KR20220102046A - Method for producing excreted human epidermal growth factor - Google Patents

Abstract

The present invention relates to a method for producing exocrine human epidermal growth factors and, more particularly, to a method for producing exocrine human epidermal growth factors, which manufactures recombinant P. pastoris by introducing a hEGF expression vector manufactured so that the produced protein is secreted out of the cell to P. pastoris, and in which the recombinant P. pastoris is sequentially cultured in a growth medium for cell growth and a medium for hEGF production, and thus the hEGF production can be improved by optimizing cell density, methanol concentration, and methanol injection interval in culture using a carbon source used in the growth medium and the hEGF production medium.

Description

Method for producing exocrine human epidermal growth factor

The present invention relates to a method for producing exocrine human epidermal growth factor, and more particularly, introducing a hEGF expression vector prepared to secrete the produced protein out of cells into P. pastoris to prepare a recombinant P. pastoris , and the recombinant P. pastoris. pastoris is sequentially cultured in a growth medium for cell growth and a medium for hEGF production, and cell density, methanol concentration and methanol injection interval are optimized in culture using the carbon source used for the growth medium and the medium for hEGF production Thus, it relates to a production method of exocrine human epidermal gland factor capable of improving hEGF production.

Human epidermal growth factor (hEGF) is a single-chain polypeptide consisting of 53 amino acids and having a molar mass of 6.2 kDa. It is widely used.

As described in the following patent documents, the hEGF is generally produced using E. coli , which is inexpensive and easy to genetically manipulate.

<Patent Literature>

Patent No. 10-0102993 (registered on Aug. 02, 1996) "Human epithelial cell growth factor expression vector and manufacturing method of hEGF using the same"

However, the conventional method for producing human epidermal growth factor using E. coli requires cell destruction and refolding steps because human epidermal growth factor is generated within cells, resulting in poor production efficiency and high production cost. have. Therefore, research is being carried out to produce human epidermal growth factor using P. pastoris, but the method using P. pastoris also has a problem in that satisfactory hEGF production cannot be obtained.

Accordingly, there is an increasing need for a manufacturing method capable of improving hEGF production by optimizing various variables affecting hEGF production.

The present invention has been devised to solve the above problems,

The present invention prepares a recombinant P. pastoris by introducing a hEGF expression vector prepared so that the production protein is secreted out of the cell into P. pastoris, and uses the recombinant P. pastoris in a growth medium for cell growth and a medium for hEGF production. An object of the present invention is to provide a method capable of producing hEGF by sequentially culturing.

In addition, the present invention provides a method for producing hEGF capable of improving hEGF production by optimizing cell density, methanol concentration, and methanol injection interval in culture using a carbon source used for a growth medium and a medium for hEGF production. There is this.

The present invention is implemented by an embodiment having the following configuration in order to achieve the above object.

According to an embodiment of the present invention, in the method for producing human epidermal growth factor according to the present invention, a recombinant Pichia pastoris for hEGF production is produced by introducing a hEGF expression vector prepared so that the production protein is secreted out of cells into Pichia pastoris. A recombination step to prepare, a cell growth step of culturing the recombinant Pichia pastoris for the production of hEGF in a growth medium for cell growth, and a recombination for hEGF production in which the culture for cell growth is completed in the cell growth step It is characterized in that it comprises a protein production step of culturing Pichia pastoris in a medium for hEGF production to produce hEGF.

According to another embodiment of the present invention, in the method for producing human epidermal growth factor according to the present invention, the growth medium is characterized in that glucose is used as a carbon source.

According to another embodiment of the present invention, in the method for producing human epidermal growth factor according to the present invention, the recombinant medium for producing hEGF is characterized in that it contains methanol.

According to another embodiment of the present invention, in the method for producing human epidermal growth factor according to the present invention, the protein production step comprises adding recombinant Pichia pastoris for hEGF production to a cell density of 8 to 12OD ₆₀₀ in a medium for producing hEGF. supply, and additionally injecting 0.65 to 0.75% of methanol into the hEGF production medium at intervals of 7 to 8 hours and culturing.

According to another embodiment of the present invention, in the method for producing human epidermal growth factor according to the present invention, the recombination step comprises a promoter for hEGF expression, a signal sequence for extracellular secretion after hEGF expression, and a polynucleotide encoding hEGF. A cassette production step of preparing a DNA cassette for hEGF production comprising the sequence, an expression vector production step of preparing a hEGF expression vector by inserting the DNA cassette for hEGF production into an expression vector, and the hEGF expression vector in Pichia pastoris It is characterized in that it comprises a transformation step of introducing a recombinant Pichia pastoris for production of hEGF.

According to another embodiment of the present invention, in the method for producing human epidermal growth factor according to the present invention, the promoter for hEGF expression is a promoter induced by methanol and inhibited by glucose is used.

The present invention can obtain the following effects by the configuration, combination, and use relationship described below with the present embodiment.

The present invention prepares a recombinant P. pastoris by introducing a hEGF expression vector prepared so that the production protein is secreted out of the cell into P. pastoris, and uses the recombinant P. pastoris in a growth medium for cell growth and a medium for hEGF production. By sequentially culturing, there is an effect that can produce hEGF.

In addition, the present invention has the effect of improving hEGF production by optimizing cell density, methanol concentration, and methanol injection interval in culture using the carbon source used for the growth medium and the hEGF production medium.

1 is a schematic schematic diagram of a DNA cassette for hEGF production.
2 is a schematic diagram of a hEGF expression vector map into which a DNA cassette for hEGF production is introduced.
3 is a view showing the production amount of hEGF according to the carbon source supplied to the growth medium.
4 is a view showing the production of hEGF according to the initial cell density supplied to the hEGF production medium.
5 is a diagram showing the production of hEGF according to the concentration of methanol supplied to a medium for hEGF production.
6 is a view showing the production of hEGF according to the injection interval of methanol supplied to the medium for hEGF production.

The production method of exocrine human epidermal growth factor according to the present invention will be described in detail with reference to the drawings. Unless otherwise defined, all terms in this specification have the same general meaning as understood by those of ordinary skill in the art to which the present invention belongs, and if they conflict with the meaning of the terms used in this specification, the According to the definition used in the specification. In addition, detailed descriptions of well-known functions and configurations that may unnecessarily obscure the gist of the present invention will be omitted. Throughout the specification, when a part "includes" a certain element, it means that other elements may be further included, rather than excluding other elements, unless otherwise stated.

When the production method of exocrine human epidermal growth factor according to an embodiment of the present invention is described with reference to FIGS. 1 to 6, the production method introduces the hEGF expression vector prepared so that the production protein is secreted out of the cell into Pichia pastoris. a recombination step of producing recombinant Pichia pastoris for hEGF production, a cell growth step of culturing the recombinant Pichia pastoris for hEGF production in a growth medium for cell growth, and a cell growth step in the cell growth step. Recombination for hEGF production after culture has been completed It includes a protein production step of culturing Pichia pastoris in a medium for hEGF production to produce hEGF. As previously seen, the conventional method for producing hEGF using P. pastoris has a low production efficiency of hEGF, so the present invention is a recombinant hEGF expression vector prepared so that the production protein is secreted out of the cell by introducing it into P. pastoris . Prepare P. pastoris , and sequentially culture the recombinant P. pastoris in a growth medium for cell growth and a medium for hEGF production, influence the carbon source of the growth medium, and cells in culture using the medium for hEGF production By analyzing the effects on density, methanol concentration and methanol injection interval, production conditions were optimized to maximize hEGF production.

The recombination step is a step to prepare a recombinant Pichia pastoris for hEGF production by introducing a hEGF expression vector prepared so that the production protein is secreted out of the cell into Pichia pastoris, a promoter for hEGF expression, hEGF expression and then extracellular secretion A cassette production step of preparing a DNA cassette for hEGF production comprising a polynucleotide sequence encoding a signal sequence and hEGF for and a transformation step of introducing a hEGF expression vector into Pichia pastoris to produce recombinant Pichia pastoris for hEGF production. The Pichia pastoris is a GRAS (Generally recognized as safe) strain that expresses and secretes peptides and proteins, and grows using methanol in addition to glucose, glycerol, and the like.

The cassette manufacturing step is a step of preparing a DNA cassette for hEGF production including a promoter for hEGF expression, a signal sequence for extracellular secretion after hEGF expression, and a polynucleotide sequence encoding hEGF. As the promoter for the hEGF expression, a known promoter suitable for expression in Pichia pastoris may be used, for example, the AOX1 promoter induced by methanol and repressed by glucose may be used, and the hEGF sequence is inserted after the AOX1 promoter to By supplying methanol, it becomes possible to produce hEGF. As the signal sequence, a known signal sequence that can be secreted out of cells after expression of hEGF may be used, for example, an α-mating factor signal sequence derived from S. cerevisiae may be used. The DNA cassette for hEGF production includes a sequence DDDDK recognized by Enterokinase (the cleavage enzyme recognizes the sequence DDDDK and cuts after K) for final hEGF separation and purification, His-tag, an affinity tag for high-efficiency separation and purification, and It may further include a termination sequence (Terminator).

The expression vector production step is a step of preparing the hEGF expression vector by inserting the DNA cassette for hEGF production into the expression vector. As the expression vector into which the DNA cassette for hEGF production is inserted, a known expression vector may be used, for example, pPinkα -HC can be used.

The transformation step is a step for preparing recombinant Pichia pastoris for hEGF production by introducing the hEGF expression vector into Pichia pastoris.

The cell growth step is a step of culturing the recombinant Pichia pastoris for hEGF production in a growth medium for cell growth, and the growth medium may be a conventional medium used for cell growth of yeast, but preferably For example, a medium using glucose as a carbon source may be used. Since glucose, glycerol, etc. inhibit the methanol pathway including the AOX1 promoter, the cell density was increased by culturing recombinant Pichia pastoris for hEGF production in a growth medium, and then cultured in a methanol-injected medium for hEGF production to hEGF. can improve the production of

The protein production step is a recombinant hEGF production step in which the culture for cell growth is completed in the cell growth step. In a step of culturing Pichia pastoris in the hEGF production medium to produce hEGF, for example, recombinant Pichia pastoris for hEGF production is supplied to the hEGF production medium at a cell density of 8 to 12OD ₆₀₀ , and 0.65 to 0.75% ( v/v) concentration of methanol is additionally injected every 7 to 8 hours and cultured to generate hEGF. As the recombinant medium for producing hEGF, various conventional medium including methanol used for protein production in yeast may be used.

Hereinafter, the present invention will be described in more detail through examples. However, these are only for describing the present invention in more detail, and the scope of the present invention is not limited thereto.

<Example 1> Preparation of recombinant P. pastoris for hEGF production

1. Preparation of DNA Cassette for hEGF Production

Promoter for hEGF expression, signal sequence for extracellular secretion after hEGF expression, His-tag as affinity tag for high-efficiency separation and purification, and Enterokinase for final separation and purification of hEGF A DNA cassette for hEGF production as shown in FIG. 1 was prepared, including a sequence DDDDK, a polynucleotide sequence encoding hEGF (SEQ ID NO: 1), and a terminator sequence. As the promoter, the AOX1 promoter using methanol induction was used, and the α-mating factor signal sequence derived from S. cerevisiae was used as the signal sequence.

2. Preparation of hEGF expression vector and transformation of P. pastoris

(1) The hEGF production cassette was inserted into the expression vector pPinkα-HC (Invitrogen) to prepare a hEGF expression vector (pBJY_hEGF) as shown in FIG. 2 .

(2) The hEGF expression vector was digested with AflII for linearization, and 5 μg of the digested hEGF expression vector was mixed with P. pastoris competent cells (Pichia EasyComp Kit (K1730-01), Invitrogen) and transformed to produce hEGF A recombinant P. pastoris strain was obtained.

(3) Recombinant P. pastoris strains for hEGF production were selected on a PAD (-Adenine) plate. On the other hand, in order to confirm the insertion of the hEGF expression vector into the genome of the recombinant P. pastoris strain for hEGF production, Colony PCR (primers used in Colony PCR are forward: 5'-GACTGGTTCCAATTGACAAGC-3' [sequence code: 2] and reverse : 5'-CCAAAACCTTCTCAAGCAAGGTTTTCA-3' [SEQ ID NO: 3]) was performed, and as a result, bands were not confirmed in the P. pastoris strain as a negative control on the agarose gel, but the P. pastoris strain into which the hEGF expression vector was inserted was The band was confirmed, and it was confirmed that the hEGF expression vector was introduced into the P. pastoris strain.

<Example 2> Production of hEGF using recombinant P. pastoris for hEGF production

1. Cultivation of recombinant P. pastoris for hEGF production

(1) The recombinant P. pastoris strain 1 colony for hEGF production was inoculated into 80 mL growth medium in a 250 mL baffled flask, and then seed culture was performed in a shaking incubator at 30° C. and 250 rpm for 24 h.

(2) The seed cultured P. pastoris was inoculated into 80 mL growth medium in a 250 mL baffled flask at a concentration of 5% (v/v), and cultured for cell growth was performed at 30° C. and 250 rpm in a shaking incubator for 24 h. The growth medium contains 10 g of yeast extract, 20 g of peptone, 13.4 g of YNB, 100 mM potassium phosphate at pH 6.0, 0.0004 g of biotin, and 10 g of carbon source per 1 L of distilled water.

(3) The cell density of recombinant P. pastoris for hEGF production, which has been cultured for cell growth, was adjusted to a certain optical density (OD600), respectively, and centrifuged at 3,000 × g in a centrifuge for 5 minutes to obtain a cell pellet. ) was resuspended in 30mL hEGF production medium in a 250mL baffled flask, incubated in a shaking incubator at 30°C and 250rpm for 96h, and methanol at a certain concentration was additionally injected at regular time intervals to generate hEGF. The hEGF production medium contains 10 g of yeast extract, 20 g of peptone, 13.4 g of YNB, 100 mM potassium phosphate at pH 6.0, 0.0004 g of biotin, and 5 g of methanol per 1 L of distilled water. That is, in the culture of recombinant P. pastoris for hEGF production, the carbon source, cell density, methanol concentration and injection interval were set as variables.

2. Purification and analysis of hEGF from culture of recombinant P. pastoris for hEGF production

(1) Since the produced hEGF is his-tag attached, it can be purified with dynabeads (Dynabeads His-Tag Isolation & Pulldown, Invitrogen), so 2X Binding/Wash buffer (100mM sodium phosphate pH 8.0, 600mM NaCl, 0.02%) tween-20) and His elution buffer (300mM imidazole, 50mM sodium phosphate pH 8.0, 300mM NaCl, 0.01% tween-20) were prepared, 2X Binding/Wash buffer was diluted to 1X, and 50μL dynabead was poured into a 1.5mL microtube. After transfer, separated with a magnet for 2 minutes, the supernatant separated with a magnet is removed using a pipette, and 350 μL and 350 μL of the sample finally obtained in Example 2 1 and 350 μL 1X Binding/Wash Buffer are placed in a microtube and 5 using a roller After mixing at room temperature for a minute, the mixed solution was separated with a magnet for 2 minutes, and the supernatant was removed. Using 300μL 1X Binding/Wash Buffer, place the beads in the microtube on a magnet for 2 minutes, discard the supernatant, and wash 4 times. After adding 100μL His-Elution Buffer to the microtube containing beads, Mix the microtube well using a roller for 1 minute, place the microtube on a magnet for 2 minutes to separate beads, and finally purify hEGF through the supernatant.

(2) After hEGF purification, qualitative analysis was performed using SDS-page to confirm that hEGF was produced from the cultured recombinant P. pastoris for hEGF production, and the amount of hEGF produced was confirmed through HPLC analysis. For HPLC analysis, mobile phase A was prepared by dissolving 0.1% (v/v) trifluoroacetic acid (TFA) in water for HPLC, and mobile phase B was prepared by dissolving 0.1% (v/v) TFA in acetonitrile for HPLC, followed by Agilent 1260 infinityⅡ HPLC. The analysis was performed on a Zorbax 300SB-C18 column (300A 5μ, agilent) using the system.

3. Review of hEGF production according to carbon source

(1) Using glycerol, glucose, sucrose, and sorbitol as carbon sources, cell density of 10OD _600, methanol concentration of 0.5% (v/v), and methanol injection interval of 12 hours, 1 and 2 of Example 2 process, the cell density was measured after culture for hEGF production, and the amount of hEGF produced was analyzed using HPLC, and the results are shown in FIG. 3 .

(2) Since glycerol inhibits the methanol pathway including the AOX1 promoter, the cell density is increased by culturing P. pastoris in a growth medium, and then cultured in a methanol-infused hEGF production medium to improve the production of hEGF. In other words, the production of hEGF can be improved by increasing the cell density through optimization of the growth medium.

(3) Referring to FIG. 3, it can be seen that glucose produces the most hEGF (33.32 mg/L) and the cell density ( 30.63OD ₆₀₀ ) is high. can be seen to be effective.

4. Review of hEGF production according to initial cell density

(1) The initial cell density was 2, 6, 10, 14, and 18OD ₆₀₀ , respectively, using glucose as a carbon source, methanol concentration of 0.5% (v/v), and methanol injection interval of 12 hours. Processes 1 and 2 of Example 2 were performed, cell density was measured after culture for hEGF production, and the amount of hEGF produced was analyzed using HPLC, and the results are shown in FIG. 4 .

(2) Referring to FIG. 4 , as the initially injected cell density gradually increased, the cell density after culturing for hEGF production increased, but the hEGF production showed little change after the initial injected cell density was 10OD ₆₀₀ . As it can be confirmed, it can be seen that _10OD600 is the initial cell density capable of producing optimal hEGF.

5. Review of hEGF production according to methanol concentration

(1) The methanol concentration was 0.1, 0.3, 0.5, 0.7, 0.9, 1.1% (v/v), respectively, using glucose as a carbon source, cell density of 10OD ₆₀₀ , and methanol injection interval of 12 hours, Processes 1 and 2 of Example 2 were performed, cell density was measured after culture for hEGF production, and the amount of hEGF produced was analyzed using HPLC, and the results are shown in FIG. 5 .

(2) Referring to FIG. 5, both cell density and hEGF production have a maximum value at a methanol concentration of 0.7% (v/v), and it can be confirmed that cell density and hEGF production are suppressed after 0.7% (v/v). there was. This is because as the amount of the inducer methanol increases, the production increases due to overexpression of the hEGF-producing gene, but excessive methanol produces toxic substances such as hydrogen peroxide and formaldehyde that negatively affect cell growth and protein production. .

6. Review of hEGF production according to methanol injection interval

(1) The methanol injection interval was 4, 8, 12, 16, 20 h, glucose was used as the carbon source, the cell density was 10OD _{600, and the} methanol concentration was 0.7% (v/v). Processes 1 and 2 were performed, cell density was measured after culture for hEGF production, and the amount of hEGF produced was analyzed using HPLC, and the results are shown in FIG. 6 .

(2) Referring to FIG. 6 , the maximum hEFG production (46 mg/L) was obtained when the injection interval was 8 hours, and the cell density was low when the injection interval was 4 hours, but the hEGF production amount compared to the case where the injection interval was 16 hours. was high, and it was confirmed that the cell density and hEGF production decreased after the injection interval was 8 hours. Therefore, the appropriate injection interval was 8 hours at which methanol had the most positive effect on hEGF production as an inducer without inhibiting the growth of the strain. You can tell it's time

In the above, the applicant has described various embodiments of the present invention, but these embodiments are only one embodiment that implements the technical idea of the present invention, and any changes or modifications are not allowed as long as the technical idea of the present invention is implemented. should be construed as falling within the scope of

<110> Kwangwoon University Industry-Academic Collaboration Foundation <120> Method for producing excreted human epidermal growth factor <130> PDAHJ-20220 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> hEGF <400> 1 Asn Ser Tyr Pro Gly Cys Pro Ser Ser Tyr Asp Gly Tyr Cys Leu Asn 1 5 10 15 Gly Gly Val Cys Met His Ile Glu Ser Leu Asp Ser Tyr Thr Cys Asn 20 25 30 Cys Val Ile Gly Tyr Ser Gly Asp Arg Cys Gln Thr Arg Asp Leu Arg 35 40 45 Trp Trp Glu Leu Arg 50 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 2 gactggttcc aattgacaag c 21 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 3 ccaaaacctt ctcaagcaag gttttca 27

Claims (6)

A recombination step of introducing a hEGF expression vector prepared so that the production protein is secreted out of the cell into Pichia pastoris to prepare a recombinant Pichia pastoris for hEGF production, and growing the recombinant Pichia pastoris for hEGF production for cell growth The cell growth step of culturing in a medium, and the recombinant for hEGF production in which the culture for cell growth is completed in the cell growth step. A method for producing human epidermal growth factor comprising the step of culturing Pichia pastoris in a medium for hEGF production to produce hEGF. According to claim 1,
The growth medium is a method for producing human epidermal growth factor, characterized in that using glucose as a carbon source. 3. The method of claim 2,
The recombinant medium for hEGF production is a method for producing human epidermal growth factor, characterized in that it contains methanol. 4. The method of claim 3,
In the protein production step, recombinant Pichia pastoris for hEGF production is supplied to the hEGF production medium at a cell density of 8 to 12OD ₆₀₀ , and methanol at a concentration of 0.65 to 0.75% is added to the hEGF production medium at intervals of 7 to 8 hours. A method for producing human epidermal growth factor, characterized in that it is additionally injected and cultured. 4. The method of claim 3,
The recombination step includes a cassette production step of preparing a DNA cassette for hEGF production comprising a promoter for hEGF expression, a signal sequence for extracellular secretion after hEGF expression, and a polynucleotide sequence encoding hEGF, and the hEGF production DNA cassette An expression vector production step of preparing a hEGF expression vector by inserting the Method for producing human epidermal growth factor. 6. The method of claim 5,
The promoter for hEGF expression is a method for producing human epidermal growth factor, characterized in that a promoter induced by methanol and suppressed by glucose is used.

Images