KR20230111871A - synthesis method of EGF using E. coli - Google Patents

Abstract

The present invention relates to a method for synthesizing EGF using E. coli, and according to a manufacturing method according to one aspect, by expressing an EGF protein through E. coli, it is possible to obtain the EGF protein with high purity and efficiency. One aspect provides the method for manufacturing an EGF protein, which comprises the steps of: cloning the gene for EGF into an expression vector; inserting the expression vector into a host cell; and culturing the host cells in a medium to express the EGF protein.

Description

Manufacturing method of EGF using E. coli {synthesis method of EGF using E. coli}

It relates to a method for producing EGF using E. coli.

The skin is a tissue that accounts for 15-20% of the total body mass, and as the primary barrier, it protects from external environmental stimuli such as changes in temperature and humidity, ultraviolet rays and pollutants, and plays an important role in maintaining homeostasis of the human body. The skin can be divided into three layers: epidermis, dermis, and subcutaneous fat tissue.

When a wound occurs in such skin tissue, mesenchymal stem cells secrete various growth factors and cytokines such as epidermal growth factor (EGF) and fibroblast growth factor (FGF) to stimulate the production of collagen from fibroblasts to regenerate the skin.

EGF is a potent cell division promoter of various epithelial cells originating from ectoderm and mesoderm. It is widely distributed in body fluids and is present in high concentrations in urine and breast milk (Capenter, 1985). It is a polypeptide with a molecular weight of 6,200 daltons (Capenter and Cohen, 1979; Capenter and Wahl, 1990). EGF acts as a strong promoter of epithelial cell, endothelial cell, and fibroblast cell proliferation, and has excellent wound healing effect because it promotes epithelial cell migration and proliferation in case of epithelial cell defect.

Based on these characteristics of EGF, EGF is widely used as a wound healing agent. Therefore, it is important to produce EGF with high efficiency and high purity, and research on this is required.

Korean Patent Publication 10-2021-0145705

In one aspect, cloning (cloning) the gene of epidermal growth factor (epidermal growth factor: EGF) into an expression vector; inserting the expression vector into a host cell; and culturing the host cells in a medium to express the EGF protein.

In one aspect, cloning the gene of EGF into an expression vector; inserting the expression vector into a host cell; and culturing the host cells in a medium to express the EGF protein.

According to one embodiment, the expression vector may be pPOPINTRX, pET32a, pET15b, pET22b or pET27b. It may also include artificial plasmids suitable for gene expression in bacterial cells.

Molecular cloning techniques are known to those skilled in the art and are described in “Molecular Cloning: A Laboratory Manual” by Sambrook and Russell. A recombinant vector can be created by inserting the EGF or FGF gene into an expression vector by cloning techniques.

As used herein, the term "recombinant vector" can be used as an expression vector of a target polypeptide capable of expressing the target polypeptide with high efficiency in an appropriate host cell when the coding gene of the target polypeptide to be expressed is operably linked, and the recombinant vector can be expressed in the host cell.

According to one embodiment, the host cell may be a microbial cell, such as a bacterium, archaea, yeast or fungus. For example, it may be Escherichia coli ( E. coli ).

The step of inserting the expression vector into the host cell may be accomplished using a method commonly known to those skilled in the art, for example, transformation or electroporation.

According to one embodiment, the preparation method may further include isolating the expressed recombinant EGF protein. Recombinant EGF protein can be obtained through the above separation step. The separation may include a centrifugation step of centrifuging the host cells to obtain a cell pellet, and may further include resuspending the cell pellet in a buffer.

The EGF gene may include an EGF mutant gene.

As used herein, the term "variant" refers to a gene sequence consisting of a DNA sequence in which the DNA sequence of a gene is randomly mutated, and includes a functional equivalent of a nucleic acid molecule, for example, a variant in which some base sequences have been modified by deletion, substitution, or insertion, but can functionally perform the same function as the existing nucleic acid molecule. Such variants may include nucleotide sequences having sequence homology of 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more, respectively, with the existing nucleotide sequence. The "percentage of sequence homology" for polynucleotides is determined by comparing two optimally aligned sequences with a region of comparison, wherein a portion of the polynucleotide sequence in the region of comparison may contain additions or deletions (i.e., gaps) relative to the reference sequence (not including additions or deletions) for the optimal alignment of the two sequences.

The step of expressing the protein may be induced by adding an inducer.

In one embodiment, the derivative may be isopropyl β-D-1-thiogalactopyranoside (IPTG), and the final concentration of IPTG added is 0.001 to 1 mM, for example, 0.01 to 1 mM, 0.05 to 1 mM, 0.1 to 1 mM, 0.2 to 1 mM, 0.3 to 1 mM , 0.4 to 1 mM, 0.5 to 1 mM, 0.6 to 1 mM, 0.7 to 1 mM, 0.8 to 1 mM, 0.9 to 1 mM, 0.01 to 0.9 mM, 0.01 to 0.8 mM, 0.01 to 0.7 mM, 0.01 to 0.6 mM, 0.01 to 0.5 mM, 0.01 to 0 0.4 mM, 0.01 to 0.3 mM, 0.01 to 0.2 mM or 0.01 to 0.1 mM.

The IPTG is at a temperature of 15 to 30 ℃, for example, 15 to 25 ℃, 16 to 25 ℃, 17 to 25 ℃, 18 to 25 ℃, 19 to 25 ℃, 20 to 25 ℃, 21 to 25 ℃, 22 to 25 ℃, 23 to 25 ℃, 24 to 25 ℃, 15 to 20 ℃, 16 to 25 ℃ It may be added at 20 ° C, 17 to 20 ° C, 18 to 20 ° C, 19 to 20 ° C, 17 to 23 ° C, 18 to 23 ° C, 19 to 23 ° C, 20 to 23 ° C, 21 to 23 ° C or 22 to 23 ° C.

According to one embodiment, the preparation method may further include purifying the expressed EFG protein.

The purification may be carried out using a purification method known in the art, for example, centrifugation, size exclusion chromatography, ion exchange chromatography, electrophoresis, affinity chromatography, filtration and high performance liquid chromatography (HPLC), or a combination thereof. The centrifugation may be, for example, ultracentrifugation, and the filtration may be, for example, diafiltration.

In addition, a gene encoding a tag for separation and purification may be additionally included to facilitate purification of the target protein. These types of tags are amino acid sequences that allow for affinity purification, solubilization, chromatographic separation and/or immunodetection of the fusion protein.

As used herein, the term "fusion protein" refers to a protein formed from the combination of at least two different proteins or protein fragments, and is encoded by a recombinant DNA molecule.

The tag for separation and purification includes chitin binding protein (CBP), biotin carboxyl carrier protein (BCCP), maltose binding protein (MBP), glutathione-S-transferase (GST), poly-His, thioredoxin (Trx), small ubiquitin -like modifier: SUMO) protein, NusA, FLAG-tag, V5-tag, Myc-tag, HA-tag, S-tag, SBP-tag, Sftag 1, Softag 3, Tc-tag, Xpress-tag, Strep-tag, Spy-tag, Ty-tag, Nus-tag, or a combination thereof, but is not limited thereto. In addition, those skilled in the art can use an appropriate tag for separation and purification according to the purpose. For example, in the case of the maltose binding protein, the wide hydrophobic gaps made by a hydrophobic amino acid involved in the parts involved in the formation of the dipper effectively hide the hydrophobic site of the newly synthesized protein, preventing the purpose protein from becoming insoluble aggregate, and thiore single helps to bond the daisulfide of the purpose protein. In the case of high, NUSA, when it is expressed in exceeding E. coli, the ability to fold into active type is very excellent, which can induce the correct folding of the objective protein.

According to one embodiment, the fusion protein may be (His) ₆ -Trx-EGF fusion protein.

According to the manufacturing method according to one aspect, there is an effect of obtaining EGF protein with high purity and high efficiency by expressing the EGF fusion protein through E. coli and going through a purification process.

1 is a graph showing the mass spectrometry results of the central fraction of purified EGF protein.
Figure 2 is a graph showing the mass spectrometry results of the side fraction of the purified EGF protein.

It will be described in more detail through the following examples. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

Example 1. E. coli Preparation of EGF using

1.1. EGF gene cloning

After the EGF gene represented by SEQ ID NO: 1 was amplified through PCR, the EGF gene was inserted into the MCS site of the vector. and e. coli to induce transformation. At this time, N-terminal His-tag and TRX were used as tags for separation and purification. As vectors, pPOPINTRX and pET32a were used.

Specifically, after cutting the pPOPINTRX and EGF genes using restriction enzymes, pPOPINTRX and EGF genes were linked by dna ligation to prepare a recombinant plasmid. Thereafter, heat shock was applied to DH5a at a temperature of about 42° C. for 30 seconds to introduce the plasmid to induce transformation and secure a recombinant pPOPINTRX-EGF plasmid.

Recombinant pET32a-EGF plasmid was obtained by cloning pET32a in the same manner as in the pPOPINTRX vector.

1.2. EGF protein production and purification

Each of the recombinant plasmids obtained in Example 1.1 was transformed into BL21 DE3, and cultured in LB medium containing 100 mg/mL of ampicillin at 30 °C until the OD ₆₀₀ reached 0.8. Thereafter, the BL21 DE3 was incubated for 2 hours at 37° C. in a flask, and IPTG, an expression inducer, was added to a final concentration of 1 mM at 20° C. to induce EGF expression. Thereafter, the culture medium was centrifuged at 3500 g for 30 minutes at 4 ° C to harvest the cell pellet, and resuspended in Tris (20 mm), NaCl (500 mm), imidazole (5 mm) and Tween20 (0.2%, v / v, pH 8.0) and frozen. After thawing, the resulting supernatant was incubated with Ni-NTA (Qiagen) by centrifuging the lysate at 48° C. for 30 minutes at 35,000 g after disruption by ultrasonication for 1 to 3 hours. After that, it was washed with buffer and (His) ₆ -Trx-EGF fusion protein was eluted with imidazole (400 mM). The (His) _6- Trx-EGF fusion protein was further purified on a Superdex 75 column in sodium phosphate (20 mm), and cleaved overnight by adding 1 mg of HRV3C protease per 50 mg of fusion protein under the conditions of NaCl (250 mm), EDTA (0.2 mm) and sodium azide 0.01% (w/v), pH 7.4, at 48 ° C. After cleavage, the supernatant was treated with Ni-NTA resin to remove the (His) ₆ -Trx tag. And after recovering through the flow-through fraction, it was further purified through size exclusion chromatography on a Superdex 75 column in NMR buffer to obtain EGF protein. After measuring the molecular weight of the purified EGF protein by mass spectrometry through electrospray analysis, the results are shown in FIGS. 1 and 2 .

1 is a graph showing the mass spectrometry results of the central fraction of purified EGF protein.

Figure 2 is a graph showing the mass spectrometry results of the side fraction of the purified EGF protein.

As shown in Figures 1 and 2, it was confirmed that the mass of the purified EGF protein was identical to that of the EGF monomer.

Claims (9)

Cloning the gene of epidermal growth factor (EGF) into an expression vector;
inserting the expression vector into a host cell; and
EGF protein production method comprising the step of culturing the host cell in a medium to express the EGF protein. The method of claim 1,
The expression vector is pPOPINTRX, pET32a, pET15b, pET22b or pET27b, EGF protein production method. The method of claim 1,
The host cell is Escherichia coli ( E. coli ), EGF protein production method. The method of claim 1,
The step of expressing the protein is induced by the addition of an inducer, EGF protein production method. The method of claim 4,
The derivative is isopropyl β-D-1-thiogalactopyranoside (isopropyl β-D-1-thiogalactopyranoside: IPTG), EGF protein production method. The method of claim 5,
The final concentration of the IPTGT is 0.001 to 1 mM, EGF protein production method. The method of claim 1,
The expression vector further comprises a gene encoding a tag for separation and purification, EGF protein production method. The method of claim 7,
The tag for separation and purification includes chitin binding protein (CBP), biotin carboxyl carrier protein (BCCP), maltose binding protein (MBP), glutathione-S-transferase (GST), poly-His, thioredoxin (Trx), small ubiquitin -like modifier: SUMO) protein, NusA, FLAG-tag, V5-tag, Myc-tag, HA-tag, S-tag, SBP-tag, Sftag 1, Softag 3, Tc-tag, Xpress-tag, Strep-tag, Spy-tag, Ty-tag, and Nus-tag. The method of claim 1,
EGF protein production method further comprising the step of purifying the expressed EFG protein.