JPS61257931A - Recovery of interleukin 2 polypeptide - Google Patents

Abstract

PURPOSE:To recover the titled substance from an aqueous solution of guanidine in high recovery, by contacting lysozyme with microbial cell accumulating interleukin 2 polypeptide in the cell in granular form, disintegrating the cell by ultrasonic vibration and precipitating the objective substance by gravitational force. CONSTITUTION:Microbial cell (E.coli pT9-11/HB101) accumulating interleukin 2 (IL-2) polypeptide in the cell is made to contact with lysozyme and disintegrated by ultrasonic vibration. The disintegrated product is subjected to gravitational field, the precipitate is collected and exposed to a weak oxidizing condition in a 1-6M aqueous solution of guanidine, and the IL-2 polypeptide dissolved in the aqueous solution is separated. A soluble IL-2 can be recovered in high activity and recovery as a polypeptide having IL-2 activity and essentially free of foreign materials. The aqueous solution of guanidine is preferably maintained at 6-10pH and 5-45 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for recovering interleukin-2 (IL-2), and more particularly, to a method for recovering interleukin-2 (IL-2), and more specifically, it relates to a method for recovering interleukin-2 (IL-2). Regarding how to recover.

IL-2 is a type of lymphokine that acts as a -Ta cell growth factor, and is expected to be used as a medicine.

BACKGROUND ART As one of the methods for producing IL-2, a method using microorganisms created by recombinant DNA technology is known (European Patent Application Publication No. 0091539). When attempting to produce IL-2i using such microorganisms, IL-2,
f? Polypeptides are often accumulated in granular form within microbial cells. Conventionally, cumbersome steps were required to solubilize the IL-2 polypeptide accumulated in granules and recover it as a polypeptide with II,-2 activity, and the specific activity of the resulting polypeptide was also low. Furthermore, the polypeptide recovery rate was high and Fi was not achieved.

In order to collect the proteins accumulated in microbial cells in the form of granules, the microbial cells are first brought into contact with lysozyme, then the cells are crushed by ultrasound, and the protein granules are separated by centrifugation. (For example, for bovine growth hormone, Biotenology
)+151-154.See February 1985). It is also known to solubilize the obtained protein granules with guanidine hydrochloride etc. (for example,
For interferon, U.S. Patent No. 4,476,0
(See No. 49). Furthermore, it is also known that two thiol residues in general soluble protein molecules are oxidized with all glutathione to form a disulfide bond (for example, Biochemistry 9
(1970) 5015-5022).

However, IL accumulated in granular form within microbial cells
Regarding the -2 polypeptide, it is not known what kind of recovery method is suitable for obtaining it with a higher recovery rate than all polypeptides with higher specific activity.

In particular, IL-2 polypeptide is less soluble in water than other proteins. In addition, when a granular polypeptide is solubilized, three thiol residues are generated in the molecule, and such polypeptide does not have IL-2 activity. Therefore, it is considered necessary to form a cydisulfide bond between these three thiol residues, but the disulfide bond must be formed selectively between any two thiol residues. From these points, it is expected that recovery of IL-2 polypeptide will be more difficult than conventional proteins accumulated in granular form.

Problems to be Solved by the Invention Therefore, an object of the present invention is to convert IL-2 polypeptides accumulated in microbial cells into granules into polypeptides having soluble IL-2 activity with high specific activity. The goal is to find a way to recover the waste with a higher recovery rate.

Means to Solve the Problems Under such circumstances, the present inventors have discovered that microbial cells that accumulate IL-2 polybezotide in granular form,
After contacting with lysozyme, the cells are disrupted by ultrasonication, then the crushed material is collected in a gravity field to collect the precipitate, the precipitate is placed in a 1M to 6M guanidine aqueous solution, and this is placed under weak oxidative conditions, Finally, to collect the Ru2/ripeptide dissolved in the guanidine aqueous solution,
It has been found that polypeptides with high specific activity can be obtained with a high recovery rate.

A microorganism capable of producing IL-2 created by recombinant DNA technology and a method for culturing the microorganism to produce IL-2 polypeptide are disclosed in European Patent Publication No. 009.
No. 1539.

The method of bringing the IL-2 polypeptide into contact with the microbial cell lysozyme accumulated in the form of granules includes the method of contacting the microbial cells with p? Suspend in 16 to 8 hypotonic buffer or culture solution, add lysozyme t-2,500 to so, ooo units/lnt, and keep preferably in the range of 0 to 30°C for usually 30 minutes or more. Bye. 0 in suspension
．． Favorable results may be obtained with additions of 1 to 100 mμ of EDTA t-.

Cells contacted with lysozyme are disrupted by ultrasound at 9 to 25 kHz. As for the device, it is sufficient to use an ultrasonic generator that is normally used for disrupting and enlarging cells and tissues, and the horn that transmits ultrasonic waves to the sample can be either an immersion type or a cup type. . The ultrasonic output may be any value suitable for the ultrasonic generator used, but typically 20 W to 1 kW is used. Processing time depends on the amount of processing liquid.

It may be selected appropriately depending on the output, the horn used, etc. The crushed state of cells can be easily observed under a microscope. When proper disruption is performed, cellular components other than granules of IL-2 polypeptide (cell wall) are removed.

cell membranes, etc.) become minute fragments and do not retain their original shape. Furthermore, since ultrasonic treatment is accompanied by heat generation, cell disruption is usually carried out at as low a temperature as possible, 4° C., to prevent thermal denaturation of the suspension.

The IL-2 polypeptide granules obtained by crushing are placed in a gravity field and collected as a sediment. Centrifugation is IL-2
It can be carried out under conditions that allow the polypeptide granules to settle, but usually 2,000 to 30,000 OOX! i', preferably 10
,000X#, will be performed for about 5 minutes. Alternatively, density gradient centrifugation of sucrose or the like may be performed. The precipitate thus obtained usually contains 5 Oes or more of IL-2 polypeptide.◎ The precipitate consisting of the obtained granular IL-2 polypeptide is placed in a guanidine solution ranging from 1M to 6M. Preferably, it is suspended or dissolved to a concentration of o, oi to 0.211/l. When the guanidine solution is in the range of 4 to 6M, the recovery rate of IL-2 polypeptide may be higher if the granules are sufficiently dissolved and then diluted with water to a concentration of 1 to 4M. - of guanidine aqueous solution is 6 to 1
It is in the range of 0, more preferably in the range of 7 to 9. Further, the temperature of the aqueous guanidine solution in which the granular IL-2 polypezotide is suspended or dissolved is preferably kept at a temperature in the range of 5°C to 45°C.

Guanisine is commonly used as the hydrochloride salt since guanidine hydrochloride is readily available, but it may be in other salts or in free form.

A 1 to 6 M aqueous solution of guanidine in which the granulated IL-2 polypezotide is suspended or dissolved is placed under mildly oxidizing conditions. To place it under weak oxidizing conditions, air or a gas containing oxygen may be bubbled through the guanidine aqueous solution, and glutathione, cysteine, etc.

Weak oxidizing agents such as mercaptoethanol, dithiothreitol, and other thiol compounds (and oxygen or other oxidizing agents) and their disulfides may be added to the aqueous solution. A weak oxidizing agent may be added to the guanidine aqueous solution at the same time as the granular IL-2 polypegated, but IL-2y
j? After the liputide has dissolved to some extent, especially from 4 to 6
M'guanidine may be added after diluting with water.

Weak oxidizing agents are used in large excess amounts, usually from 1 to 100
A concentration range of 10 to 20 mM is preferably used. When using a disulfide compound as an oxidizing agent, it is best to mix it with its thiol form.1For example, when using glutathione, reduced glutathione (GSB
) and oxidized glutathione (G55G), preferably 5
: Mixed in a ratio of 1 to 20:1. When only the reduced form is used, it is recommended to flow air through the guanidine aqueous solution.

Oxidation is continued until the peak of active IL-2 stops increasing, as determined by taking samples of one reaction solution at appropriate time intervals and analyzing them by reverse phase HPLC.

The IL-2 polypeptide thus dissolved in the aqueous guanidine solution is recovered in a conventional manner.

For example, a guanidine aqueous solution is desalted by gel filtration,
Subject to column chromatography using a cation exchange resin. By increasing the salt concentration, the adsorbed I
The L-2 polypeptide was eluted and the eluate was subjected to reverse phase HPL.
It is purified by direct loading onto C. In this way, IL-2 polybezotide substantially free of contaminants can be obtained.

Functions and Effects of the Invention According to the method for collecting IL-2 of the present invention, IL-2/IJ peptides with high specific activity can be obtained at a high recovery rate.

Example I Obtaining IL-2-producing microorganisms Escherichia coli that can accumulate IL-2 polypeptide in granular form within cells
) pT9-11/I (BIOI was obtained as follows (see Figure 1).

That is, pIL2-5OA (European Patent Application Publication No. 91539) containing the entire IL-2 cDNA, (Deposited Escherichia
Cori (Escher i ah i a co 11
) #1776/p IL2-5OA, AJ1199
6 jFERMBP-226)
Interleukin-2cD was cleaved with E-Pstl.
The NA fragment was obtained and further digested with DraI to obtain IL-2cD.
A-T, G- present in the 3' non-structural gene of the NA gene
A 530 base pair fragment was obtained by removing the approximately 300 base pair fragment containing the C homopolymer. And this fragment and B
amHI linker and Ps t I of pBR322
The larger EcoRI fragment (the EcoRI site was made blunt-ended with Flenow) was ligated with T4 DNA ligase, creating a BamH1 cleavage site approximately 50 base pairs downstream of the interleukin-2 structural gene after the nonstructural gene. A plasmid was constructed. This plasmid was then digested with I (giAl) and Hg i containing the IL-2 gene.
An Al fragment was obtained, which was treated with DNA & Limerase (Flenow) to remove the nucleotides in the single-stranded portion that protruded to the 3' side, resulting in a blunt end. And then add this fragment to B
Digestion with amHI yielded a fragment of about 450 base pairs.

Furthermore, pDR720 was used as a plasmid carrying the trp promoter. pDR720 is pKO-1
(Rus+se1. D, R, and Bennett,
G.N., Gene+ 20 +231 (1982)
) SmaI cleavage site 1ctrp promoter%.

It includes an operator fragment. pDR72
0 was digested with Hpal and BamHI, and the large Hpa
I-BamHI fragment was obtained, and a part of the trp promoter, the SD sequence AAGG, and the protein synthesis initiation codon A were obtained.
TG and a synthetic oligomer containing OCA encoding the N-terminal alanine of mature IL-24 ripeptide were ligated as shown in FIG. 1 to select pMI-9.

On the other hand, pTrpA was obtained by ligating the large Pvul and 5ail cleavage fragment of pBR322 with the synthesized DNA having the trpA terminator sequence. pM
I-9 and pTrpA together with EcoR'l and B amH
pMI-9 is a fragment containing the IL-2 gene, and pTrpA is a fragment containing the trpA terminator. The two are ligated to create pT9-
I got 11. Therefore, pT'9-11 was used as Escherichia coli HBOI (F-, hsds20 (rB-).

mB-), recA13. ara-14+ pr
oA2.1icY1. galK2+rpsL20(S
mr), xyl-5, m+4-1.5upE44. λ
-) introduced into L tepT9-11/'HBIOI t obtained fc.

Preparation of IL-2 polypeptide granules The cells were (2 Ticademinoic acid, 0.2 Ti yeast extract.

0,596 NH4Cl, 2% G#:r-su,
0.1% KH2PO4#0, 05% MgSO4
・7H20, 0,005SC&C12-2H20.

0.8 Da/1ttt L-Leucine, 0.8 Da/1ttt L-Proline.

Bq/l thiamine, 100 μl/- ampicillin.

25 μl/streptomycin) in a medium 30°C at 31°C while adjusting the temperature to 6.2 with ammonia.
The cells were cultured under aeration for 13 hours. in the middle.

660nm 0. When D reaches approximately 5.0.

Add 25 μl/- of 3-indoleacrylic acid.

The IL-2 gene was expressed.

Thereafter, the cells were collected and suspended in 20 mM lithium-hydrochloric acid buffer (pH 7.5) to which 50 mM EDTA was added to give a wet weight of 0.02.9/-, and then suspended to a wet weight of 0.02. lysozyme (Department of Biochemical Industries, egg white, specific activity>5
0.0 OOU/1 mg) and kept at 10°C for 30 minutes.

Next, 20 d of these were subjected to ultrasonication at 4°C, 50 W, for 10 minutes (using Otake Seisakusho 5onicator) to disrupt the cells, and centrifuged at 12,0 OOX for 9,5 minutes to collect IL-2 polyligtide granules. did.

Recovery of active IL-2 polypeptide Obtained IL-2yj? The lJ peptide granules were dissolved in 6M guanidine hydrochloride (Gun-H('t) 20-), and the amount of IL-2/ripegutide was measured by liquid chromatography.Meanwhile, the cells after completion of culture were treated with lysozyme, 6MGun-H directly without cell disruption using sound waves
A C2K solubilized product was prepared, and the amount of IL-2 polypeptide was measured by liquid chromatography. These results are shown in Table 1.

Table 1 The obtained pellet containing IL-2 poly-eftide.

An IL-2 concentration of 0 was added to a 0.1 M) Lis-HCl buffer (pH 8.0, hereinafter abbreviated as A) containing 6 M guanidine hydrochloride.
．． 3119/-. Then 0.1
M) Diluted 3 times with Lis-HCl buffer (pH 8.0, hereinafter abbreviated as B), added GSH and G55G to final concentrations of 10 mM and 1 nM, respectively, and then diluted with dilute caustic soda. The pH was adjusted to 8.

After standing still at room temperature for 12 hours, the reaction solution was subjected to column chromatography using Sephadex G-25 equilibrated with 0.05 M acetic acid buffer (pH 5.0) to remove low molecules. After passing the solution through a column packed with CM-Seph7dex C-25 equilibrated with 0.05 M sodium acetate buffer (pH 5,0) and adsorbing IL-2, 0.5 M sodium acetate buffer was added. (pH 5
,0) at.

IL-2 was eluted.

The obtained IL-2 polyligtide fraction was subjected to reverse phase HPLC (using the method described in JP-A-59-225195), and the IL-2 fraction was separated. Obtained IL
-2 polypezotide showed a specific activity of 4.8 x 10'U/■ in an activity assay using a cytotoxic T-phosphorus lumbar strain. In addition, the peptide map method (Cys-5
It was found to be between Cys-8 and Cys-105.

Moreover, the recovery rate from IL-2 polypezotide contained in the pellet was 86.

Example 2 When diluting the pellet solubilized with Buffer A described in Example 1 with Buffer B, the repeptide concentration and guanidine hydrochloride concentration relative to the pellet amount were adjusted to the values shown in Table 1. A solution was prepared.

This was done in the same manner as described in Example 1.

Treated with GSH and assa, and directly subjected the treated solution to reverse phase HPLC
The conversion rate of the active form of IL-2, that is, IL-2 having a disulfide bond between Cys-58Cys-105, with respect to total IL-2 poly2ftide was determined.

The results are shown in Table 2. ◎ Table 2 Conversion rate of IL-2 polypeptide to active form (H) Example 3 The pellet containing IL-2 polypeptide obtained by the method described in Example 1 was dissolved in a buffer solution. A VCrL-2 concentration 0
．． 311/-, then diluted with buffer B
Diluted twice. The solution was stirred for 12 hours while gently bubbling air. Thereafter, the method described in Example 2 was followed by desalting, ion exchange chromatography, and reverse phase H
An IL-2 fraction was obtained through a PLC process. Obtained IL
-2 exhibited a specific activity of 4.9X10 U/ap, and
What is the disulfide bond position?

It was confirmed that it was between Cy@-58-Cys-105. The recovery rate from IL-2 polypezotide contained in the pellets was 72%.

Example 4 IL-2yR obtained by the method described in Example 1
3M guanidine hydrochloride,
0.1 containing 10mM GSH, 1mM G55G
M Suspended in Tris-HCl buffer (pH 8,0) at room temperature.

Stir gently for 24 hours. After removing the insoluble fraction by centrifugation, desalting, ion exchange chromatography, and reverse phase HPLC were performed by the method described in Example 2.
IL-2 fraction was obtained. The obtained IL-2 was 48X1
0 U/immediate specific activity, disulfide bond is changed to Cys
-58-105. Furthermore, the recovery rate from the IL-2 polypeptide contained in -(left) was 65%.

Example 5 The pellet containing the IL-2 polypeptide obtained by the method described in Example 1 was dissolved in
．． The suspension was suspended in 1 M) lithium-hydrochloric acid buffer (pH 8,0) and stirred for 24 hours at room temperature while bubbling with air. After removing the insoluble fraction by centrifugation.

Desalting, ion exchange chromatography, and reverse phase HPLC were performed according to the method described in Example 2 to obtain an IL-2 fraction. The obtained IL-2 exhibited a specific activity of 5.0×10 U/da and had a disulfide bond between Cys-58 and 105. Furthermore, the recovery rate from IL-2 polypeptide P contained in the pellet was 52, which is impressive.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram of the acquisition progress of interleukin-2 producing microorganisms.

Claims (1)

[Claims] The first step is to contact lysozyme with microbial cells that have accumulated interleukin-2 polypeptide in the form of granules, the second step is to crush the cells that have been contacted with lysozyme using ultrasound, and the crushed product is placed in a gravitational field. The third step is to collect the precipitate, the fourth step is to place the precipitate in a 1M to 6M guanidine aqueous solution under weak oxidizing conditions, and the fifth step is to collect the interleukin-2 polypeptide dissolved in the guanidine aqueous solution. Method for collecting interleukin 2 polypeptide.