JPH03219890A - Method for purifying adenylate kinase-fused peptide - Google Patents

Abstract

PURPOSE:To enable to prepare the subject peptide in high purity, yield and amount by dissolving a specific crude product in the aqueous solution of guanidine chloride or urea. CONSTITUTION:A DNA comprising an amino acid sequence coding a portion (or all portions) of the amino acid sequence of adenylate kinase (or an analogue thereof) is combined with a DNA comprising an amino acid sequence of a peptide to be fused to prepare a manifestation vector (A). The component A is introduced into an microorganism or cell, which is cultured to manifest an adenylate kinase-fused peptide(AKP). The cultured microorganisms or cells are crushed and subsequently subjected to a solid-liquid separation process to recover a solid content comprising a crude product (B) containing AKP. The component B is dissolved in a 10-100mM tris hydrochloric acid buffer solution (pH: 7.5-8.5) containing 4-6M guanidine hydrochloride or 3-7M urea and cysteine, etc., to prepare a solution (C). The solution C is dialyzed with a collodion membrane, etc., to reduce the concentration of urea, etc., to 0.1-10M, and the deposited solid content is separated and removed. The remained solution is purified by a chromatography method, etc., to produce the adenylate kinase- fused peptide.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing a peptide. In particular, the present invention relates to a crude product containing the adenylate kinase fusion peptide, such as a method for recovering and purifying the adenylate kinase fusion peptide from a culture in which the adenylate kinase fusion peptide is expressed.

[Prior Art] When producing a heterologous peptide using E. coli by genetic recombination means, etc., the expressed peptide is degraded by the protease of E. coli, so the desired peptide is often not obtained. There is. The present inventor discovered and proposed that such a peptide can be produced by expressing it in a fused state with adenylate kinase (Japanese Patent Application No. 1-207200,
1-271250).

Conventionally, peptides expressed by such methods form insoluble peptides in E. coli, so after disrupting the bacterial cells,
Soluble proteins were separated by centrifugation, solid content was collected, dissolved in guanidine hydrochloride, urea, etc., and purified using means such as gel filtration and ion exchange. However, this method has problems such as poor purification efficiency and difficulty in processing large quantities.

[Problems to be Solved by the Invention] The present invention aims to solve the above-mentioned problems, and an object of the present invention is to propose a method for purifying adenylate kinase fusion peptide in large quantities and with high yield. In particular, this makes it possible to produce useful polypeptides with high purity and in large quantities.

[Means for Solving the Problems] In order to solve the above problems, the present inventors have conducted extensive research and have surprisingly found that the above adenylate kinase fusion peptide can be dissolved once in a guanidine hydrochloride or urea solution. It was then discovered that even if the concentration of guanidine hydrochloride or urea was lowered, it would not precipitate as a solid, it could be easily separated from other peptides, and the fusion peptide could be highly concentrated.

The present invention is based on this finding, that is, when an adenylate kinase fusion peptide is dissolved in guanidine hydrochloride or urea and the concentration of the guanidine hydrochloride or urea in this solution is reduced, the adenylate kinase fusion peptide exists in a solution state. This was based on the knowledge that other peptides precipitate as solids, and that the two can be efficiently separated using this difference in properties.

That is, the present invention involves dissolving a crude product containing an adenylate kinase fusion peptide in an aqueous solution of guanidine hydrochloride or urea, reducing the concentration of guanidine hydrochloride or urea in this solution, and separating and removing the precipitated solid content. , a method for purifying an adenylate kinase fusion peptide comprising collecting a solution.

In the present invention, a culture expressing an adenylate kinase fusion peptide is used as the crude product containing the adenylate kinase fusion peptide. In particular, an expression vector in which DNA consisting of a base sequence encoding part or all of the amino acid sequence of adenylate kinase or its analogue is ligated to DNA consisting of the amino acid sequence of the peptide to be fused is used in microorganisms or The adenylate kinase fusion peptide is introduced into cells, the resulting microorganisms or cells are disrupted, and the solid content is recovered by solid-liquid separation.

It is desirable to use Escherichia coli as such a microorganism or cell. Furthermore, the method of the present invention is preferably used to purify an adenylate kinase fusion peptide in which the peptide to be fused is hirudin or an analog thereof.

The above-mentioned adenylate kinase is an enzyme that transfers the terminal phosphate group of adenylic acid to the hydroxyl group, carboxyl group, phosphate group, etc. of a compound that becomes an acceptor, and catalyzes phosphate compounds. or contained in yeast, plants, bacteria, etc. The amino acid sequence of this enzyme is widely known and consists of 194 amino acids. All of these adenylate kinases can be used in the present invention. This is because all of these enzymes are insoluble in E. coli when expressed in E. coli, and furthermore, when dissolved in a solution such as guanidine hydrochloride or urea, this adenylate kinase assumes its original three-dimensional structure. This is because they have a common property that they do not precipitate even if the concentration of guanidine hydrochloride, urea, etc. is lowered.

In the present invention, it is not necessary to use the entire amino acid sequence of this adenylate kinase, and it goes without saying that fragments thereof can also be applied to the present invention as long as they retain the above properties. This property is completely retained in any peptide consisting of amino acids up to 65 from the amino terminus, and can be used satisfactorily if the peptide consists of at least 65 amino acids from the amino terminus. Moreover, it goes without saying that analogs of these can be used as long as they have the above properties.

On the other hand, the peptide to be fused is a peptide that has the desired physiological activity, and although there is no particular restriction, the expressed peptide is degraded by the protease of microorganisms such as E. coli or cells. For example, hirudin and cell growth factor (TGF-α) that cannot be produced.
) etc. are preferably used.

An expression vector in which a DNA consisting of a base sequence encoding part or all of the amino acid sequence of the adenylate kinase of the present invention or its analogue is linked to a DNA consisting of the amino acid sequence of the peptide to be fused is introduced into Escherichia coli. A specific method for expressing a denylate kinase fusion peptide is described in Japanese Patent Application No. 1-2011, in which pig adenylate kinase is used as the adenylate kinase and hirudin or TGF-α is used as the fused peptide.
It is described in detail in No. 7200 and No. 1-271250.

Specifically, Japanese Patent Application No. 1-207200 contains a fragment of the tac promoter or trp promoter, a fragment of the origin of replication (Ori) of the commercially available plasmid pUc18, and a fragment encoding part or all of the amino acid sequence of porcine adenylate kinase. D consisting of the base sequence
A DNA comprising a base sequence encoding the amino acid sequence of NA and hirudin or an analog thereof, and in which both of the above DNAs are linked to part or all of the amino acid sequence of porcine adenylate kinase and hirudin or an analog thereof. A method is described in which E. coli is transduced with a hirudin expression vector designed to express a fusion protein with hirudin, the E. coli is cultured, the hirudin fusion protein is expressed, and the hirudin fusion protein is collected.

Furthermore, Japanese Patent Application No. 1-271250 describes a fragment of the tac promoter, a fragment of the origin of replication (Ori) of the commercially available plasmid pUc18, and a nucleotide sequence encoding part or all of the amino acid sequence of porcine adenylate kinase. and a DNA consisting of a base sequence encoding the amino acid sequence of TGF-α, and both DNAs are linked, and a part or all of the amino acid sequence of porcine adenylate kinase and a DNA consisting of a base sequence encoding the amino acid sequence of TGF-α are combined.
A method is described in which E. coli is transduced with a TGF-α expression vector designed to express a fusion protein with TGF-α, the E. coli is cultured, the TGF-α fusion protein is expressed, and the TGF-α fusion protein is collected.

In the present invention, the E. coli etc. cultured in this manner are crushed using a crusher in the culture solution as is. This crushing can be performed by conventional means such as a homogenizer, ultrasonic crushing, or French press. This fracture has an optical density of 60
It is preferable to carry out until the reduction is ~90%. in this case,
The adenylate kinase fusion peptide is present in solid form. Therefore, the crushed bacteria are subjected to solid-liquid separation to remove soluble peptides and collect the solid content containing the adenylate kinase fusion peptide. This solid-liquid separation is facilitated by using means such as centrifugation or filtration. The collected solids are washed and dissolved in 10-100 mM Tris-HCl buffer (pH 7.5-8.5) containing 4-6M guanidine hydrochloride or 3-7M urea. In this case, dithiothretol, β-mercaptoethanol,
Addition of a reducing agent such as glutathione or cysteine facilitates dissolution. To this solution, add 5 to 2
It is preferable to dissolve it so that it becomes 0% by weight. Next, the concentration of guanidine hydrochloride or urea in this solution is reduced. As a method for reducing this concentration, dialysis, dilution, etc. are simple and preferred. The dialysis method is preferably carried out against water using a semipermeable membrane such as cellulose, collodion membrane, or hollow fiber. In addition, in the case of dilution, it is preferable to use water, Tris-HCl buffer, or the like. This concentration reduction is 0.1 to 1. It is best to do this until the OM concentration is reached. As a result, most of the peptide precipitates as a solid, but the adenylate kinase fusion peptide exists in a dissolved state. Separate this into solid and liquid, collect the solution, and use cyanozene bromide etc.
The adenylate kinase fusion peptide is cleaved and separated into adenylate kinase and the fused peptide. The obtained fusion peptide is treated with dithiothretol, β-
The desired peptide can be obtained by folding with mercaptoethanol, glutathione, cysteine, etc., and purifying with ion exchange chromatography, gel filtration chromatography, or high performance liquid chromatography.

[Example] A more preferred embodiment of the present invention will be described using an example in which hirudin is used.

E. coli transformed with plasmid pMTAKHV1 was obtained by the following method described in Japanese Patent Application No. 1-207200.

That is, the P tac site region obtained by cutting commercially available plasmid pKK223-3 (manufactured by Pharmacia) with restriction enzymes Pvu I and Nru I, and the replication origin (
Ori) and the site containing the ampicillin resistance gene were joined using 14 ligase. This was introduced into E. coli strain JM109, cultured, and screened for ampicillin resistance to obtain a vector having the tac promoter and the origin of replication (Ori) of pUc18. This plasmid was named pMK2. This plasmid pliK21
0 μg was digested with 30 units of EcoRI and Eco47III to remove the fragment containing the tac promoter, and the fragment containing the replication origin was collected by agarose gel electrophoresis.

On the other hand, the base sequence of the trp promoter was synthesized using a DNA synthesizer. This was purified by high performance liquid chromatography using a reverse phase C18 column, and each
were annealed to obtain double-stranded DNA. This fragment and the aforementioned pMK2 were digested with EcoRI and Eco47n, and the fragments were reacted overnight at 16°C with T4 DNA ligase. This was used to transform E. coli JM109 strain,
trp promoter and pMK2 replication origin (Ori)
A vector with the following was prepared. The base sequence of this plasmid was confirmed by the method of Sanger et al. and named pMT1. 11 μg of this plasmid pMT was digested with 10 units of restriction enzymes EcoRI and Hindm, and a portion of the vector was recovered by agarose gel electrophoresis. On the other hand, pMA
KHVI 1oμg 43o units of EcoRI and Hin
After digestion with dIII, the gene fragment encoding the fusion protein was recovered from the gel. This fragment and Eco of the above pMT1
The fragment digested with RI and EcoR47m was converted into T4 DN
The mixture was reacted with A ligase at 16°C overnight. Escherichia coli strain JM109 was transformed using this to obtain a bacterial strain containing the desired recombinant plasmid pMTAKHV1. This bacterial species has accession number FERM BP No. 2538 (FERM BP
-2538) and has been deposited with the National Institute of Microbial Technology.

Escherichia coli transformed with the plasmid pMTAKHV 1 obtained by the method of Japanese Patent Application No. 1-207200 [accession number FERM BP-2538]
2538)] containing 50 μg/ml ampicillin.
9 medium (glucose 5g/l, casamino acids 5g/l,
NaJPO46g/l, KH2P0゜3g/l, Na
C10,5g/l, NH4Cl 0.5g/l, thiamine hydrochloride 10mg/l, Mg5O,io1g/l, Ca
Cl2100. cz g/l, tryptophan 50mg
/l, pH 7,2) and cultured overnight at 37°C.

Fusion peptide times.

After culturing, the culture solution was heated at 5-10℃ and 450-50℃.
0kg/crrr, feeding speed 200~300m1/m
homogenizer (Manton Gau
(manufactured by Lin Laboratory) about 3 times to disrupt the E. coli, and solid-liquid separation is performed using a centrifuge.

The obtained solid content is redispersed in a 1% Triton X-100 aqueous solution 101 containing 1 mM ethylenediaminetetraacetic acid, washed, and centrifuged to obtain a pellet. This washed pellet was dispersed in an aqueous solution (pH 8.5) containing 6M guanidine hydrochloride or urea, 50mM Tris-HCl and 1mM ethylenediaminetetraacetic acid, and β-mercaptoethanol was added to this to a concentration of 0.1M. The mixture was stirred at room temperature for 1 hour to carry out the reduction. Add this to water or 50
Dilute the guanidine hydrochloride or urea to 0.5M with mM Tris-HCl buffer (pH 7,0), or
Alternatively, place it in a semi-permeable membrane, use 200I water as the target solution, exchange this twice and dialyze for about 18 hours until the concentration of guanidine hydrochloride or urea is 0.3M, and centrifuge the dialyzed solution. , the precipitated solid content is removed, and the solution bone is concentrated using hollow fibers to a concentration of 21 or less.

Hirudin cycle 1 To the above concentrated solution, add formic acid to a final concentration of 70% to make a 5-101 aqueous solution, and to this, add 1 of the fusion peptide.
/ Add about 2 amounts of cyanocene bromide and heat at room temperature for 12 hours.
Incubate for ~18 hours. This solution was concentrated to dryness at 40°C using an evaporator, and an aqueous solution (pH 8, 5) containing 6M urea, 50rnM Tris-HCl and 1mM ethylenediaminetetraacetic acid was added to this to give a peptide concentration of 10mg10+1. In addition to this,
Add β-mercaptoethanol to 0.1M. This was mixed with an aqueous solution containing 10 to 20 times the amount of 50 mM Tris-HCl and 1 mM ethylenediaminetetraacetic acid (pH 8).
, 5) at room temperature for 12 to 18 hours. Furthermore, 200-300! Dialysis is performed using water as the target solution at 4° C., replacing the water 3 to 4 times, and taking 6 to 12 hours each time. The solution after dialysis was centrifuged to remove the precipitated solid matter, and then added to a 20mM Tris-HCl buffer (pH 7,5) using a column packed with DEAE Sephacel (manufactured by Pharmacia).
) is added with sodium chloride to obtain a developing solution, and the solution is eluted and separated by anion exchange chromatography while adjusting the sodium chloride concentration from 0.1 to 0.4M. A fraction containing hirudin is taken and freeze-dried.

Dissolve this in water and use a C18 reverse phase HPLC (manufactured by Vydac) packed column to gradate acetonitrile from 15 to 30% in a 0.065% trifluoroacetic acid aqueous solution, or
．． The sample is eluted and separated by high performance liquid chromatography using a 065% trifluoroacetic acid aqueous solution.

(Experiment example) Plasmid pM obtained by the method of Japanese Patent Application No. 1-207200
Escherichia coli transformed with TAKHV 1 [Accession number FERM BP"2538] was added to M9 medium 15 containing 50 pg/ml ampicillin.
1 and cultured overnight at 37°C.

After culturing, this culture solution was heated at 10℃ and 450 kg/cr.
rl' and a feed rate of 300 ml/min, the E. coli was crushed by passing it through a homogenizer three times, and solid-liquid separation was performed using a centrifuge. The obtained solid content was redispersed in 1% Triton X-100 aqueous solution 101 containing 1 mM ethylenediaminetetraacetic acid, washed, and centrifuged to obtain pellets.

Repeat the above operation to obtain 5 types of pellets, each with a peptide concentration of 10 to 15 g/l.
M guanidine hydrochloride (Experiments Nos. 1 to 3) or 6 M urea (Experiments Nos. 4 to 5), 50 mM Tris-HCl and 1
An aqueous solution containing mM ethylenediaminetetraacetic acid (pH 8.5
) 121 and dissolved therein. Of these, three types of guanidine hydrochloride solutions and one type of urea solution (experiment N04) were placed in a semipermeable membrane, 2001 water was used as the target liquid, the water was exchanged twice and dialyzed, and the dialyzed liquid was centrifuged. The supernatant was collected. Also,
The other urea solution (experiment N05) was diluted 12 times with water, centrifuged, and the supernatant was collected.

The fusion peptide in the pellet and supernatant was analyzed by 17.5% 5DS-polyacrylamide gel electrophoresis, measured using a densitometer, and quantified. The results and recovery rates are shown in Table 1.

Table 1 (Comparative Experimental Example) Regarding pellet No. 3 of the above experimental example, an aqueous solution containing 6M guanidine hydrochloride, 50mM Tris-hydrochloric acid, and 1mM ethylenediaminetetraacetic acid (pH 8, 5) Perform gel filtration of the solution dispersed and dissolved in 121 using a column packed with Sephacryl S-200 (manufactured by Pharmacia) to obtain a fraction containing the fusion peptide. The amount of fusion peptide inside was measured and the recovery rate was determined. As a result, when the concentration of the fusion peptide in the fraction was increased to 42%, the recovery rate was only 36%, and conversely, the recovery rate was 83%.
%, the concentration of the fusion peptide was found to be only 25%.

[Effects of the Invention] The present invention provides that once an adenylate kinase fusion peptide is dissolved in an aqueous solution of guanidine hydrochloride or urea, and when the concentration of guanidine hydrochloride or urea in this solution is reduced, it does not precipitate and can maintain a solution state. On the other hand, other peptides precipitate as their concentrations decrease, and this difference in the properties of both peptides is used to separate them.

In the present invention, the adenylate kinase fusion peptide can be purified and collected with high purity and good yield by separating the two by taking advantage of the difference in their properties. In the method of the present invention, when a heterologous peptide such as hirudin is expressed in E. coli etc. by genetic recombination means, the peptide is prevented from being degraded by the flotease of E. coli etc., and the peptide is efficiently digested. It has the effect of being able to be purified and collected.

Claims (3)

[Claims] (1) Dissolve the crude product containing the adenylate kinase fusion peptide in an aqueous solution of guanidine hydrochloride or urea, reduce the concentration of guanidine hydrochloride or urea in this solution, separate and remove the precipitated solid content, and dissolve the solution. A method for purifying an adenylate kinase fusion peptide, the method comprising: collecting an adenylate kinase fusion peptide. (2) The crude product containing the adenylate kinase fusion peptide is a DNA consisting of the amino acid sequence of the peptide to be fused to a DNA consisting of a base sequence encoding part or all of the amino acid sequence of adenylate kinase or its analogue. Introducing the expression vector linked with into microorganisms or cells to express the adenylate kinase fusion peptide,
The method for purifying an adenylate kinase fusion peptide according to claim 1, wherein the obtained microorganism or cells are crushed and solid-liquid separation is performed to recover the solid content. (3) The method for purifying an adenylate kinase fusion peptide according to claim (1) or (2), wherein the adenylate kinase fusion peptide is a peptide in which adenylate kinase and hirudin or an analog thereof are fused.