JPH02425A - Cultivation of recombinant protein manifesting cell - Google Patents

Abstract

PURPOSE:To limit an amount of oxygen dissolved in a culture medium to <=a given value and to selective obtain polypeptide free from N-end methionine in cultivating a host cell transformed with a directly manifesting vector integrated with gene of useful polypeptide. CONSTITUTION:A host cell such as Escherichia coli transformed with a directly manifesting vector integrated with a gene of useful polypeptide such as interleukin 1 (IL-1) is cultivated under the following condition. Namely, the culture is carried out by limiting the amount of oxygen dissolved in a culture medium comprising various kinds of synthetic mediums and natural mediums to <=about 20% saturated concentration. The culture is preferably carried out under conditions suitable for the above-mentioned transformant, usually at pH5.5-8.5 at 18-40 deg.C by aerated spinner culture. A desired polypeptide from which methionine (MET) corresponding to initiation codon of manifestation protein is removed is obtained by the culture.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention provides a new method for culturing recombinant protein-expressing cells, in particular, by controlling the amount of dissolved oxygen in the medium during culture of the cells, a translation initiation codon is added to the amine terminus of the cell. The present invention relates to a new method for culturing the above-mentioned cells, which can selectively obtain useful polypeptides that do not have a methionine residue (hereinafter referred to as Met) corresponding to .

Conventional technology In recent years, advances in the field of biochemistry, especially genetic recombination technology, have been remarkable, and there are many genetic engineering methods that can obtain large quantities of physiologically active substances such as lymphokines, which were previously available in trace amounts. It is being researched and developed.

Among them, the gene of a particularly useful polypeptide is inserted into a microbial vector such as E. coli, and the gene is replicated within the microbial cell.
The technique of producing a large amount of a desired useful polypeptide by transcription, translation, and expression is known as the most typical and advantageous method.

In the production of useful polypeptides by such genetic engineering techniques, translation of the polypeptides is carried out by Me, regardless of whether the host cells used are eukaryotic or prokaryotic.
Starts from the translation start codon (A-D-G) corresponding to t,
Therefore, the amine terminal <N-terminus> of the polypeptide produced
becomes Met.

This N-terminal Met is known to be cleaved in the host cell's intracellular aminobebutidase cell in the case of polypeptides etc. naturally produced by microbial cells [Molecular Biology of Genes]
(above) 3rd edition, written by J, D, Shocho SON, translated by Kinichi Miura et al., 375 pages (1977)].

However, in the case of cells transformed with vectors containing foreign peptides, such as interferon-α (IFN-α), growth factor (GH), interleukin-2 (IL-2), etc., When attempting to produce a large amount of useful polypeptide by culturing the host cell, the enzyme present in the host cell cannot fully exert its action, and the N-terminal M
A large amount of Iζ voripebudide is produced leaving et (Na
True.

293.408 (1981) etc.]. Thus, polypeptides with such N-terminal Met, e.g.
1α, ll-1β is known to have lower biological activity than the natural type, that is, the polypeptide that does not have Met, and may have antigenicity as a 1171 substance different from the natural type. Polypeptides containing such substances may be subject to various restrictions when used as pharmaceuticals.

Recently, methionine aminopeptidase (ABEN-BAS
SAT et al., J. Bact., 169.
751-757 (1987); C, G, Hi
ller et al,,P,N,8.

S, Nakagawa et al, B
io, /Techno1. .

824 (1987)), etc.
A method of removing terminal Met has been proposed, but this method can only be applied to polypeptides with a specific sequence, and cannot be applied to a wide variety of polypeptides.Moreover, the enzyme itself is expensive, making it impractical for practical use. There are problems to be solved.

Problems to be Solved by the Invention In view of the above-mentioned current situation, the present inventors have proposed that the invention can be similarly applied to a wide variety of useful polypeptides regardless of the type of polypeptide, is easy to apply, and is suitable for industrial implementation. We have conducted extensive research with the aim of providing a means for removing the above N-terminus to 1et that is both economical and satisfactory. As a result, the production rate of the desired Met-free polypeptide depends on the amount of dissolved oxygen in the culture medium during host cell culture, and by appropriately controlling this amount of dissolved oxygen, it was found that the desired amount of N It has been found that polypeptides that are completely free of iMet can be selectively obtained.

The present invention was completed based on this knowledge.

Means for solving the problem, that is, the present invention is to reduce the amount of dissolved oxygen in the culture medium to approximately the normal concentration when culturing host cells transformed with a direct expression vector into which a gene for a useful polypeptide has been incorporated. N le corresponding to the start of the expressed protein, limited to 20?6 or less.
The present invention relates to a method for incubating and cultivating white fleas, which is characterized by iqing useful polypeptides from which t has been removed.

The useful polypeptide 1 to which the method of the present invention can be applied is not particularly limited in its type, and may be any of the various polypeptides whose amino acid sequence or the DNA sequence encoding the same has been conventionally determined. Examples of such substances include, for example, interleukin-1 (II-1>, interleukin-2 (IL-2)
, interleukin-3 (IL-3>, interleukin-4 (II-4), interleukin-5 (IL-5)
), interleukin-6 (IL-6>, tumor necrosis factor (TNF>), colony stimulating factor (MC3F, G~1-C
3F, G-C3F, etc.), Lymph 4 toxin (LT), and various other lymphoids.

In addition, various types of host cells known to be usable in this type of direct expression system, that is, a protein expression system that utilizes a vector in which a gene for a useful polypeptide is linked immediately after the translation initiation codon, such as Escherichia coli, can be used. , Bacillus subtilis, diplococcus pneumoniae, yeast, actinomycetes, etc. can all be used.

The method for transforming secondary host cells can be carried out in accordance with a publicly known method, and the expression vector used for producing the transformant, the gene encoding the useful polypeptide constituting the vector, and the expression of the gene. Origin vectors for incorporating various regulatory elements such as promoters, ribosome binding sites, transcription termination signals, etc., are known or can be easily prepared according to known methods, and various types of vectors used at that time are known. The methods and operations used in this type of genetic presynthesis technology can be adopted.

The method of the invention particularly provides recombinant IL-1α, IL-1β
It can be advantageously applied to the expression of derivatives such as and the like in E. coli.

Hereinafter, the present invention will be described in further detail by taking this direct expression system of IL-1 using Escherichia coli as an example, but the same can be applied to other cases as well.

The IL-1α gene or IL-1β gene used in the method of the present invention includes known natural IL-1α
and the amino acid sequence of IL-1β using conventional methods, such as the phosphite Trieste/L/method (Nature,
310, 105 (1984), etc., or by extraction and isolation from cells, etc., using general methods.

Moreover, each of the above-mentioned IL-1 genes has already been established, and these can also be used. Specific examples of the above genes are described, for example, in the following documents.

oNishida et al,,B,B,R,C1,
143,345oFrutani et al.
Nucleic Ac1d Res,, 1
3゜OEP Patent Publication No. 237967 @ OEP Patent Publication No. 237073 @ Various known promoters for expression of the above genes, such as λP1, λPR1trp, 1ac
Any promoter such as c, tufB, reCA, lpp etc. may be used, but the trp promoter is particularly advantageous.

Examples of direct expression vectors that have the above IL-1 gene and promoter and can be advantageously used in the present invention include those described in the above-mentioned documents as well as those described in the following documents.

oKikumoto et al., B.B.R.C.
, 147, 315 <1987> The secondary plasmid is introduced into E. coli as a host cell to transform the E. coli according to a conventional method.

The medium for culturing the transformant thus obtained may be any of various synthetic media, natural media, and other media commonly used for the growth of E. coli and the production of the desired polypeptide. Commonly used carbon sources include, for example, glucose, fructose, lactose, glycerol, mannitol, sorbitol, etc.; nitrogen sources include, for example, ammonium chloride [NH4cQ], fil ammonium [(NH<>2 SO4], casamino acids, yeast extract, Other nutritional sources include polypeptone, meat extract, batat tryptone, corn steep liquor, etc.2 HPO4, KH2PO4, NaCQ, MgSO4,
Examples include vitamin BT and rVIQ0922. When culturing E. coli using an expression vector having a t-pump promoter, it is preferable to use a medium having the following composition, for example.

Na2HPO& ・12H206CJ/QKH2PO4
3CJ/Q NaCQ O,5Q/QNH4C
Q 1CJ/(1!Bacto Casamino Acid 10!;]/Q Bacto Yeast Extract 0.5Q/QL-Cystine/HCQ
75mg/QL-Proline
75 m (1/QL-leucine
75 m (1/Q The above medium is 4N NaOH
After adjusting the DH to 7.4, autoclaving at 121 °C for 30 minutes or steam heat sterilization at 123 °C for 20 minutes, then adding the following ingredients aseptically at the time of inoculation. be done.

1 M Mg5Oa 67 H202mQ/QH
vl CaC92・2H200, 1mQ/Q7, 5m
g,/mQ Thiamine-HCQ 1 7/Q40% Glucose 18.75mQ10 The transformant is cultured using the above medium under conditions suitable for the growth of the transformant, usually in the range of pH 5.5 to 8.5, Temperature 18-40℃
It is preferable to carry out aerated agitation culture within this range.

In the present invention, the above culture is carried out to saturate the culture medium with dissolved oxygen.
It is important to limit the dissolved oxygen to about 20% or less, preferably about 0 to 15%, more preferably about 0 to 5%. The period when proteins begin to be expressed, that is, the logarithmic growth phase,
In particular, it may be carried out at the end of the logarithmic growth phase. This dissolved oxygen concentration can be controlled, for example, by appropriately controlling ventilation conditions and stirring conditions.
~ Can be carried out by rolling. The details are as shown in Examples below.

In this way, a desired useful polypeptide from which the Met corresponding to the initiation codon of the expressed protein has been removed can be obtained.

Confirmation of the production of the useful polypeptide 1 above is carried out according to a conventional method, such as enzyme immunoassay (CE IA method), etc. (q
Ru.

In addition, extraction operations for isolation and purification of useful polypeptides within bacteria can be carried out using conventional methods, such as ultrasonication and Laemmli's method using 5DS-buffer (Laemmeli, U.S.
, K., Nature, 227.680 (19
70)) etc. can be followed.

According to the method of the present invention, it is possible to selectively obtain a desired useful polypeptide from which Mei has been removed, but there is a risk that polypeptides having Met may be present in the mixture. 3P-5PW
(7,5x75mn+column, 1-Sor Co., Ltd.)) to measure the absorbance at 2801m of each fraction. This can be determined by measuring the N-terminus to 1et content using a method such as the following.

As described above, the desired useful polypeptide (q) can be purified according to various general processing operations utilizing the physical, chemical properties, etc. 1259, 1st edition No. 1
See Inkeisu, June 23, 1980, published by Tokyo Kagaku Dojin Co., Ltd.]. In particular, when extracting polypeptides for fj from host cells, it is recommended to use methods that employ mild conditions, such as osmotic shock methods (from the perspective of preserving higher-order polypeptides). Preferred.As for the above purification method, specific examples of the above purification method include, for example, using a conventional protein precipitant, ultrafiltration, molecular sieving, and molecular sieving.
Combinations of methods such as gel filtration, liquid chromatography, centrifugation, electrophoresis, affinity chromatography, dialysis, etc. can be employed.

More specifically, the above operation can be performed, for example, as follows. That is, first, a polypeptide of interest is partially purified in advance from a supernatant obtained by extraction from cells. This partial purification can be carried out using, for example, acetone, methanol, ethanol, propatool, dimedylformamide (DMF
Processing using organic solvents such as acetic acid, perchloric acid (to PC), trichloroacetic acid (dichloroacetic acid) as protein precipitants, ammonium sulfate, sodium bM acid, phosphoric acid, etc.
This is carried out by a treatment using a salting-out agent for thulium and/or by a method using a dialysis membrane, a flat plate membrane, a hollow fiber membrane, or the like. The operations and conditions for each of these treatments may be the same as those for ordinary methods of this type.

Next, the crude product obtained above is subjected to gel purification to obtain both components containing the activity of the target substance. There are no particular limitations on the gel filtration agent used here, such as dextran gel, polyacrylamide gel,
Various types of materials such as agarose gel, polyacrylamide-agarose gel, and cellulose can be used.

Specific examples include Sephadex G type and Sephadex L type.
H type, Ceph 10-type, Cephacryl type (
Methylmasia), Cellulofine (Chisso)
, Biogel P type, Biogel type (Bio-Rad)
, Ultrogel (LKB Co., Ltd.), TSK-G type (Toso Co., Ltd.), and other commercially available products.

The target polypeptide can be obtained by subjecting the active fraction obtained by the gel filtration to affinity chromatography using a hydroxyapatite column, ion exchange column chromatography such as the DEAE method, CM method, or SP method, or chromatofocusing method. , reverse phase high performance liquid chromatography, etc., or by a combination of these operations, it can be further purified and isolated as a homogeneous substance.

The above chromatofocusing method can be carried out by various known methods. For example, PBE94 (Methylmacia) can be used as the column, imidazole-hydrochloric acid, etc. can be used as the starting buffer, and polybuffer 74 (manufactured by Methylmacia)-hydrochloric acid (pH 4.0) can be used as the eluent.

The above-mentioned reversed-phase high performance liquid chromatography is performed using, for example, a C4 high-bore one-reversed-phase HPLC column (Bio-Rad Co., Ltd.
-Rad Laboratories) ) etc.,
Acetonitrile, trifluoroacetic acid (TF
A>, water and a mixed solvent such as these can be used. In this way, the desired Met-free polypeptide can be isolated and obtained.

The resulting polypeptide has the same physiological activity as the naturally occurring polypeptide of this type, and is suitable for various uses such as the same pharmaceuticals as the polypeptide.

Such pharmaceuticals are usually prepared and utilized in the form of a pharmaceutical composition by blending the polypeptide with a suitable pharmaceutical carrier. As the pharmaceutical carrier, any excipients or diluents such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc., which are commonly used in the preparation of pharmaceuticals according to the usage form, are used. can. The form of the pharmaceutical composition is not particularly limited as long as it effectively contains the polypeptide of the present invention.
For example, it may be in the form of a solid preparation such as a tablet, powder, granule, or emulsion, but it is usually preferable to take the form of an injection such as a liquid preparation, suspension, or emulsion. It can also be made into a dry product which can be made liquid by adding a suitable carrier before use. Both secondary formulation compositions can be prepared according to conventional methods.

The pharmaceutical preparation 1q can be administered by an appropriate administration route depending on the form of the pharmaceutical composition, for example, a pharmaceutical preparation in the form of an injection can be administered intravenously, intramuscularly, subcutaneously, intradermally, intraperitoneally,
Pharmaceutical preparations in solid form can be administered orally or enterally.

The amount of active ingredient in a pharmaceutical preparation and the dosage of the preparation are appropriately selected depending on the administration method, dosage form, purpose of use, and symptoms of the patient to whom the preparation is applied, and are not constant, but are usually effective. It is desirable to prepare a formulation containing about 1 to 80 mm% of the ingredient and administer this formulation in such a range that the amount of the active ingredient contained therein is about 0.1 μg to 10 mq per adult per day. . The administration need not be once per day, but can also be divided into 3 to 4 times per day.

EXAMPLES Hereinafter, the method of the present invention will be explained in more detail with reference to Examples. Note that the measurement of physiological activity is performed by the following method.

<Measurement of physiological activity> (1) Measurement of IL-1α activity Oppenheim et al. (J, J, 01) penheim
et at) method (J, Immunol, .
116.1466<1976>), 03H/H
e Expressed by LAF activity measured using thymocytes of J mouse.

(2>Measurement of IL-1β activity (GIF activity) 0.1 ml of diluted sample solution (2) at various concentrations was placed in a 96-well microplate (Corning), and then in each well
0.1 mf2 of Eagles N4 EM suspension containing 10% FC3 containing melanoma follicle A375 at a concentration of 2 x 10']/- is added and cultured for 4 days in a carbon dioxide incubator (manufactured by Nafco).

After the culture is completed, 0.05% Neutral Red (manufactured by Wako Shikuso) is added to each well and cultured at 37°C for 2 hours. After removing the supernatant, add 0.3 ml of phosphoric acid saline. Wash the wells by gently adding to each well. After removing the wash solution, add 1 to 1 liters of phosphoric acid to each well.
Add 0.1 mG of a mixture of equal parts of 100% aluminum and ethanol, shake for several minutes with a micromixer, and measure the amount of dye taken into the cells using a 96-L Microtie 1~Ration Prey I~ photometer ( Using Titer Check Multiscan (manufactured by Flora Laboratories), the absorbance was 540 mμ.
to determine the growth inhibitory activity. The reciprocal of the dilution rate of the test group showing 50% inhibition of cell proliferation of the control group, that is, the test group showing 1/2 of the measured absorbance of the control group, is calculated as the GIF activity. Unit. Therefore, for example, when this GIF activity is 10 units, this test solution still has the activity of inhibiting cell proliferation by 50% even if it is diluted 10 times.

Example 1 Human IL-1β direct expression vector p trpGIF
-1 Escherichia coli χl 776/p trp GIF-α:F
ERM BP949: EP Equity Publication No. 23796
E. coli strain H8101 (Esch
erichia coli HBIOI/ptrpGI
F-α: FERM BP-
2089)) - Platinum loop was added to LB medium 200 with the following composition.
m (1!) and cultured overnight at 37°C to obtain a culture solution.

[Formation of IB medium] Hakuto Tributone (Difco) 10a/Q Baku1~-Gose 1~Extract (Difco) 5(]/QNa
CQ (Wakosha) 10 (1/QDH
7,5 A predetermined amount of the above preculture solution was inoculated into a predetermined amount of a production medium having the composition shown below, and the aeration speed was varied using a jar fermenter under the conditions shown in Table 1 below. Production culture was carried out.

[Production medium composition] Na2HPOa ・12H206'J/QKH2Pot
3 tJ/Q\aCQ
0.5q/QNH, 1CQ1
Q/Q Baku1~-Casamino acids 10g/9 Bakuto yeast extract 0.5 (J/QL-Cystine・HC(1! 75mg/QL-Proline 75mg/9L-Leucine
After adjusting the DH to 7.4 with 75 mg/Q4N NaOH, autoclave at 121°C for 30 minutes or steam heat sterilize at 123°C for 20 minutes, and then aseptically inoculate the culture medium with the reduced bacterial solution of each of the following components. Added.

IM MQSO4・7H202mQ/Q1M Ca
CQ2 "2H200," ImQ/Q7, 51+1 (1
/1110 Thiamine-HCQ 1 mQ/940%
Glucose 18.75 mQ/Q During the culture, the oxygen concentration was monitored using a dissolved oxygen meter (manufactured by Marubishi Bio-Engineering Co., Ltd.). Q, 25vvm is the lowest 0% saturation (approximately 5~
0% saturation concentration), Q, 5vvm is the minimum 20% saturation amount (
approximately 25-20% saturation concentration).

Table 1 After completing the above production culture, test the bacterial solution 10 times at 10,000XΩ.
Centrifuge for 0 minutes to collect bacteria, and add 1M Na2HPOt
(Wako Co., Ltd. >1+r+I2), and after standing at 4°C for several hours, this suspension was disrupted by ultrasonic waves.The Kokutai crushed product was then centrifuged at 10,000XCI for 10 minutes to collect the supernatant.

Add 500uQ of 100mM CH3C to 500μQ of this supernatant.
After adding OONa (pH 5.5) and leaving it at 4°C for 30 minutes, the precipitate was removed using a 0.22 μm filter (manufactured by Millipore), and then filtered using liquid chromatography [UItrochrom GTi (L K 8 companies) used; column TSK gel 5P-5PW, 7.5 x 10 mm
;Adsorption buffer 50mHCH3C0ONa (pH 5
,5): Elution 50mM CH3COONa-0,58Na
Met by CQ (pH 5,5 > gradient end)
(+) IL-1β/Met(-) IL-1
From the area ratio of the chart obtained by fractionating β and 1q, Met
The (+)/Met(-) ratio was calculated.

The results are shown in Table 2.

From Table 2 above, it is clear that the lower the aeration rate, the more the production ratio of Met(+) decreases, and it is possible to selectively produce Met(-).

Example 2 Cystine (Cys) at position 71 of human IL-1β
Direct expression vector for IL-1β derivatives with substitutions for otl and +on (Ser) p trp GI F-α-71
E. coli H8101 strain (Escher
ichia coli HBIOI/ptrp GIF
-α-713: FERM BP1296: EP Patent Publication No. 23796 Tan (200 (1!) is used, the ventilation amount is Q, 5vvm
The cells were cultured in the same manner as in Example 1, and the same treatment was repeated, followed by the same liquid chromatography operation.

The obtained taromad chart is shown in FIG. In the figure, (1) represents Met(+), and (2) represents Met(-).

From FIG. 1 above, it can be seen that Met(-) can be selectively obtained according to the method of the present invention.

[Brief explanation of the drawing]

FIG. 1 is a chart showing the results of liquid chromatography analysis of a useful polypeptide obtained according to Example 2 of the present invention. (that's all)

Claims (1)

[Claims] (1) When culturing host cells transformed with a direct expression vector incorporating the gene of a useful polypeptide, the amount of dissolved oxygen in the culture medium is limited to approximately 20% or less of the saturation concentration, and the expressed protein is 1. A method for culturing recombinant protein-expressing cells, the method comprising obtaining a useful polypeptide from which a methionine residue corresponding to an initiation codon has been removed.