 strain was
The cells were inoculated into 0 ml of LB medium (containing 100 μg / ml of ampicillin) and cultured at 37 ° C. overnight. After completion of the culture, the cells were collected by centrifugation and washed with 10 ml of 100 mM potassium phosphate buffer (containing 5 mM β-mercaptoethanol).
The suspension was suspended in 10 ml of the same buffer. Subsequently, the cells were crushed using an ultrasonic homogenizer (Sonifier 250: manufactured by Branson), and centrifuged to collect a cell lysate.

Cellulose KC flock W-300 (manufactured by Sanyo Kokusaku Pulp) is crushed (Takano, M. et al., J. Ferment. Bi
oeng., 73, 79-88 (1992)), 5 ml of ball mill cellulose (BMC) adjusted to 3% concentration with 100 mM potassium phosphate buffer containing 5 mM β-mercaptoethanol and 5 ml of the previously prepared cell lysate Were mixed and left on ice for 10 minutes. After centrifugation (1,000 × g, 10 minutes), the supernatant was removed. Cellulose was suspended in 5 ml of 100 mM potassium phosphate buffer (containing 5 mM β-mercaptoethanol) and centrifuged again (1,000 × g, 10 minutes) to wash the cellulose. After washing the cellulose three times, it is suspended in 1% cellobiose dissolved in 5 ml of 100 mM potassium phosphate buffer (containing 5 mM β-mercaptoethanol), and centrifuged (1,000 × g,
20 minutes) to collect the supernatant containing the fusion protein.

Table 1 shows the results obtained by measuring the protein amount and cellulase activity of the cell lysate and the recovered solution. The protein concentration was measured using a commercially available protein assay kit (manufactured by Bio-Rad), and the activity was measured according to the literature (Karita, S. et al., J. Ferment. Bioeng., 8).
1, 555-558 (1996)).
The amount releasing 1 μmol of glucose per minute was defined as 1 unit.

[0053]

[Table 1]

As a result, 39% of the fusion protein was recovered by purification using this cellulose carrier.
It was confirmed that the specific activity increased 15-fold. Also,
As a result of performing SDS-PAGE electrophoresis to examine the purity, it was confirmed that the obtained fusion protein was almost a single protein (see lane 2 in FIG. 4).

Example 4 EGIV-CBD by Trypsin Digestion
Specific cleavage of the fusion protein In this example, the specific protease trypsin was used to excise CBD from the fusion protein,
V was obtained. The trypsin was allowed to act at a low concentration and a low temperature to specifically hydrolyze the carboxy-terminal portion of the arginine residue present in the linker sequence between EGIV and CBD.

1 μg of trypsin (manufactured by Wako Pure Chemical Industries, Ltd.) was allowed to act on 350 μg of the EGIV-CBD fusion protein at 4 ° C. overnight, and a portion was subjected to SDS-PAGE. As a result, E
Two bands corresponding to GIV and CBD were confirmed (see lane 3 in FIG. 4).

Further, in order to examine the cleavage site of the fusion protein, the smaller band estimated to correspond to CBD was electroblotted onto a ProBlott membrane (manufactured by PE Applied Biosystems). Using this as a sample, the model
N-terminal amino acid sequence analysis was performed using a 476A protein sequencer (manufactured by PE Applied Biosystems).

As a result, the N of the excised protein
The terminal amino acid sequence was confirmed to begin with SSPVPAPGDN. That is, despite the presence of a large number of tryptic and target arginic acid residues and lysine residues in the amino acid sequence of the EGIV-CBD fusion protein, arginine residues present in the linker sequence between EGIV and CBD Shows that specific hydrolysis was carried out at the carboxy terminus. This is because a linker consisting of a sequence rich in hydrophilic amino acids is used to make EGIV and C
It was presumed that the arginine residue in the linker sequence was preferentially a target of trypsin in order to act as a flexible hinge connecting the BD and to expose it on the fusion protein surface.

Example 5 Purification of EGIV by Ion Exchange Column Chromatography In this example, the target EGIV was isolated by performing ion exchange column chromatography on a tryptic digest of the fusion protein. The trypsin digest obtained in Example 4 was applied to HitrapQ (manufactured by Pharmacia), and ion exchange column chromatography was performed. A part of the obtained fraction corresponding to EGIV was subjected to SDS-PAGE electrophoresis, and as a result, a single band corresponding to EGIV was confirmed (see lane 4 in FIG. 4). In FIG. 4, lane M: size marker, lane 1: lysate of a culture of recombinant E. coli JM109 / pCsCBD2 strain, lane 2: EGIV-CBD fusion protein purified by BMC, lane 3: EGIV-CBD fusion Tryptic digest of protein, lane 4: EG purified by ion exchange chromatography
Shows IV.

[0060]

The CBD of the present invention has a higher affinity for cellulose than conventional CBD and is not eluted with Tris-HCl buffer at all, so that a large amount of the target protein can be adsorbed on the cellulose carrier. In addition, elution of the protein from the cellulose carrier of the protein containing the CBD is characterized by no denaturation of the protein and no destruction of the cellulose under mild conditions using an aqueous solution of sugar. Therefore, according to the present invention, a recombinant protein can be efficiently and economically produced and purified.

[0061]

[Sequence list]

SEQ ID NO: 1 Sequence length: 126 Sequence type: amino acid Number of chains: single-chain Topology: linear Sequence type: polypeptide Sequence characteristics Clostridium stercorarium-derived xylanase A CBDI constitutes. Sequence Ile Arg Arg Asp Ala Phe Ser Ile Ile Glu Ala Glu Glu Tyr Asn Ser 1 5 10 15 Thr Asn Ser Ser Thr Leu Gln Val Ile Gly Thr Pro Asn Asn Gly Arg 20 25 30 Gly Ile Gly Tyr Ile Glu Asn Gly Asn Thr Val Thr Tyr Ser Asn Ile 35 40 45 Asp Phe Gly Ser Gly Ala Thr Gly Phe Ser Ala Thr Val Ala Thr Glu 50 55 60 Val Asn Thr Ser Ile Gln Ile Arg Ser Asp Ser Pro Thr Gly Thr Leu 65 70 75 80 Leu Gly Thr Leu Tyr Val Ser Ser Thr Gly Ser Trp Asn Thr Tyr Gln 85 90 95 Thr Val Ser Thr Asn Ile Ser Lys Ile Thr Gly Val His Asp Ile Val 100 105 110 Leu Val Phe Ser Gly Pro Val Asn Val Asp Asn Phe Ile Phe 115 120 125

SEQ ID NO: 2 Sequence length: 132 Sequence type: number of amino acid chains: 1 chain Topology: linear Sequence type: polypeptide Sequence characteristics Constructs CBDII of xylanase A derived from Clostridium stercolarium . Sequence Asp Asn Thr Arg Asp Ala Tyr Ser Ile Ile Gln Ala Glu Asp Tyr Asp 1 5 10 15 Ser Ser Tyr Gly Pro Asn Leu Gln Ile Phe Ser Leu Pro Gly Gly Gly 20 25 30 Ser Ala Ile Gly Tyr Ile Glu Asn Gly Tyr Ser Thr Thr Tyr Lys Asn 35 40 45 Ile Asp Phe Gly Asp Gly Ala Thr Ser Val Thr Ala Arg Val Ala Thr 50 55 60 Gln Asn Ala Thr Thr Ile Gln Val Arg Leu Gly Ser Pro Ser Gly Thr 65 70 75 80 Leu Leu Gly Thr Ile Tyr Val Gly Ser Thr Gly Ser Phe Asp Thr Tyr 85 90 95 Arg Asp Val Ser Ala Thr Ile Ser Asn Thr Ala Gly Val Lys Asp Ile 100 105 110 Val Leu Val Phe Ser Gly Pro Val Asn Val Asp Trp Phe Val Phe Ser 115 120 125 Lys Ser Gly Thr 130

SEQ ID NO: 3 Sequence length: 10 Sequence type: number of amino acid chains: 1-chain Topology: linear Sequence type: oligopeptide Sequence characteristics between CBDI and CBDII of xylanase A derived from Clostridium stercolarium Constitute the linker present in

SEQ ID NO: 4 Sequence length: 432 Sequence type: nucleic acid (DNA) Number of strands: double-stranded Topology: linear Sequence type: DNA Sequence characteristics CDB II of xylanase A derived from Clostridium stercolarium And configure the linker. Sequence CGA AGT TCA CCA GTG CCT GCA CCT GGT GAT AAC ACA AGA GAC GCA TAT 48 Arg Ser Ser Pro Val Pro Ala Pro Gly Asp Asn Thr Arg Asp Ala Tyr 1 5 10 15 TCT ATC ATT CAG GCC GAG GAT TAT GAC AGC AGT TAT GGT CCC AAC CTT 96 Ser Ile Ile Gln Ala Glu Asp Tyr Asp Ser Ser Tyr Gly Pro Asn Leu 20 25 30 CAA ATC TTT AGC TTA CCA GGT GGT GGC AGC GCC ATT GGC TAT ATT GAA 144 Gln Ile Phe Ser Leu Pro Gly Gly Gly Ser Ala Ile Gly Tyr Ile Glu 35 40 45 AAT GGT TAT TCC ACT ACC TAT AAA AAT ATT GAT TTT GGT GAC GGC GCA 192 Asn Gly Tyr Ser Thr Thr Tyr Lys Asn Ile Asp Phe Gly Asp Gly Ala 50 55 60 ACG TCC GTA ACA GCA AGA GTA GCT ACC CAG AAT GCT ACT ACC ATT CAG 240 Thr Ser Val Thr Ala Arg Val Ala Thr Gln Asn Ala Thr Thr Ile Gln 65 70 75 80 GTA AGA TTG GGA AGT CCA TCG GGT ACA TTA CTT GGA ACA ATT TAC GTG 288 Val Arg Leu Gly Ser Pro Ser Gly Thr Leu Leu Gly Thr Ile Tyr Val 85 90 95 GGG TCC ACA GGA AGC TTT GAT ACT TAT AGG GAT GTA TCC GCT ACC ATT 336 Gly Ser Thr Gly Ser Phe Asp Thr Tyr Arg Asp Val Ser Ala Thr Ile 100 105 110 A GT AAT ACT GCG GGT GTA AAA GAT ATT GTT CTT GTA TTC TCA GGT CCT 384 Ser Asn Thr Ala Gly Val Lys Asp Ile Val Leu Val Phe Ser Gly Pro 115 120 125 GTT AAT GTT GAC TGG TTT GTA TTC TCA AAA TCA GGA ACT TAA GGG TAC 432 Val Asn Val Asp Trp Phe Val Phe Ser Lys Ser Gly Thr *** 130 135 140

SEQ ID NO: 5 Sequence length: 28 Sequence type: nucleic acid (DNA) Number of strands: single strand Topology: linear Sequence type: synthetic oligonucleotide TTTTCGAAGT TCACCAGTGC CTGCACCT 28

SEQ ID NO: 6 Sequence length: 24 Sequence type: nucleic acid (DNA) Number of strands: single strand Topology: linear Sequence type: synthetic oligonucleotide TTGGTACCCT TAAGTTCCTG ATTT 24

[Brief description of the drawings]

FIG. 1 is a diagram showing the construction of an expression plasmid encoding an EGIV-CBD fusion protein.

FIG. 2 is a view showing base and amino acid sequences corresponding to the vicinity of the amino terminal of the linker and the vicinity of the carboxy terminal of CBD, and the annealing sites of primers 1 and 2.

FIG. 3 is a diagram showing base and amino acid sequences corresponding to the vicinity of the junction between EGIV and a linker.

FIG. 4 is a photograph replacing a figure showing the results of SDS-PAGE electrophoresis.

Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12N 1/21 C12N 1/21 9/24 9/24 11/12 11/12 C12P 21/02 C12P 21/02 C // (C12N 15 / 09 ZNA C12R 1: 145) (C12N 1/21 C12R 1:19) (C12N 9/24 C12R 1: 145) (C12P 21/02 C12R 1:19)

Claims (44)

[Claims] Claims: 1. A solution containing a protein having a cellulose-binding domain of a cellulolytic enzyme is allowed to act on a cellulose carrier, whereby the protein is adsorbed on the carrier, and then a saccharide is allowed to act on the carrier to convert the protein from the carrier. A protein purification method characterized by elution. 2. The method for purifying a protein according to claim 1, wherein the cellulolytic enzyme is xylanase. 3. The method for purifying a protein according to claim 1, wherein the cellulolytic enzyme is xylanase A produced by Clostridium stercorarium. 4. The method according to claim 1, wherein the cellulose binding domain is SEQ ID NO: 1.
2. The method for purifying a protein according to claim 1, wherein the polypeptide chain comprises at least 40% of a homologous amino acid sequence with respect to the continuous portion of the amino acid sequence shown in 2. 5. The method according to claim 5, wherein the cellulose binding domain is SEQ ID NO: 1.
2. The method for purifying a protein according to claim 1, wherein the polypeptide chain comprises at least 60% of a homologous amino acid sequence with respect to a continuous portion of the amino acid sequence shown in 2. 6. The method according to claim 6, wherein the cellulose binding domain is SEQ ID NO: 1.
2. The method for purifying a protein according to claim 1, wherein the method is a polypeptide chain comprising any of the amino acid sequences shown in 2. 7. The method for purifying a protein according to claim 1, wherein the protein having a cellulose-binding domain of a cellulolytic enzyme is a fusion protein of a protein to be purified and a polypeptide chain containing a cellulose-binding domain of the cellulolytic enzyme. 8. The method according to claim 7, wherein the protein to be purified is a recombinant protein. 9. The method according to claim 1, wherein the cellulose carrier is insoluble cellulose. 10. The method for purifying a protein according to claim 9, wherein the insoluble cellulose is non-crystalline cellulose. 11. The method according to claim 1, wherein the saccharide is cellobiose, maltose, glucose or xylose. 12. The method according to claim 1, wherein the saccharide is cellobiose. 13. The protein purification method according to claim 12, wherein the concentration of cellobiose is used in a range of 1 to 10%. 14. The eluted fusion protein may further comprise:
The protein purification method according to claim 7, wherein a polypeptide chain containing a cellulose binding domain is separated from the fusion protein by treating with a specific protease, and only the protein is recovered. 15. The fusion protein comprises (a) a protein to be purified, (b) a linker consisting of the amino acid sequence shown in SEQ ID NO: 3, further (c) a cellulose-binding domain of a cellulolytic enzyme, and further comprising (d) 8. The method for purifying a protein according to claim 7, comprising a site cleaved at said linker portion by a specific protease. 16. The method according to claim 15, wherein the specific protease is trypsin. 17. A fusion protein comprising (a) a protein to be purified and (b) a polypeptide chain containing a cellulose-binding domain of a cellulolytic enzyme. 18. The fusion protein according to claim 17, wherein the cellulolytic enzyme is xylanase. 19. The fusion protein according to claim 17, wherein the cellulolytic enzyme is xylanase A produced by Clostridium stercorarium. 20. The fusion protein according to claim 17, wherein the cellulose binding domain is a polypeptide chain containing at least 40% homologous amino acid sequence to a continuous portion of the amino acid sequence shown in SEQ ID NO: 1 or 2. 21. The fusion protein according to claim 17, wherein the cellulose binding domain is a polypeptide chain containing at least 60% homologous amino acid sequence to a continuous portion of the amino acid sequence shown in SEQ ID NO: 1 or 2. 22. The fusion protein according to claim 17, wherein the cellulose binding domain is a polypeptide chain comprising the amino acid sequence shown in SEQ ID NO: 1 or 2. 23. (a) a protein to be purified,
(B) a fusion protein comprising a linker consisting of the amino acid sequence shown in SEQ ID NO: 3, further (c) a cellulose binding domain of a cellulolytic enzyme, and (d) a site cleaved at the linker by a specific protease. 24. The fusion protein according to claim 23, wherein the specific protease is trypsin. A recombinant DNA encoding the fusion protein according to any one of claims 17 to 24. 26. A recombinant vector comprising a recombinant DNA encoding the fusion protein according to claim 25. 27. A host cell transformant transformed with the recombinant vector according to claim 26. 28. The host cell transformant according to claim 27, wherein the host cell is Escherichia coli. 29. A vector encoding a gene encoding a protein and a gene encoding a polypeptide chain containing a cellulose binding domain of a cellulolytic enzyme,
A host cell is transformed with the vector, the host cell is cultured, and a fusion protein of a polypeptide chain containing a protein and a cellulose-binding domain of a cellulolytic enzyme is expressed in the host cell. A method for producing a fusion protein, comprising collecting the fusion protein. 30. A host cell extract containing a fusion protein with a protein and a polypeptide chain containing a cellulose-binding domain of a cellulolytic enzyme is allowed to act on a cellulose carrier, whereby the fusion protein is adsorbed on the carrier. 30. The method for producing a fusion protein according to claim 29, wherein the fusion protein is eluted from the carrier by reacting a saccharide. 31. The method according to claim 29, wherein the cellulolytic enzyme is xylanase. 32. The method for producing a fusion protein according to claim 29, wherein the cellulolytic enzyme is xylanase A produced by Clostridium stercorarium. 33. The production of a fusion protein according to claim 29, wherein the cellulose binding domain is a polypeptide chain containing at least 40% homologous amino acid sequence to a continuous portion of the amino acid sequence shown in SEQ ID NO: 1 or 2. Method. 34. The production of a fusion protein according to claim 29, wherein the cellulose binding domain is a polypeptide chain containing at least 60% homologous amino acid sequence to a continuous portion of the amino acid sequence shown in SEQ ID NO: 1 or 2. Method. 35. The method for producing a fusion protein according to claim 29, wherein the cellulose-binding domain is a polypeptide chain containing any of the amino acid sequences shown in SEQ ID NOS: 1 and 2. 36. The method for producing a fusion protein according to claim 29, which is an enzyme, an antibody or a hormone. 37. The method for producing a fusion protein according to claim 30, wherein the cellulose carrier is insoluble cellulose. 38. The method according to claim 38, wherein the insoluble cellulose is a non-crystalline cellulose. 39. The method for producing a fusion protein according to claim 29, wherein the saccharide is cellobiose, maltose, glucose or xylose. 40. The saccharide is cellobiose.
The method for producing a fusion protein according to the above. 41. The method for producing a fusion protein according to claim 29, wherein the concentration of cellobiose is used in a range of 1 to 10%. 42. The fusion protein comprises (a) a protein to be produced, (b) a linker consisting of the amino acid sequence shown in SEQ ID NO: 3, further (c) a cellulose binding domain of a cellulolytic enzyme, and further comprising (d) 30) The method for producing a fusion protein according to claim 29, comprising a site cleaved at the linker portion by a specific protease. 43. The eluted fusion protein according to claim 42, which is further treated with a specific protease to separate a polypeptide chain containing a cellulose binding domain from the fusion protein, and recover only the protein. A method for producing a protein, comprising: 44. The method for producing a protein according to claim 43, wherein the specific protease is trypsin.