JP2012044888A - Method for producing water-soluble protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a large amount of target proteins as a water-soluble protein while suppressing the formation of an insoluble fraction (inclusion body), when producing the target proteins in an Escherichia coli.SOLUTION: The method for producing the target proteins as the water-soluble protein includes a first step of proliferating and culturing the Escherichia coli that maintains a gene that encodes the target protein, and a second step of exchanging culture medium after proliferation culturing, and culturing the Escherichia coli in the target protein inducing the expression, wherein the cell wet weight of the Escherichia coli in the culture medium during culturing of the second step is 2.5-5.0 g/100 ml.

Description

  The present invention relates to a method for producing a soluble protein in, for example, E. coli.

  Conventionally, when recombinant proteins are produced in E. coli, many proteins become insoluble fractions called inclusion bodies (inclusion bodies) and cannot be obtained as active soluble proteins.

  Known methods for avoiding the insolubilization include a low nutrient medium such as a minimal medium and a culture condition under a low temperature (for example, 20 ° C. or lower). However, since this method is based on the premise that the protein expression level itself is reduced in order to suppress the formation of inclusion bodies, very few soluble proteins are obtained.

  In addition, examples of modifying host cells, expression vectors, gene sequences and the like are known as methods for avoiding the insolubilization. For example, Patent Document 1 uses a polynucleotide containing a polynucleotide encoding a secretory signal peptide, a polynucleotide encoding a polypeptide rich in basic amino acids, and a polynucleotide encoding a target protein in this order. Disclosed is a method for producing a target protein as a soluble protein by expressing the protein in a host cell such as Escherichia coli. Patent Document 2 discloses a method of synthesizing a protein that is soluble and retains its function by synthesizing a protein using a ribosome mutant E. coli strain. However, the amount of protein produced by these methods is not necessarily large, and optimization is required for each protein to be expressed.

JP 2007-325521 A JP 2007-300858

  In view of the above-described circumstances, the present invention provides a method for producing a target protein in large quantities as a soluble protein by suppressing the formation of an insoluble fraction (inclusion body) when the target protein is produced in E. coli.

  As a result of earnest research to solve the above problems, in the production of the target protein in E. coli, the E. coli culture period was divided into two periods, the E. coli growth period and the target protein expression induction period. By culturing in the above manner, it was found that the formation of an insoluble fraction (inclusion body) can be suppressed and the target protein can be produced in large quantities as a soluble protein, and the present invention has been completed.

  That is, the present invention comprises a first step of growing and cultivating E. coli retaining a gene encoding the target protein, and a second step of culturing the E. coli under induced expression of the target protein after the growth and culture. And the target protein is produced as a soluble protein, wherein the wet weight of Escherichia coli in the medium in the culture in the second step is 2.5 to 5.0 g / 100 ml.

  Examples of the culture conditions in the first step include 16 to 24 hours at a temperature of 16 to 40 ° C. Moreover, as culture conditions in a 2nd process, 16 to 24 hours are mentioned under the temperature of 16 to 37 degreeC.

  Examples of the medium used for the culture include a medium containing 10 to 30 g of yeast extract, 10 g to 30 g of Trypton, and 0 ml to 4 mL of glycerol per liter of the medium.

  Furthermore, the induction of target protein expression may be performed by adding isopropyl-1-thio-β-D-galactoside (IPTG) to the medium.

  According to the present invention, in Escherichia coli, the formation of an insoluble fraction (inclusion body) can be suppressed, the target protein can be produced in large quantities as a soluble protein, and many proteins including proteins that have not been conventionally obtained as a soluble fraction can be produced. Industrial production of proteins can be achieved.

2 is a graph showing the amount of formate dehydrogenase (FDH) soluble protein (mg / L) per culture volume produced by the culture methods shown in Comparative Examples 1 and 2 and Example 1. In Example 1, when the first half of the main culture is carried out with TB medium and the latter half of the main culture is carried out with the medium shown, the wet cell weight after completion of the main culture (g / 100 mL) and the amount of soluble FDH protein (FDH It is a graph which shows the relationship with the light absorbency (A438) in OD438nm which shows the amount of yellow formazan based on activity. In Example 1, when the first half and the second half of the main culture were carried out with LB or TB medium at the indicated concentrations, the wet cell weight (g / 100 mL) after the completion of the main culture and the amount of soluble FDH protein (based on the FDH activity) It is a graph which shows the relationship with the light absorbency (A438) in OD438nm which shows the amount of yellow formazan.

  Hereinafter, the present invention will be described in detail.

  The present invention includes a first step of growing and cultivating E. coli retaining a gene encoding the target protein, and a second step of culturing the E. coli under induction of expression of the target protein, after the growth and culture, exchanging the medium. This is a method for producing a target protein as a soluble protein (hereinafter referred to as “the present method”). In this method, the wet weight of Escherichia coli in the medium is controlled to 2.5 to 5.0 g / 100 ml (preferably 3.0 to 4.0 g / 100 ml) in the second step culture. According to the present invention, formation of inclusion bodies can be suppressed, and the target protein can be produced in a large amount (for example, 400 mg or more per 1 L culture volume) in a solubilized state.

  In this method, first, E. coli holding a gene encoding the target protein is prepared. The target protein may be any protein, but is not limited to, for example, a structure-related protein (keratin, collagen, etc.), an enzyme (oxidoreductase, transferase, hydrolase, isomerase, Lyases, ligases, etc.), proteins involved in information transmission (peptide hormones, receptors, etc.), antibodies, fluorescent proteins (GFP, etc.) and the like.

  For example, by preparing a vector in which a gene encoding a target protein is placed under the control of an inducible promoter, and introducing the vector into E. coli, E. coli holding the gene encoding the target protein can be prepared. Such an inducible promoter is not particularly limited, and conventionally known promoters can be used. For example, an inducible promoter exhibiting transcriptional activity in the presence of isopropyl-1-thio-β-D-galactoside (IPTG) can be used. Examples of such promoters include Trp promoter, Lac promoter, Trc promoter, Tac promoter, T7 promoter and the like. In addition, other promoters that exhibit transcriptional activity in the presence of inducers other than IPTG, and other promoters that exhibit transcriptional activity according to culture conditions such as medium components and temperature (for example, low temperature) are also used as inducible promoters. can do.

  The vector is not particularly limited as long as it can be replicated in E. coli, and may be any of a plasmid vector, a phage vector, and the like. Specific examples of the vector include pCDF series, pRSF series, and pET series. For example, when the expression vector is a pET (having T7 promoter) system, E. coli BL21 (DE3) can be used. As a method for introducing the above-described vector into Escherichia coli, various methods generally known as transformation methods can be applied. As a specific method, for example, a calcium phosphate method, an electroporation method, a lipofection method, or the like can be applied. The transformation may be either transient or stable.

  In addition, E. coli carrying the plasmid pLysS (BL21 (DE3) pLysS: manufactured by Invitrogen) may be used as E. coli. Plasmid pLysS expresses T7 ribozyme at low levels. Since T7 ribozyme inhibits T7 RNA polymerase, the expression of the target protein before the induction of expression by IPTG can be suppressed.

  Furthermore, in E. coli, a chaperone or the like may be co-expressed to support correct folding of the target protein.

  Next, in this method, the prepared Escherichia coli holding the gene encoding the target protein is subjected to preculture, and the cells for main culture in the first step and the second step described above are prepared. The preculture is performed, for example, in ampicillin-containing LB medium (Difco) (1 × composition (per 1 L): Yeast extract 5.0 g, NaCl 5.0 g, Tryptone 10.0 g, ampicillin 50 mg).

In this method, the culture solution (preculture solution) obtained in the preculture is used to perform the main culture in the first step and the second step described above. The main culture may be any of batch culture and continuous culture. The main culture may be either stationary culture or shaking culture, but shaking culture is preferred. As a medium used in the main culture, a medium having high nutritional value can be used. Examples of such a highly nutritious medium include, for example, 5 to 50 g of yeast extract, preferably 10 to 30 g, 5 to 50 g of tryptone, preferably 10 to 30 g, and 0 to 10 ml of glycerol, preferably 0 to 4 ml per liter of the medium. A culture medium is mentioned. In addition to glycerol, sugars such as glucose may be used as the carbon source. Also, commercially available LB medium (Difco) (1 × composition (per liter): Yeast extract 5.0 g, NaCl 5.0 g, Tryptone 10.0 g) and TB medium (Difco) (1 × composition) (Per liter): tryptone 12 g, yeast extract 24 g, K ₂ HPO ₄ 9.4 g, KH ₂ PO ₄ 2.2 g, glycerol 4 ml) and the like can be used. LB medium is used at a concentration of x1 to 5, and TB medium is used at a concentration of x0.5 to 1.5.

  In the main culture, E. coli holding a gene encoding the target protein is first subjected to a growth culture in which only the growth is performed without inducing the expression of the target protein (first step). Examples of the culture conditions in the first step include 16 to 24 hours at a temperature of 16 to 40 ° C. (for example, 37 ° C.).

  Next, after completion of the first step, the supernatant is removed from the culture by a method such as centrifugation or filter filtration, and a predetermined medium used in the second half of the main culture (second step) is added to the culture from which the supernatant has been removed. Add and suspend. By this operation, medium exchange is performed. After exchanging the medium, Escherichia coli holding the gene encoding the target protein is cultured under the expression of the target protein expression (second step). When the gene encoding the target protein is placed under the control of a predetermined inducible promoter as described above, the induction of expression of the target protein is performed by adding an inducer that induces transcriptional activity from the inducible promoter. Is started. For example, when an inducible promoter exhibiting transcriptional activity in the presence of IPTG is used, expression of the target protein is initiated by the addition of IPTG to the medium. When other promoters exhibiting transcriptional activity are used depending on the culture conditions such as medium components and temperature, expression of the target protein is started by supplying E. coli holding the gene encoding the target protein to the culture condition. Will be.

  The culture conditions in the second step include 16-24 hours at a temperature of 16-37 ° C. However, in the second step, the wet weight of Escherichia coli in the medium is controlled to 2.5 to 5.0 g / 100 ml (preferably 3.0 to 4.0 g / 100 ml). The wet cell weight can be determined by subjecting the culture solution to centrifugation and measuring the cell weight after removing the supernatant.

  After the main culture, Escherichia coli cells can be disrupted to prepare a crude protein suspension. In this method, since the formation of inclusion bodies is suppressed and the obtained target protein is soluble, the crude target protein suspension contains a target protein having a predetermined activity and function. Therefore, the obtained crude target protein suspension can be used as it is as the target protein. The target protein can also be isolated and purified from the obtained crude target protein suspension. In this case, general biochemical methods (for example, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc.) used for protein isolation and purification can be used alone or in appropriate combination. The isolated and purified target protein can be used in a state suspended in a buffer solution having a predetermined pH, for example.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.

1． Reagent (including medium)
The reagent containing the culture medium used in the following Examples and Comparative Examples is as follows.
Unless otherwise specified, reagents manufactured by Nacalai Tesque were used.
(1) TALON Buffer Kit (manufactured by CLONTECH)
5 × Equilibration / Wash Buffer: diluted 5 times (used as “1 × E / W Buffer”).
10 × Elution Buffer: diluted 10 times.
(2) TALON CellThru Resin and TALON CellThru 10-ml Disposable Columns (CLONTECH)
(3) Ampicillin (Amp) (SIGMA)
Used at a final concentration of 50 μg / ml.
(4) LB medium (Difco)
After adjusting to 20 g / L, it was used by autoclaving.
When ampicillin is added, it is expressed as “LB-Amp”.
1 × LB (Normal concentration: In the following, when simply referred to as “LB”, it indicates “1 × LB”)
(1x composition (per liter): Yeast extract 5.0g, NaCl 5.0g, Tryptone 10.0g)
0.5 x LB (diluted to normal 1/2 concentration)
2 x LB (normal double concentration).

(5) TB medium (Difco)
It was adjusted to 47.6 g / L, and after adding 4 ml of glycerol, it was used by autoclaving.
When ampicillin is added, it is expressed as “TB-Amp”.
1 x TB (Normal concentration: In the following, when simply referred to as "TB", it indicates "1 x TB")
(1x composition (per liter): tryptone 12g, yeast extract 24g, K ₂ HPO ₄ 9.4g, KH ₂ PO ₄ 2.2g, glycerol 4ml)
0.5 x TB (diluted to normal 1/2 concentration)
2 x TB (normal double concentration).

(6) M9YE medium
M9YE medium composition:
M9 Min medium salts (MP Biomedicals) 11.2g / L
MgSO ₄ 2mM
CaCl ₂ 0.1 mM
Yeast extract (Difco) 1g / L
Lactose 4g / L

(7) IPTG (Takara Bio)
After dissolving at 1M in sterilized water, it was sterilized with a 0.22 μm filter.
(8) Potassium phosphate buffer (KPB) pH 7.5 (100 mM)
16 ml of Solution A (0.2 M KH ₂ PO ₄ 5.4 g / 200 ml) and 84 ml of Solution B (0.2 MK ₂ HPO ₄ 7.0 g / 200 ml) were mixed to prepare KPB (100 ml).
(9) Sodium formate 1.62M (prepared with 100 mM KPB pH 7.5)
(10) NAD16.2mM (prepared with 100mM KPB pH7.5)
(11) Methoxy PMS (mPMS) (DOJINDO)
Prepared with H ₂ O to 0.5 mg / ml.
(12) WST1 (DOJINDO)
Prepared with H ₂ O to 8 mg / ml.
(13) Protein Assay (BIORAD)
(14) MinElute PCR Purification Kit (QIAGEN)
(15) QIAquick PCR Purification Kit (QIAGEN)
(16) Ligation-Convenience Kit (manufactured by Nippon Gene)
(17) QIAprep Spin Miniprep Kit (QIAGEN)

(18) YPD medium
YPD medium composition:
Yeast extract 10g
Bacto pepton 20g
Glucose 20g
Water was added to make 1 L, the pH was adjusted to 7, and then autoclaved for use.

(19) YP-Methanol medium
YP-Methanol medium composition:
Yeast extract 10g
Bacto pepton 20g
Water was added to 1 L, and the pH was adjusted to 7, followed by autoclaving. After autoclaving, 20 ml methanol was added and used.

2． Preparation of expression vector of Gibberella zeae (G. zeae) -derived formate dehydrogenase (FDH) gene The expression vector of G. zeae-derived FDH gene used in the following Examples and Comparative Examples was prepared as follows.

  G. zeae (NBRC4474) purchased from the National Institute for Biological Resources (NBRC) is cultured overnight in YPD medium and then inoculated into 5 ml of YP-Methanol medium using methanol as a single carbon source. And aerobically shaking culture at 25 ° C.

  After the culture, Total RNA was prepared from the obtained cells using RNeasy Plant Mini Kit (manufactured by QIAGEN). Further, cDNA was synthesized from the obtained total RNA using RNA PCR Kit (TaKaRa). The composition of the reaction solution and the reaction cycle are as follows.

Reaction solution composition:
Final concentration
MgCl ₂ 5mM
RT buffer 1 ×
dNTP mixture 1mM
RNase Inhibitor 0.5U
AMV Reverse Transcriptase XL 0.25U
Oligo dT-Adapter primer 0.13μM
Total RNA 5μg
RNase free H ₂ O was added so that the liquid volume became 10 μl.
Reaction cycle:
50 ° C (2 hours) → 99 ° C (5 minutes) → 4 ° C

  PCR was performed using the synthesized cDNA as a template and the following primers, reaction solution composition and reaction conditions.

Primer:
pET14b-F primer: 5'-TAT ACA TAT GGT CAA GGT TCT TGC AGT-3 '(SEQ ID NO: 1)
pET23b-R primer: 5'-CCG CAA GCT TTT TCT TCT CAC GCT GAC CAT-3 '(SEQ ID NO: 2)
Reaction solution composition:
10XBuffer 5μl
dNTP (2 mM each) 5 μl
MgSO ₄ (25 mM) 2 μl
pET14b-F primer (10 pmol/μl) 1.5μl
pET23b-R primer (10 pmol/μl) 1.5μl
KOD-Plus-DNA Polymerase (1U / μl) 1μl
Template (cDNA) 0.5μl
Sterile water 33.5μl
50 μl
Reaction cycle:
94 ℃: 2 minutes → (94 ℃: 15 seconds → 68 ℃: 1 minute 30 seconds) × 30 → 68 ℃: 2 minutes → 4 ℃

  The amplified fragment (PCR product) of about 1.1 kb was purified with the MinElute PCR Purification Kit. The purified product and vector pET23b were then digested with NdeI / HindIII and purified using the QIAquick PCR Purification Kit.

  Furthermore, after subjecting the DNA fragment purified using the Ligation-Convenience Kit and the vector to ligation, Escherichia coli JM109 strain was transformed according to the protocol using the obtained recombinant vector. The obtained recombinant was cultured in 4 ml LB medium + Amp, and then the plasmid was extracted using QIAprep Spin Miniprep Kit. The extracted plasmid was used as an FDH gene expression vector. In the FDH gene expression vector, the FDH gene is arranged so that the FDH protein to be expressed has a His tag at the C-terminus. In the FDH gene expression vector, the FDH gene is arranged under the control of the T7 promoter.

[Comparative Example 1] Culture method 1 without medium change
The E. coli (BL21 (DE3)) transformant containing the FDH gene expression vector was cultured overnight in 5 ml of LB-Amp medium, then inoculated 1% in 100 ml of the same composition medium, until OD600 reached about 0.5. Incubated at 0 ° C.

  After culturing, IPTG (isopropyl-1-thio-β-D-galactoside) was added to the medium to a final concentration of 1 mM, and further cultured for 4 hours from the FDH gene expression vector introduced into the FDH gene expression vector. FDH protein expression was induced.

The expressed FDH was purified and evaluated by the method described in Example 1.
FIG. 1 shows the amount of FDH protein (mg / L) per culture volume obtained by the conventional method. In FIG. 1, the “prior art method 1” bar graph shows the amount of FDH protein obtained by the conventional method.

[Comparative Example 2] Culture method 2 without medium change
An E. coli (BL21 (DE3)) transformant containing the FDH gene expression vector was cultured overnight in 5 ml of LB-Amp medium, then 1% inoculated into 100 ml of the same composition medium, and cultured with shaking at 37 ° C. for 10 hours. .

  After culturing, IPTG was added to the medium to a final concentration of 1 mM, and further cultured at 20 ° C. for 16 hours to induce expression of FDH protein from the FDH gene introduced into the FDH gene expression vector.

Purification and evaluation of the expressed FDH were carried out by the method described in Example 1.
FIG. 1 shows the amount of FDH protein (mg / L) per culture volume obtained by the conventional method. In FIG. 1, the “Conventional method 2” bar graph shows the amount of FDH protein obtained by the conventional method.

[Example 1] Culture for medium exchange (method according to the present invention)
As a preculture, an E. coli (BL21 (DE3)) transformant containing the FDH gene expression vector was cultured in 5 ml of LB-Amp medium at 37 ° C. for 17 hours with shaking (130 rpm).

  Then, as the first half of the main culture, 1.5% of the preculture solution was inoculated into each medium, followed by shaking culture (130 rpm) at 37 ° C. for 16 hours.

  Furthermore, after the end of the first half of the main culture, the culture solution is transferred to a 300 ml centrifuge tube sterilized by autoclaving and centrifuged at 2500 xg for 10 minutes (20 ° C, Avanti HP-26 XPI / Beckman). Qing was removed. Each medium used in the latter half of the main culture was added to the culture excluding the supernatant, and suspended by vortexing. Next, as the latter half of the main culture, IPTG was added to the suspension to a final concentration of 1 mM, followed by shaking culture (130 rpm) at 20 ° C. for 24 hours. The expression of FDH protein was induced from the FDH gene introduced into the FDH gene expression vector by the addition of IPTG.

  After completion of the second half of the main culture, the culture solution is cooled on ice and centrifuged at 4000 xg for 10 minutes in a 300 ml centrifuge tube (4 ° C, Avanti HP-26 XPI / Beckman) to obtain the supernatant. The wet weight of the obtained cells was measured.

  In addition, a crude extract was prepared from the cells thus obtained, and the amount of soluble FDH was estimated by measuring the FDH activity. The procedure is shown below.

1. Crude extract preparation and FDH activity measurement procedure
(1) Add 23 g of crushing glass beads (0.1 mm) to the cells.
(2) Add 23 ml of 1 × E / WB (0.024% PEG) (first add about 5 ml to suspend the cells).
(3) The cells are crushed using a multi-bead shocker (manufactured by YASUI KIKAI).
1) 6000rpm shaking: 90 seconds
2) Interval: 60 seconds
Repeat 1) and 2) 6 times.
(4) Centrifuge at 7000 × g for 20 minutes (4 ° C., Avanti HP-26XPI, JA-12 rotor manufactured by Beckman) to collect the supernatant.
(5) Activity measurement

  The reaction solution shown below was reacted at 37 ° C. for 1.5 minutes, and the absorbance (yellow formazan amount) was measured at OD438 nm (using a Nunc 96-well flat bottom plate and Tecan InfiniteM200). The tetrazolium salt WST1 generates yellow formazan by the reduction reaction of dehydrogenase when 1-Methoxy PMS is used as an electron carrier.

Reaction solution composition:
1.62M sodium formate 15μl
16.2mM NAD 15μl
100 mM KPB (pH 7.5) 113 μl
0.5mg / ml mPMS 1μl
8mg / ml WST1 5μl
1 μl of crude extract
150μl

  Furthermore, purification of soluble FDH and protein quantification were performed from the above crude extract using a TALON column (Clontech). The procedure is shown below.

2. Purification by TALON column 15.5 ml of crude enzyme solution (3.6 g wet weight) using TALON CellThru Resin (CLONTECH) and TALON CellThru 10-ml Disposable Columns (CLONTECH) Was purified.

(1) Suspend Resin stock solution.
(2) Immediately after suspension, 20 ml of Resin suspension is transferred to a plastic 250 ml V bottom centrifuge.
(3) Let stand for 2 minutes. After confirming the precipitation of Resin, remove the supernatant.
(4) Add 100ml of 1X E / W Buffer and mix.
(5) Let stand for 1-2 minutes, confirm the precipitation of Resin, and then remove the supernatant.
Repeat steps (4) and (5).
(6) The crude extract is added to Resin and stirred at room temperature for 20 minutes (TAITECH shaker NR-3).
(7) Let stand for 1-2 minutes, confirm the precipitation of Resin, and then save the supernatant as a pass fraction.
(8) Add 100 ml of 1 × E / W Buffer and stir at room temperature for 10 minutes (TAITECH shaker NR-3).
(9) Let stand for 1-2 minutes, confirm the precipitation of Resin, and then remove the supernatant.
Repeat steps (8) and (9).
(10) Add 10 ml of 1 × E / W Buffer and stir.
(11) Transfer suspended Resin to Column (discard Buffer as flow-through).
(12) Wash with 50 ml of 1 × E / W Buffer.
(13) Add 15 ml of 1X Elution Buffer to elute FDH protein (elution twice).

3． Desalination and concentration The elution sample obtained above was transferred to Amicon Ultra15 30000NMWL (Millipore) and subjected to centrifugation at 4500 × g for 20 minutes, thereby desalting and concentrating the liquid volume to 1.1 ml.

Four. Measurement of FDH protein concentration FDH protein concentration in the concentrated sample obtained above was measured according to the procedure of Protein Assay reagent manufactured by BIORAD. The FDH protein concentration was calculated from the relational expression between the absorbance of the standard BSA and the protein concentration.

  The amount of FDH protein (mg / L) per culture volume obtained by this method is shown in FIG. In FIG. 1, the “present method” bar graph shows the amount of FDH protein obtained by this method. According to the method shown in this example, the concentration of the purified FDH protein in the concentrated sample was 39.3 mg / ml. As shown in FIG. 1, since the amount of the solution is 1.1 ml, about 43 mg of soluble FDH protein (the amount of FDH protein per culture volume: about 430 mg / L) is obtained from a 100 ml culture (wet cell weight 3.6 g). It will be.

  Fig. 2 shows the wet weight (g / 100mL) and soluble FDH protein after completion of the main culture when the first half of the main culture is performed in TB medium and the latter half of the main culture is performed in the medium indicated. FIG. 5 shows the relationship with (absorbance at OD 438 nm (A438) indicating the amount of yellow formazan based on FDH activity).

  Furthermore, FIG. 3 shows the wet weight (g / 100 mL) and the amount of soluble FDH protein (FDH activity) after completion of the main culture when the first half and the second half of the main culture were carried out in the LB or TB medium having the concentrations shown. 3 shows the relationship with the absorbance at OD438nm (indicated by A438) indicating the amount of yellow formazan based on the above. In FIG. 3, “XX-YY” indicates that XX represents the medium in the first half of the main culture, and YY represents the medium in the second half of the main culture.

Claims (2)

A first step of growing and culturing Escherichia coli carrying a gene encoding the target protein;
After the growth culture, a second step of exchanging the medium and culturing the E. coli under the expression of the target protein,
A target protein as a soluble protein, wherein the wet weight of Escherichia coli in the medium in the culture in the second step is 2.5 to 5.0 g / 100 ml.   The method according to claim 1, wherein the medium used for the culture contains 10 to 30 g of yeast extract, 10 g to 30 g of tryptone, and 0 ml to 4 mL of glycerol per liter of the medium.

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