JP2000083694A - Purification of polypeptide and kit reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of purifying polypeptide in high yield even for highly hydrophobic polypeptide. SOLUTION: This method of purifying polypeptide from cells capable of producing the aimed polypeptide comprises the following procedure: the polypeptide is made to directly adsorb to a magnetic support to which a ligand having the affinity for the polypeptide is bound, without the need of removing the spalled pieces from a solution where the cells are spalled, and the aimed polypeptide is subsequently recovered.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The method for purifying a polypeptide of the present invention comprises adsorbing the fusion protein by adding a magnetic carrier on which a specific ligand is immobilized as it is to a lysate of cells producing the target polypeptide. Or a method for purifying the polypeptide, wherein the step of disrupting cells and the step of adsorbing the target protein to a magnetic carrier are performed simultaneously. In a more specific embodiment, the polypeptide of interest is produced in the form of a fusion protein of a polypeptide having an affinity for a specific ligand and a biologically active polypeptide, for example, by genetic recombination techniques. The present invention relates to a purification method in which a lysate of a host cell of a fusion protein is directly used for adsorption of the fusion protein with the magnetic carrier, and a purification method of the fusion protein in which cell crushing and adsorption to the magnetic carrier are performed simultaneously.

[0002]

2. Description of the Related Art Today, a wide variety of methods, materials, and methods for separating specific proteins in biological material from other components are known.
And approaches exist. One example of a common approach is the non-specific affinity of a protein for a carrier. For example, proteins can be separated by ion exchange chromatography based on molecular charge.
That is, the protein mixture is added to the oppositely charged chromatography matrix, and various proteins bind to the matrix by reversible electrostatic interactions. Proteins bound to the matrix are eluted in the order of weak to strong binding by increasing the ionic strength or changing the pH of the elution buffer.

Another common approach is to use the physical properties of proteins as a means of separation. For example, proteins can be separated based on protein size using gel filtration. In this method, the protein mixture is added to a gel filtration column packed with a chromatography matrix having pores of a predetermined size.
The protein is then eluted, usually with a buffer, and collected and analyzed as individual chromatographic fractions.

A third general approach utilizes the specific affinity of a protein for a purification reagent. For example, the protein can be purified using an antibody specific to the protein. Alternatively, the antibody may be purified by an antigen specific to it. Usually, the antibody or antigen is bound to the column substrate, and a solution containing that particular antigen or antibody can be added to the column to form an immune complex. Subsequently, the bound components of the immune complex are eluted by destabilizing the antigen-antibody complex, for example, by exposure to a buffer of very high ionic strength or high or low pH.

[0005] As described above, affinity chromatography is a very effective method for purifying a protein utilizing a specific interaction between a protein to be purified and a ligand immobilized on a solid phase. In general, solid phase ligands have some unique scientific properties, thereby selectively adsorbing proteins to be separated. Other contaminating proteins (cont
aminant protein) does not bind to the solid phase ligand,
Alternatively, it is removed by washing the solid phase ligand with an appropriate solution.

[0006] The possibility of preparing hybrid genes by genetic techniques has opened new avenues in the production of recombinant proteins. By combining a gene sequence encoding a desired protein with a gene sequence encoding a protein fragment (affinity peptide) having a high affinity for a ligand, the desired recombinant protein can be obtained in one step using the affinity peptide. Can be purified in the form of a fusion protein. It is also possible to introduce a specific chemical or enzymatic cleavage site at the junction between the affinity peptide and the desired recombinant protein by site-restricted mutation, so that the fusion protein can be transformed with an appropriate affinity resin. After purification, the desired recombinant protein can be recovered by chemical or enzymatic cleavage.

[0007] For example, Japanese Patent No. 2686090 discloses that as a recombinant protein, six histidine residues (hereinafter referred to as 6 × His) at the N-terminal or C-terminal of a target protein.
Are described, and affinity purification is performed using a specific Ni ligand. However, in this method, specificity between the affinity peptide and the ligand is low, so that purification may not be performed with high purity.

Further, a target protein is produced as a CBD fusion protein using a cellulose-binding region (hereinafter referred to as CBD) of xylanase A obtained from Clostridium stercorarium, and affinity purification is performed. J.Ferment.
Bioeng. 81, 553-556 (1996)
Has been reported to. However, this method requires a CBD
May greatly affect the properties of the target protein.

[0009] In addition to the above, such purification methods include, for example,
Science Vol. 198, pp. 1056-1063 (197
7 years) (by Itakura et al.), Proc. Natl. Acad. Sci. US
A. Vol. 80, pp. 6848-6852 (1983) (G
ermino et al.), NucleicAcids Res.
151-162 (1985) (by Nilsson et al.), And Gene 32, 321-327.
(1984) (by Smith et al.). However, even in these methods, CBD
Similarly to the above, the affinity peptide region may greatly affect the properties of the target protein.

[0010]

As described above, affinity chromatography using a fusion protein is a very excellent purification method, but it is mainly used for purification of a soluble protein which is hydrophilic, and is relatively hydrophobic. In some cases, the yield may be low for a protein having a high level of strength. Therefore, there is a long-felt need for a purification method capable of obtaining a high yield even with a highly hydrophobic fusion protein.

[0011]

Means for Solving the Problems The present inventors have conducted intensive studies in view of the above circumstances, and found that a magnetic carrier is added to a cell disruption suspension as it is and a target polypeptide is adsorbed thereon, thereby obtaining a relatively hydrophobic substance. It has been found that even a protein having a high affinity can adsorb the target fusion protein with high affinity to the immobilized ligand without the polypeptide non-specifically adsorbing to cell debris and causing loss. It was completed.

That is, the present invention has the following configuration. (1) In a method of purifying a polypeptide from a cell producing the polypeptide of interest, a ligand having an affinity for the polypeptide is bound as it is without removing debris from a solution in which the cell is disrupted. A method for purifying a polypeptide, comprising recovering the polypeptide after adsorbing the polypeptide on a magnetic carrier. (2) The purification method of (1), wherein the step of disrupting cells and the step of adsorbing the polypeptide to a magnetic carrier to which a ligand having affinity for the polypeptide are bound are performed simultaneously. (3) The purification method according to (1) or (2), wherein the polypeptide is a fusion protein. (4) The polypeptide is a fusion protein in which a polypeptide region having affinity for a ligand is directly or indirectly bound to an amino-terminal amino acid or a carboxy-terminal amino acid of the polypeptide (1) to (3). )). (5) The ligand is a nickel ion (1) to (4)
Any of the purification methods. (6) The purification method according to any one of (1) to (5), wherein the magnetic carrier is a magnetized agarose carrier or a magnetized cellulose carrier. (7) The polypeptide is a fusion protein in which a polypeptide region having an affinity for cellulose is directly or indirectly bound to an amino-terminal amino acid or a carboxy-terminal amino acid of the polypeptide (1) to (3). )). (8) The purification method of (7) using a magnetized cellulose carrier or a cellulose-bound magnetic carrier. (9) The purification method according to any one of (1) to (8), wherein the cell producing the polypeptide is a microorganism. (10) The cell producing the polypeptide is Escherichia
Any one of (1) to (8), which is Escherichia coli. (11) The purification method according to any one of (1) to (10), wherein the cells producing the polypeptide are disrupted by ultrasonic waves. (12) The purification method according to any one of (1) to (10), wherein the cells producing the polypeptide are disrupted by an enzyme. (13) The purification method according to (12), wherein the enzyme is lysozyme or β-1,3-glucan laminalipentaohydrolase. (14) A method for purifying a polypeptide, comprising removing the polypeptide obtained by purification by any of the methods (1) to (13) by selective cleavage. (15) The purification method of (12), wherein a protease is used for selective cleavage of the polypeptide obtained by purification by any of the methods of (1) to (13). (16) The method according to (14) or (15), wherein the protease is at least one selected from the group consisting of thrombin, Factor Xa, and enterokinase. (17) A magnetic carrier having a ligand that binds to the target polypeptide, a cell disruption / adsorption solution, a washing solution used for removing unnecessary substances, and an eluate used for separating the target polypeptide from the carrier A kit reagent used for the purification method according to any one of (1) to (16), wherein (18) The kit reagent according to (17), wherein the pH of at least one of the cell disruption / adsorption solution and the washing solution is 6 to 8. (19) The kit reagent according to (17) or (18), wherein the cell disruption / adsorption solution and the washing solution have the same composition. (20) A substance that antagonizes specific adsorption of the target polypeptide to the magnetic carrier is added to the eluate (1).
(7) The kit reagent according to any one of (19) to (19). (21) The kit reagent according to (20), wherein the substance that antagonizes specific adsorption of the target polypeptide to the magnetic carrier is imidazole. (22) The kit reagent according to (20), wherein the substance that antagonizes specific adsorption of the target polypeptide to the magnetic carrier is cellobiose. (23) The kit reagent according to any one of (17) to (22), wherein the cell disruption / adsorption solution, the washing solution, and the eluate contain an alkali metal salt. (24) The kit reagent according to (23), wherein the alkali metal salt is NaCl or KCl.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION In the method for purifying a polypeptide of the present invention, a ligand having an affinity for the polypeptide is bound as it is without removing debris from a solution in which cells producing the polypeptide have been disrupted. After adsorbing the polypeptide on a magnetic carrier, the polypeptide is recovered. At this time, the step of crushing the cells and the step of adsorbing the polypeptide to the magnetic carrier to which the ligand having an affinity for the polypeptide is bound are performed at the same time in order to eliminate the time lag from crushing to adsorption. preferable. In addition, when crushing and adsorption are performed simultaneously, the operation time required for purification can be reduced.

Usually, when a fusion protein is purified in a batch system using a non-magnetic carrier, the carrier is subjected to a solid-liquid separation by a centrifugal separation operation. This separation method utilizes gravity, and it is necessary to remove cell debris without fail before adsorption. If the cell lysate is used as it is, it will not be possible to separate the non-magnetic carrier and cell lysates again.

However, when a magnetic carrier is used,
Only the magnetic carrier can be separated by magnetic force. Also,
Cell disruption and adsorption to the carrier can be performed simultaneously. Such a purification method does not require a step of removing the disrupted cell debris by centrifugation as compared with a conventional method using a non-magnetic carrier, and the purification operation time can be shortened.

The purification method of the present invention is particularly useful when the polypeptide is a fusion protein, and the fusion protein has a polypeptide region having an affinity for a ligand (hereinafter, referred to as an affinity peptide). Preferred is a fusion protein directly or indirectly linked to the amino or carboxy terminal amino acid of the polypeptide. The method for obtaining the fusion protein is not particularly limited, but is preferably produced by a microorganism using a gene recombination technique.

The fusion protein can be directly or indirectly linked to the polypeptide. If a single affinity peptide is used, the same affinity peptide can be attached to either the amino or carboxy terminal amino acids of the polypeptide. If two affinity peptides are used, one of them will be linked to the amino terminal amino acid of the polypeptide and the other will be linked to the carboxy terminal amino acid of the polypeptide.

In the case of indirect binding, the affinity peptides contain a suitable selective cleavage site, through which they are bound to the desired polypeptide. The selective cleavage site is preferably an amino acid sequence-(Asp) _n -Lys- (n
Represents 2, 3 or 4) or -Ile-Glu-Gly-Arg-. It can be specifically recognized by the proteases enterokinase and aggregate Factor Xa, respectively. Such affinity peptides can then be cleaved enzymatically by methods known per se.

In the case of direct binding, the affinity peptide remains bound to the desired polypeptide. That is, the affinity peptide cannot be cleaved chemically or enzymatically. Thus, this form of binding is advantageous when the activity of the desired polypeptide is not adversely affected by the presence of the affinity peptide.

As the above ligand, nickel ion, cellulose, glutathione and the like are preferable because the affinity peptide has a low molecular weight and high specificity. Further, as the magnetic carrier, magnetized agar allose, magnetized cellulose and the like are preferable in terms of price, strength and particle size.

In another embodiment of the purification method of the present invention, a polypeptide region (CBD) having an affinity for cellulose binds directly or indirectly to an amino-terminal amino acid or a carboxy-terminal amino acid of the polypeptide. And a purification method using the fusion protein. In this case, a magnetized cellulose carrier may be used alone as the magnetic carrier, or a magnetic carrier such as a magnetized agarose carrier in which cellulose is bound as a ligand may be used.

The cells producing the polypeptide are preferably microorganisms. Examples of the microorganism include bacteria, yeast, and the like, and among them, Escherichia coli (Esch
E. coli) is preferred.

The disruption of the cells producing the polypeptide is preferably carried out using ultrasonic waves or enzymes. When an enzyme is used, a lytic enzyme such as β-1,3-glucan laminaripentaohydrolase such as lysozyme or zymolyase (manufactured by Kirin Brewery) is particularly preferred.

It is preferable that the polypeptide obtained by purification by the above method is removed by selective cleavage. In that case, it is preferable to use a protease.
Thrombin, Factor Xa, enterokinase and the like are particularly preferred.

The kit reagent of the present invention used in the above-described purification method includes a magnetic carrier having a ligand binding to a target polypeptide,
It consists of an adsorbing solution, a washing solution used for removing unnecessary substances, and an eluate used for separating the target polypeptide from the carrier. The eluate is preferably constituted by all or a part of the solution added to the cell disruption / adsorption solution or the washing solution in order to avoid nonspecific adsorption of the magnetic carrier.

The pH of at least one of the cell crushing / adsorbing solution and the washing solution is preferably from 6 to 8, and more preferably from 7 to 8. Further, it is more preferable that the cell disruption / adsorption solution and the washing solution have the same composition from the viewpoint of preventing the polypeptide adsorbed on the carrier from being eliminated.

The above eluate is preferably prepared by adding a substance that antagonizes specific adsorption of the target polypeptide to the magnetic carrier. The substance that antagonizes the specific adsorption of the polypeptide of interest to the magnetic carrier is preferably imidazole, cellobiose, glutathione, or the like.

Further, in order to avoid the above-mentioned non-specific adsorption, it is preferable to add an alkali metal salt to the cell disruption / adsorption solution, washing solution and eluate. Among the alkali metal salts, NaCl,
KCl and the like are particularly preferred.

[0029]

EXAMPLES The effects of the present invention will be further clarified by illustrating examples of the present invention.

Example 1 Purification of 6 × His / α-Glucosidase Fusion Protein Using Magnetized Ni-Agarose Carrier First, pUC119 was added to Bacillus stearothermophilus (Ba
cillus stearothermophilus) ATCC12016-derived α-glucosidase gene-linked plasmid pBST (Appl Mi
crobiol Biotechnol Vol. 44, pp. 629-634,
1996) as a template, a 21 nucleotide sequence complementary to the 5 'end of the α-glucosidase gene, an EcoRI recognition sequence, an initiation codon (ATG), and 6 histidine coding sequences were connected to the 5' end in this order. Primer (5'-CC
G GAA TTC ATG CAT CAC CAT CACCAT CAC TTG AAA AAA A
CA TGG TGG AAA-3 ') and a primer (5'-CGC CAG GGT TTT CCCAGT CAC GAC-3') derived from pUC119 located at the 3 'end of the α-glucosidase gene.
PC with KOD DNA polymerase (Toyobo)
R was performed.

The amplified DNA fragment (about 2.1 kbp) was cut with a restriction enzyme EcoRI, purified, and prepared in advance.
It was linked to pKK / EcoRI-SmaI (Pharmacia). Escherichia coli JM109 was transformed with the ligated plasmid pKK / 6 × His / α-glucosidase, and 6 ×
A transformant producing a His / α-glucosidase fusion protein (a fusion protein in which six histidines were linked to the N-terminal of α-glucosidase) was obtained. 6 × His / α-
Expression of the glucosidase fusion protein was confirmed by the presence or absence of α-glucosidase activity.

Next, the above transformant was treated with isopropyl-β
Induction culture was performed using -D-thiogalactopyranoside (hereinafter, referred to as IPTG; final concentration: 0.2 mM) to express a large amount of 6 × His / α-glucosidase fusion protein.

Using a magnetized Ni-agarose carrier (manufactured by Scigen, UK), the above fusion protein was adsorbed to (1) only the supernatant after cell disruption, (2) the cell disrupted solution was directly adsorbed,
(3) The cells were disrupted and purified by three methods of adsorption. Examples of conventional purification methods using a non-magnetic carrier include TALON of Clontech and N.I. of Qiagen.
Purification was carried out in the same manner as in (1) using i-NTA.

FIG. 1 shows the method of purification and the reagents used in (1) to (3). The solid-liquid separation is performed by a magnetic stand for a magnetic carrier, and a centrifugal separation for TALON and Ni-NTA. FIG. 2 shows the recovery rate and specific activity (hereinafter also referred to as SA) of α-glucosidase activity of the eluate obtained by each purification method, and FIG. 3 shows the results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Show.

As a result of the examination, as shown in FIG. 2, when only the supernatant after cell disruption was adsorbed and purified using TALON of Clontech, Ni-NTA of Qiagen and Ni-agarose magnetic carrier, The activity recovery was about 30%. However, when the crushed liquid was directly adsorbed using the magnetized Ni-agarose carrier, and when the crushed liquid was adsorbed and purified simultaneously with the crushing, the activity recovery rate doubled to about 65% and about 52%, respectively. In SA, the same tendency as the recovery rate was observed.

Also, from the results of SDS-PAGE shown in FIG. 3, when the crushed liquid is used as it is or when the crushed liquid is adsorbed and purified at the same time, the recovered amount of the 6 × His / α-glucosidase fusion protein increases. It is clear that

Example 2 Lipoprotein lipase / 6 ×
Purification of His fusion protein using magnetized Ni-agarose carrier First, PUC18 was cloned into Pseudomonas arginosa (Pseudo
Plasmid pUL11 (ARCHIVES OF BIOCHE) ligated with lipoprotein lipase gene derived from Monas aeruginousa
MISTRY AND BIOPHYSICS Vol. 296, No. 2, 505
513, 1992) as a template, a primer derived from pUC18 located at the 5 'end of the lipoprotein lipase gene (5'-CAC TTT ATG CTT CCG GCT CGT ATG T
TG-3 ') and 3' of the lipoprotein lipase gene
25 nucleotide sequences complementary to the terminal and its 5 '
BglII recognition sequence at the end, 6 histidine coding sequences,
A primer (5'-GTC AGGTCC TAG TGA TGG TGA TGG TG) connected in order of a stop codon (TGA) and an Eco47I recognition sequence
A TGA GAT CTC AGG CTG GCG TTC TTC AGG CGG TTG-
3 '), PCR was performed using KOD DNA polymerase (manufactured by Toyobo).

The amplified DNA fragment (about 1.3 kbp) was double-cut with the restriction enzymes NcoI / Eco47I, purified, and ligated to pUL11 / NcoI-Eco47I prepared in advance. The ligated plasmid pUL11 / lipoprotein lipase / 6 ×
Escherichia coli JM109 was transformed with His to obtain a transformant producing a lipoprotein lipase / 6 × His fusion protein (a fusion protein in which six histidines were linked to the C-terminal part of lipoprotein lipase).

Next, the transformant was induced and cultured in IPTG (final concentration: 0.2 mM) to express the lipoprotein lipase / 6 × His fusion protein. Expression of the lipoprotein lipase / 6 × His fusion protein is C
It was confirmed by Western blot using an antibody (manufactured by Invitrogen) recognizing 6 × His at the end. Western blot was performed according to the instruction manual of Imaging high -Western- (manufactured by Toyobo).

As in Example 1, the fusion protein was
Using an i-agarose carrier, purification was performed by two methods: (1) adsorption of only supernatant after cell disruption, and (2) adsorption simultaneously with cell disruption. The method of purification and the reagents used in (1) and (2) are the same as in FIG. FIG. 4 shows the results of SDS-PAGE of each purification step obtained by each purification method.

From the results of SDS-PAGE shown in FIG.
When only the supernatant after cell disruption was adsorbed and purified, no lipoprotein lipase / 6 × His fusion protein was recovered. However, in the case of using a magnetized Ni-agarose carrier and adsorbing and purifying at the same time as crushing, several minor bands are also recognized, but it was revealed that the target fusion protein can be specifically purified.

Example 3 α-glucosidase / Cellose
Purification of binding domain (CBD) fusion protein using a magnetized cellulose carrier Clostridium stercoralium
plasmid pXYN (Sakka, K. et a) ligating the region encoding the xylanase A gene from Rcorarium).
l., Biosci. Biotech. Biochem. 57, 237-2.
77, 1993) as a template, using Cellose binding doma
Primer (5'-CCC TCC GGA AGA TCT TCA CCA GTG) comprising a 27 nucleotide sequence complementary to the 5 'end of in (CBD) and a MroI / BglII recognition sequence connected to its 5' end.
CCT GCA CCT GGT GAT AAC-3 ') and a 20 nucleotide sequence complementary to the 3' end of the CBD gene and a primer (5 ') to which a termination codon (TAA), KpnI, EcoRI recognition sequence was connected at its 5' end. -CCG AAT TCG GTA CCC T
TA AGT TCC TGA TTT TGA G-3 ')
PCR using a polymerase (manufactured by Toyobo) was performed.

The amplified DNA fragment (about 500 bp) was double-digested with the restriction enzymes BglII / EcoRI, purified, and inserted with a restriction enzyme BglII recognition sequence at the 3 ′ end of the α-glucosidase gene by point mutation. Plasmid pBST (Ap
pl. Microbiol. Biotechnol. Vol. 44, 629-634
P. 1996, see Example 1) with the restriction enzyme BglII / Ec.
It was ligated to the one that was double cut with oRI. α-glucosidase and CBD are designed in advance so that the translation frames match. Escherichia coli JM109 is transformed with the ligated plasmid pUC18 / α-glucosidase / CBD to produce an α-glucosidase / CBD fusion protein (a fusion protein in which CBD is linked to the C-terminal part of α-glucosidase). I got the body. The expression of the α-glucosidase / CBD fusion protein was confirmed by the presence or absence of α-glucosidase activity.

Next, the transformant was induced and cultured in IPTG (final concentration: 0.2 mM) to express the α-glucosidase / CBD fusion protein in a large amount. Using this fusion protein as a magnetized cellulose carrier (Cortex, USA)
In the same manner as in Example 2, purification was performed by two methods: (1) adsorption of only the supernatant after cell disruption, and (2) adsorption simultaneously with cell disruption.

FIG. 5 shows the method of purification and the reagents used in (1) and (2). The solid-liquid separation is performed on a magnetic carrier using a magnetic stand. FIG. 6 shows the recovery rate and specific activity (SA) of α-glucosidase activity of the eluate obtained by each purification method, and FIG. 7 shows the results of SDS-PAGE.

As shown in FIG. 5, when only the supernatant after cell disruption was adsorbed and purified using a magnetized cellulose carrier, the activity recovery was 1.2%. However, in the case of using a magnetized cellulose carrier and adsorbing and purifying simultaneously with crushing, the activity recovery rate was remarkably increased to 20%. Also, SA
Also showed an increasing tendency similar to the recovery rate.

Further, from the results of SDS-PAGE shown in FIG. 7, it is clear that the amount of α-glucosidase / CBD fusion protein recovered is increased when crushing and adsorption are performed and purification is performed.

As described above, the same tendency as in Example 1 was observed. When the target protein had relatively high hydrophobicity,
When using only the crushed supernatant for the adsorption reaction with the ligand,
Due to the phenomenon that the polypeptide region having affinity goes into the target protein, the fusion protein aggregates, or the target protein nonspecifically adsorbs to the cell debris, the amount of the target protein extracted in the supernatant decreases, and the ligand decreases. It is thought that the adsorption rate of the compound decreases. Then, the recovery of the target protein becomes insufficient, which is considered to be reflected in the SA difference.

On the other hand, when the carrier is added to the crushed suspension as it is and purified, or when the crushed suspension is purified by adsorption to the carrier at the same time as the crushing suspension, the His- adsorbed nonspecifically to the cell debris is obtained.
tag fusion protein (6 × H at C-terminal or N-terminal of protein)
It is thought that the target protein can be purified by specific adsorption to a carrier, and the target protein has an increased recovery rate and an improved degree of purification (SA).

[0050]

As described above, by using the method for purifying the polypeptide of the present invention, it is possible to efficiently purify even a relatively hydrophobic fusion protein as compared with a conventional method. It became so.

[Brief description of the drawings]

FIG. 1 is a diagram showing a purification flow of a 6 × His fusion protein in Examples 1 and 2.

FIG. 2 is a view showing the recovery rate (%) and specific activity (U / mg) of α-glucosidase in Example 1.

FIG. 3 shows SDS-PAGE of each purification step of 6 × His / α-glucosidase fusion protein in Example 1.
FIG.

FIG. 4 shows SDS-PA in each purification step of 6 × His / lipoprotein lipase fusion protein in Example 2.
It is a figure showing GE.

FIG. 5: α-glucosidase / C in Example 3
It is a figure which shows the purification flow of BD fusion protein.

FIG. 6 shows the recovery rate (%) and specific activity (U / mg) of α-glucosidase in Example 3.

FIG. 7: α-glucosidase / C in Example 3
It is a figure which shows SDS-PAGE of each purification step of BD fusion protein.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) C12R 1:19) (72) Inventor Fumiyoshi Kawakami 10-24 Toyocho, Tsuruga-shi, Fukui Toyobo Co., Ltd. In-house (72) Inventor Yoshihisa Kawamura 10-24 Toyo-cho, Tsuruga-shi, Fukui Prefecture Toyobo Co., Ltd.Tsuruga Bio-Laboratory F-term (reference) 4B064 AG01 CA02 CA19 CC24 CE02 CE20 DA16 4H045 AA10 AA20 BA54 CA11 DA89 EA61 FA74 GA20 GA26

Claims (24)

[Claims] 1. A method for purifying a polypeptide from a cell producing the polypeptide of interest, wherein a ligand having an affinity for the polypeptide is bound as it is without removing debris from a solution obtained by crushing the cell. A method for purifying a polypeptide, comprising adsorbing the polypeptide on a magnetic carrier subjected to the method, and recovering the polypeptide. 2. The purification method according to claim 1, wherein the step of disrupting the cells and the step of adsorbing the polypeptide to a magnetic carrier to which a ligand having an affinity for the polypeptide are bound are performed simultaneously. 3. The purification method according to claim 1, wherein the polypeptide is a fusion protein. 4. The polypeptide according to claim 1, wherein the polypeptide is a polypeptide in which a polypeptide region having affinity for a ligand is directly or indirectly bound to an amino-terminal amino acid or a carboxy-terminal amino acid of the polypeptide. 3
The purification method according to any one of the above. 5. The purification method according to claim 1, wherein the ligand is a nickel ion. 6. The purification method according to claim 1, wherein the magnetic carrier is a magnetized agarose carrier or a magnetized cellulose carrier. 7. The polypeptide according to claim 1, wherein the polypeptide is a fusion protein in which a polypeptide region having an affinity for cellulose is directly or indirectly bound to an amino terminal amino acid or a carboxy terminal amino acid of the polypeptide.
3. The purification method according to any one of 3. 8. The purification method according to claim 7, wherein a magnetized cellulose carrier or a magnetic carrier to which cellulose is bound is used. 9. The purification method according to claim 1, wherein the cell producing the polypeptide is a microorganism. 10. The polypeptide-producing cell is Escherichia coli.
The purification method according to any one of the above. 11. The purification method according to claim 1, wherein cells producing the polypeptide are disrupted by ultrasonic waves. 12. The purification method according to claim 1, wherein cells producing the polypeptide are disrupted by an enzyme. 13. The enzyme is lysozyme or β-1,3.
The purification method according to claim 12, which is -glucan laminaripentaohydrolase. 14. A method for purifying a polypeptide, comprising removing the polypeptide obtained by purification by the method according to claim 1 by selective cleavage. 15. The purification method according to claim 12, wherein a protease is used for selective cleavage of the polypeptide obtained by purification by the method according to any one of claims 1 to 13. 16. The method according to claim 16, wherein the protease is thrombin, Factor X
The purification method according to claim 14 or 15, wherein a is at least one member selected from the group consisting of enterokinase. 17. A magnetic carrier having a ligand that binds to a target polypeptide, a cell disruption / adsorption solution, a washing solution used for removing unnecessary substances, and a method for separating the target polypeptide from the carrier. The kit reagent used in the purification method according to any one of claims 1 to 16, comprising an eluate. 18. The kit reagent according to claim 17, wherein at least one of the cell crushing / adsorbing solution and the washing solution has a pH of 6 to 8. 19. The kit reagent according to claim 17, wherein the cell disruption / adsorption solution and the washing solution have the same composition. 20. The kit reagent according to any one of claims 17 to 19, wherein a substance that antagonizes specific adsorption of the target polypeptide to the magnetic carrier is added to the eluate. 21. The kit reagent according to claim 20, wherein the substance that antagonizes the specific adsorption of the target polypeptide to the magnetic carrier is imidazole. 22. The kit reagent according to claim 20, wherein the substance that antagonizes the specific adsorption of the target polypeptide to the magnetic carrier is cellobiose. 23. The kit reagent according to claim 17, wherein the cell disruption / adsorption solution, the washing solution and the eluate contain an alkali metal salt. 24. An alkali metal salt comprising NaCl or K
The kit reagent according to claim 23, which is Cl.