JP2844484B2 - Method for producing recombinant protein - Google Patents

Description

The present invention relates to a method for efficiently producing a useful protein using Escherichia coli transformed by recombinant DNA technology.

[Conventional technology]

When it is desired to produce a large number of useful proteins derived from eukaryotes, such as various interferons, interleukins, colony stimulating factors, lymphotoxin, human serum albumin, etc. by a direct expression method using Escherichia coli using recombinant DNA technology, their expression is required. In addition to the improvement of the plasmid itself, a high production method can be considered by improving the culture method of the producing bacteria. In particular,
When the intended useful protein is used as a pharmaceutical, a useful protein having an amino-terminal methionine residue corresponding to the initiation codon for gene translation in Escherichia coli may provide antigenicity. Is a technology that can produce a large amount of the target protein with a low amino-terminal methionine in the culture stage of the production bacteria, in order to reduce the cost in the protein separation and purification steps, as well as to remove as much as possible. It is necessary to establish

Generally, the culture method of recombinant E. coli
(0.5% yeast extract, 1.0% bactotripton, 0.5% NaCl
%, Glucose 0.2%, pH 6.5-7.5) medium and M9 (0.6% Na _{2
HPO 4, 0.3% KH 2 PO} 4, 0.05% NaCl, 0.1% NH 4 Cl, 0.2% _{glucose, 0.00147% CaCl 2 · 2H 2} O, 0.05% MgSO 4 · 7H 2 O) media, further casamino acid thereto, the yeast Culture in a semi-synthetic medium containing an extract or the like is generally known.

However, these methods are not yet sufficient in terms of productivity of the useful protein to be obtained and removal of the amino-terminal methionine residue of the produced protein, and further improvement in culture conditions has been desired.

[Problems to be solved by the invention]

An object of the present invention is to improve the production amount of a product by a culturing method and to improve the efficiency of removing an amino-terminal methionine residue when producing a useful protein using Escherichia coli.

[Means for solving the problem]

The above object is achieved by the present invention described below. That is, the present invention is a recombinant
A useful protein characterized by culturing Escherichia coli transformed with an expression plasmid devised by DNA technology to produce a useful target protein, and culturing in a medium supplemented with soybean hydrolyzate as a main nutrient source. Production method.

The useful protein of the present invention is not particularly limited as long as it can be produced by recombinant DNA technology. For example, interleukin-2, human B cell differentiation factor (abbreviated as BCDF), α-, β-, Examples include lymphokines such as γ-interferon, hormones such as growth hormone, insulin and somatostatin, and blood-related proteins such as human serum albumin. In addition, human BCDF is human interleukin-6
Although it is also called (IL-6) or B cell stimulating factor (BSF-2), here, the conventionally used name of human BCDF is used.

The expression plasmid of the present invention basically comprises a DN encoding the protein such that the useful protein is produced in E. coli.
The A fragment may be a plasmid integrated under the control of a promoter together with a translation initiation signal, and a terminator may be introduced downstream of the structural gene. This plasmid is prepared by a known method (for example, Goeddel, DVeta).
l., Nucleic Acids Research, 8, 4057 (1980)).

As a method for transforming Escherichia coli with the expression plasmid, known methods (for example, Mandel, M. & Maga, A.,
J. Mol. Biol., 53, 154 (1970)).

The type of Escherichia coli as the host is not particularly limited, and examples thereof include K-12 C-600, HB101, JM105, and W3110.

Examples of the medium include a carbon source such as glucose, lactose and sorbitol, a nitrogen source such as ammonia, ammonium chloride and ammonium nitrate, an organic nutrient such as meat extract, yeast extract, casein decomposition product, and peptone, and an inorganic salt such as phosphate. A production medium containing magnesium, potassium, other trace metals and the like as appropriate can be used. However, the present inventors examined various media and found that the soybean hydrolyzate was converted to the above M9 medium (0.6% Na ₂ HPO ₄ , 0.3% KH ₂ PO ₄ , 0.05% NaC
l, 0.1% NH ₄ Cl, 0.2% glucose, 0.00147% CaCl ₂ .2H ₂ O,
0.05% MgSO ₄ · 7H ₂ O), and using a medium added in place of casamino acid, the productivity of useful proteins and the removal of methionine residues added to their amino termini Rate was found to improve. The soybean hydrolyzate includes, for example, synthetic amino acids (manufactured by Ajinomoto Co., Inc., soybean hydrochloride hydrolyzate), taste liquid, bactosoyton (manufactured by DIFCO), and the like. The concentration of soy hydrolyzate
Although it differs slightly depending on the basic medium composition and culture conditions,
Usually 1% to 10% (by weight) per medium, especially 2% to 4%
(% By weight) is preferred.

The cultivation temperature is from 20 ° C to 45 ° C, preferably from 36 ° C to 39 ° C, and the pH of the medium during culturing is from 5 to 9, preferably from 6 to 7. The culture period is usually from about 6 hours to about 2 days.

〔The invention's effect〕

Culturing Escherichia coli transformed with an expression plasmid into which a DNA fragment encoding a useful protein is incorporated, and culturing the Escherichia coli in a medium supplemented with soybean hydrolyzate in a method for producing a useful recombinant protein. The present invention provides a method for increasing the yield of a useful protein in which a methionine residue corresponding to the translation initiation codon ATG is efficiently removed at the N-terminus, and the culture method according to the present invention improves the productivity of the useful protein. At the same time, the efficiency of removing methionine residues added to the amino terminus of the target protein can be improved.

Hereinafter, the present invention will be described more specifically with reference to examples, but the usefulness of the present invention is not limited thereto.

〔Example〕

Example 1 Escherichia coli HB101 / pBSF carrying human BCDF expression plasmid
2-SD7 was compared and cultured using a 5 L. minijar in a conventional medium containing casamino acid as a main nutrient source and a medium containing soybean hydrolyzate, a synthetic amino acid (manufactured by Ajinomoto Co., Inc.) as a main nutrient source. In addition, plasmid pBSF2-SD7 was converted to plasmid pUC19 (Yanisch-Perron, C., Vieira, J. and Messing,
J .: Gene 33 (1985) 103-119) EcoRI, Hind.
It has a structure in which the trp promoter, the SD sequence of trpL, the synthetic SD sequence, the human BCDF synthetic gene, and the trpA terminator are incorporated into the III cloning site from the EcoRI side. (FIG. 1) By transforming this plasmid into Escherichia coli HB101, a human BCDF-producing bacterium was obtained. E. coli HB101 /
pBSF2-SD7 (AJ-12448) has already been deposited as FERM P-10758. (Described in Japanese Patent Application No. 1-189270) M9 casamino acid medium composition is casamino acid 1.0 g / dl, yeast extract 0.2 g / dl, ammonium chloride 0.5 / dl, magnesium sulfate heptahydrate 0.05 g / dl, calcium chloride 0.005g / d dihydrate
l, L-leucine 40mg / dl, L-proline 40mg / dl, vitamin B1
0.4 mg / dl, glucose 2.0 g / dl, and potassium phosphate 0.1 g / dl. On the other hand, the composition of M9 total amino acid medium is the same as that of casamino acid, leucine and proline except for the total amino acid (Ajinomoto Co.) (Manufactured by the company). During the culture, 100 μg / ml of ampicillin and 25 μg / ml of streptomycin were added to both media.

Then, both production media 31. were charged into a 51.-volume mini jar. L broth (Yeast extract 0.5%, Bactotripton 1.0
%, NaCl 0.5%, glucose 0.2%, pH 6.5-7.5) E. coli HB101 / pBSF2-SD7 (FERM P-1075) cultured overnight
8) Add 150 ml to the mini-jar containing each of the above media, stirring at 700 rpm, aeration 0.5 vvm, culture temperature 37 ° C, control pH
Driven at 6.7. 3-5 hours after the start of the culture,
When D 660 nm reached 4-5, indoleacrylic acid, an inducer of the trp promoter, and feed medium were added. As a feed medium, for the culture of M9 casamino acid medium, casamino acid 1.0 g / dl, L-leucine 40 mg / dl, L-proline 40 mg / dl, vitamin B1 0.4 mg / dl, (pH 6.7) , And total amino acid 1.
A composition having a composition of 0 g / dl, vitamin B1 0.4 mg / dl, (pH 6.7) was used.

Both cultures were performed for 16 hours. FIG. 2 shows the progress of each culture. The cell concentration finally reached 16 at OD 660 nm in the M9-casamino acid medium and 21 in the M9-general amino acid medium.

Next, the cells were collected from both cultures by centrifugation, and 20 mM
A suspension, lysozyme, and EDTA were added to a final concentration of 0.2 mg / ml and 0.1 M, respectively, in Tris-HCl (pH 7.5) and 30 mM NaCl, and the mixture was treated at 0 ° C. for 1 hour. Thereafter, the bacterial cell liquid was disrupted by an ultrasonic disrupter at 70 W for 20 minutes, and the inclusion bodies containing human BCDF were recovered by low-speed centrifugation at 6000 rpm for 10 minutes. The inclusion body was washed three times with the above buffer solution 20 mM Tris-HCl (pH 7.5) and 30 mM NaCl, and then suspended in 10 mM EDTA (pH 6.0).
A BCDF inclusion body suspension was obtained.

Subsequently, the inclusion body was added to 20 mM Tris-HCl (pH 8.3), 10 mM
EDTA was solubilized with 6M guanidine hydrochloride solution at room temperature for 1 hour, and the solubilized solution was subjected to reverse phase HPLC (YMC C8 column 4.6φmmX
250 mm) to isolate and purify the human BCDF fraction.

The amount of human BCDF produced in both cultures was calculated from the absorption at UV280 nm. For the N-terminal amino acid sequence, the above sample was analyzed using a protein sequencer (470A manufactured by ABI).
And tested. The results are shown in Table 1 below.

As described above, by using soybean hydrolyzate, a synthetic amino acid as a main nutrient, the production of human BCDF is about twice that of the conventional M9-casamino acid medium, and the methionine removal rate at the amino terminus is reduced. Increased from 85% to 98%. As described above, as a result of culturing in a comprehensive amino acid medium, a large amount of human BCDF from which the amino-terminal methionine was efficiently removed could be obtained.

Example 2 Escherichia coli HB101 / pT13SNco carrying a human interleukin-2 expression plasmid was subjected to comparative culturing in the same manner as in Example 1 using a 3 L. volume mini jar.

Plasmid pT13SNco for human interleukin 2 production
(Tonouchi et al., J. Biochem. 104, 30-34 (1988))
An expression plasmid designed to express human interleukin 2 under the control of a promoter. This plasmid was transformed into Escherichia coli HB101 strain and produced a human interleukin 2 producing bacterium HB101 / pT13SNco (AJ-12447) FERM P-107.
The medium composition and culture conditions already deposited as 57 are the same as in Example 1. In the case of interleukin 2, similarly to BCDF, inclusion bodies of interleukin 2 were formed in Escherichia coli. Production volume,
As a result of testing the N-terminal amino acid sequence in the same manner as in Example 1,
As shown in Table 2 below, in the case of interleukin 2, the production amount of human interleukin 2 was increased by about 1.5 times by culturing in the M9-amino acid medium as compared with culturing in the M9-casamino acid medium. In addition, the N-terminal methionine removal rate was improved from 35% to 78%. Thus, human interleukin 2 from which the amino-terminal methionine could be removed efficiently.
Could be obtained in large quantities. In Table 2, human IL-
2 is an abbreviation for human interleukin 2.

[Brief description of the drawings]

FIG. 1 shows the structure of pBSF2-SD7, a plasmid for producing human BCDF. FIG. 2 shows the progress of culturing human BCDF-producing bacteria when using a total amino acid medium and when using a casamino acid medium.

Claims (4)

(57) [Claims] 1. A method for producing useful proteins by culturing Escherichia coli transformed with an expression plasmid into which DNA encoding a useful protein has been incorporated, wherein the cells are cultured in a medium containing soybean hydrolyzate. A method for producing a recombinant protein. 2. The soybean hydrolyzate is present in an amount of 1% to 10% per medium.
The production method according to claim 1, wherein the compound is added in a concentration range of (% by weight). 3. The production method according to claim 1, wherein the useful protein is a human B cell differentiation factor. 4. The production method according to claim 1, wherein the useful protein is human interleukin 2.