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WO2019112352A2 - Procédé de production d'une protéine adhésive de moule - Google Patents

Procédé de production d'une protéine adhésive de moule Download PDF

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Publication number
WO2019112352A2
WO2019112352A2 PCT/KR2018/015451 KR2018015451W WO2019112352A2 WO 2019112352 A2 WO2019112352 A2 WO 2019112352A2 KR 2018015451 W KR2018015451 W KR 2018015451W WO 2019112352 A2 WO2019112352 A2 WO 2019112352A2
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WO
WIPO (PCT)
Prior art keywords
mussel adhesive
adhesive protein
protein
obtaining
inclusion body
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KR2018/015451
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English (en)
Korean (ko)
Other versions
WO2019112352A3 (fr
Inventor
서동식
백규원
구희관
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Amo Lifescience Co Ltd
Original Assignee
Amo Lifescience Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Amo Lifescience Co Ltd filed Critical Amo Lifescience Co Ltd
Publication of WO2019112352A2 publication Critical patent/WO2019112352A2/fr
Publication of WO2019112352A3 publication Critical patent/WO2019112352A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans

Definitions

  • the present invention relates to a method for producing a mussel adhesive protein, and more particularly, to a method for producing a mussel adhesive protein capable of obtaining a mussel adhesive protein having high purity, high yield and stability.
  • the mussel adhesive protein is a protein rich in L-dihydroxylalanine (DOPA), has excellent adhesion and has strong adhesion to various surfaces. It does not attack human cells and does not cause immune reaction. There is a high possibility of application in the field.
  • DOPA L-dihydroxylalanine
  • the conventional method for culturing and purifying mussel adhesive proteins is a method for purifying mussel adhesive proteins containing a physiologically active peptide at the terminal due to the irreversible recovery due to the purification method using a high concentration of acid and salt, In the case of purification, loss phenomenon appeared. This is also disclosed in Korean Patent Publication No. 10-2017-0017499.
  • the present invention has been conceived to solve the above-mentioned problems, and it is an object of the present invention to provide a mussel adhesive protein capable of obtaining a highly pure, high-efficiency and stable mussel adhesive protein by adjusting pH to 9.0 to 10.0 using NaCO 3 ,
  • the purpose of the production method is to provide.
  • Another object of the present invention is to provide a mussel adhesive protein capable of obtaining a high-purity, high-yield and stable mussel adhesive protein through extraction with acetic acid, n-propanol and DMSO, And to provide a production method.
  • the present invention provides a method for producing a mussel adhesive protein, comprising: expressing a mussel adhesive protein using microbial culture; Obtaining an inclusion body comprising the mussel adhesive protein from the microorganism; And obtaining the mussel adhesive protein from the inclusion body using an extraction solvent of at least one of organic acid, alcohol, chaotropic agent and sulfoxide compound.
  • the organic acid may be acetic acid.
  • the alcohol may be n-propanol.
  • the chaotropic agent may be guanidine hydrochloride.
  • the sulfoxide compound may be dimethyl sulfoxide (DMSO).
  • the extraction solvent comprises at least one of the organic acid, the alcohol, the chaotropic agent and the sulfoxide compound, and the organic acid is 15 to 50 wt%, the alcohol is 5 To 15% by weight of the sulfoxide compound, and 5% by weight of the sulfoxide compound.
  • the step of obtaining the inclusion body comprises: breaking the microorganism to obtain a disruption liquid; And centrifuging the disruption solution using at least one of a salt and a pH adjuster to obtain the inclusion body.
  • the salt may be at least one of NaCl and ammonium sulfate.
  • the pH adjusting agent may be at least one of sodium carbonate and sodium borate.
  • the step of obtaining the mussel adhesive protein comprises the steps of: obtaining the mussel adhesive protein extract solution from the inclusion body using the extraction solvent; Adding acetone to the mussel adhesive protein extraction solution to form a mussel adhesive protein precipitate; And dissolving the mussel adhesive protein precipitate in distilled water, followed by purification by ion chromatography to obtain the mussel adhesive protein.
  • the step of obtaining the mussel adhesive protein may further include desalting and lyophilizing the mussel adhesive protein.
  • the present invention increases the insolubility of the recombinant protein by increasing the pH by using a salt and NaCO 3 in the microbial disruption solution, and provides a recovery rate of 90% or more to the inclusion body.
  • Addition of a Chaotropic agent, n-propanol The protein can be extracted at a high yield compared to the conventional method of extraction and the ion chromatography method is used to remove the impurities and improve the purity of the protein, A mussel adhesive protein having high protein purity and stability can be obtained.
  • 1 is a photograph showing an outline of a method for producing a mussel adhesive protein according to an embodiment of the present invention.
  • Fig. 2 is a photograph showing that when the unmilled disrupted liquid was centrifuged, the recovery rate of the mussel adhesive protein to the pellet was increased when a salt was added to the mussel adhesive protein while the mussel adhesive protein did not substantially fall into the pellet.
  • FIG. 3 is a photograph showing that the NaCl 3 was added to the disruption solution at a ratio of 1% to raise the pH and then centrifuged to increase the recovery rate of the mussel adhesive protein falling into the pellet in the aqueous solution.
  • FIG. 4 is a photograph showing the sedimentation efficiency of mussel adhesive protein by adding acetone to the extracted mussel adhesive protein solution by volume.
  • FIG. 5 is a photograph showing the result of final purification of the mussel adhesive protein precipitated with acetone by redissolving in distilled water and using ion chromatography.
  • the method for producing a mussel adhesive protein of the present invention comprises the steps of: (a) expressing a mussel adhesive protein using microorganism culture; (b) disrupting the microorganism to obtain a disruption liquid; (c) centrifuging the above-mentioned disruption liquid using at least one of a salt and a pH adjusting agent to obtain the inclusion body; (d) obtaining the mussel adhesive protein extract solution from the inclusion body using an extraction solvent; (e) adding acetone to the mussel adhesive protein extraction solution to form a mussel adhesive protein precipitate; And (f) dissolving the mussel adhesive protein precipitate in distilled water and purifying it by ion chromatography to obtain the mussel adhesive protein. And a method for producing a mussel adhesive protein having stability, and a specific method is illustrated in FIG. 1, and a detailed description thereof is as follows.
  • the method for producing a mussel adhesive protein of the present invention is a method for producing a mussel adhesive protein by overexpressing a mussel adhesive protein having a mussel adhesive protein and a physiologically active motif (motif) linked thereto in a basic yeast culture using microorganisms (for example, Escherichia coli) do.
  • a mussel adhesive protein having a mussel adhesive protein and a physiologically active motif (motif) linked thereto in a basic yeast culture using microorganisms (for example, Escherichia coli) do.
  • microorganisms into which a mussel adhesive protein expression vector has been inserted for example, E. coli BL21 (DE3) can be produced by using IPTG in a culture medium containing LB medium containing normal ampicillin or carbon source and nitrogen source, The adhesive protein is overexpressed, and a microorganism culture solution is obtained.
  • the crushing method of the present invention belongs to a mechanical method and is a high-pressure crushing method using presses, and is a hollow cylindrical cylinder made of stainless steel widely used in a laboratory scale. After filling the microorganisms to be crushed, they are crushed by extruding under atmospheric pressure through a needle valve at the bottom of the cylinder under high pressure.
  • the high-pressure homogenizer is used, and the microorganism is disrupted at a condition of at least 800 bar to obtain a cell disruption solution containing an inclusion body containing the mussel adhesive protein.
  • the present invention provides a mussel adhesive protein extraction solution using at least one extraction solvent of an organic acid, an alcohol, a chaotropic agent and a sulfoxide compound, and shows an increase in extraction efficiency of up to 50% Respectively.
  • Table 1 shows the details of this are shown in Table 1 below when the extraction efficiency of the control (25% by weight of acetic acid) is taken as 100%, and the extraction efficiency is increased up to 150% in the composition of No. 3.
  • the organic acid is acetic acid
  • the alcohol is n-propanol
  • the chaotropic agent is Guanidine hydrochloride
  • the sulfoxide compound is DMSO (dimethyl sulfoxide).
  • the sulfoxide compound can induce protein unfolding, thereby improving extraction efficiency.
  • the extraction solvent comprises at least one of the organic acid, the alcohol, the chaotropic agent, and the sulfoxide compound, wherein the organic acid is 15 to 50 wt%, the alcohol is 5 to 15 wt%
  • the sulfoxide compound may be included in an amount of 5% by weight.
  • Table 1 shows that when one or more of acetic acid, n-propanol and DMSO is mixed as an extraction solvent to extract the mussel adhesive protein contained in the inclusion body, Efficiency can be confirmed. In addition, when 30% by weight of acetic acid and 15% by weight of n-propanol were used as an extraction solvent, it was confirmed that the highest extraction efficiency was obtained.
  • the mussel adhesive protein extract can be recovered as a pellet-shaped mussel adhesive protein precipitate using protein precipitation using acetone, and it can also remove acetic acid at high concentration.
  • the mussel adhesive protein extracted using acetic acid was lyophilized and then entered into the chromatography step, which resulted in a time loss in freeze drying.
  • the protein precipitation method using acetone it has an advantage that it can enter the chromatographic step only by centrifuging and re-dissolving.
  • the mussel adhesive protein precipitate may be dissolved in distilled water to obtain an aqueous solution, and impurities may be removed by ion chromatography.
  • the mussel adhesive protein precipitate was poured into a resin-filled column, bound to the resin, and then 1 M NaCl was added to remove the impurities. After that, only the mussel adhesive protein could be recovered using 100 mM NaOH, The resulting mussel adhesive protein can be desalted and lyophilized.
  • LB medium containing glucose (20 g / L of glucose, 5 g / L of yeast extract, 5 g / L of potassium phosphate monobasic, 3 g of potassium phosphate dibasic / L, ammonium sulfate 1.5 g / L, and magnesium sulfate 0.5 g / L), and all media contained ampicillin antibiotics at a concentration of 50 ug / ml. This is due to the presence of the ampicillin antibiotic resistance site contained in the pET-22b (+) vector used in this experiment.
  • Glucose 20 g / L, yeast extract 5 g / L, potassium phosphate monobasic 5 g / L, potassium phosphate dibasic 3 g / L, ammonium sulfate 1.5 g (L), magnesium sulfate (0.5 g / L) and a small amount of anti-form (300 ⁇ 500 ul / L) were added to each well.
  • the pH probe and DO meter were set and sealed and sterilized at 121 °C for 15 min.
  • the injection amount of air was 1 VVM
  • the temperature of the incubator was 37 ° C
  • the speed of the stirrer was 800 rpm
  • 1 liter of the seed culture was added, followed by culturing.
  • IPTG was added to induce protein expression to 0.5 mM to 1 mM, followed by further culturing for 3 to 12 hours.
  • the culture was centrifuged to recover the cells, and the cells were suspended in distilled water and homogenized.
  • the homogenized solution was homogenized using a high-pressure homogenizer twice at 800 to 1000 bar, and the pH was adjusted to 9.0 to 10.0 by adding 0.5 M NaCl and 1 to 2% sodium carbonate. After centrifugation The pellet was recovered by separating. The pellet was homogenized with a solution containing 30% of acetic acid and 15% of n-propanol. After mixing for 30 minutes or more, the protein-extracted supernatant was recovered by centrifugation.
  • the pellet was recovered by centrifugation, diluted with distilled water, and adjusted to pH 5.0 or less using acetic acid.
  • the pH of the pellet was adjusted to 5.0 or less by adding acetic acid in an amount of 2 to 3 times that of the extracted supernatant.
  • the aqueous solution was centrifuged, and the supernatant was collected and loaded onto a cation exchange resin (SP sepharose) stabilized with 20 mM sodium acetate buffer (pH 4.5), washed with the same buffer, and washed with 20 mM sodium acetate buffer pH 4.5) solution to remove impurities.
  • SP sepharose cation exchange resin
  • the column was washed with 20 mM sodium acetate buffer (pH 4.5) and 100 mM NaOH solution was flowed to elute the protein to recover high purity protein.
  • the eluted protein was adjusted to pH 5.0 or less using acetic acid, desalted, and lyophilized.
  • the method of producing mussel adhesive protein of the present invention increases the insolubility of protein by raising the pH by using a salt and / or a pH adjuster to the microbial disruption solution to increase the recovery rate to the inclusion body to almost 100% And extracting the protein with a yield of up to 50% higher than the conventional method of extracting only acetic acid using an extraction solvent of at least one of organic acid, alcohol, chaotropic agent and sulfoxide compound.
  • the freeze-drying step before the entry into the conventional chromatography step is omitted, and acetone precipitation and re-dissolution are performed, thereby enabling time reduction during the process.
  • the purified protein was purified by using ion chromatography to remove impurities and increase the purity of the protein.
  • the purification method using the existing high concentration acid and salt, high purity of the protein recovered, high yield and high stability Respectively.
  • the present invention increases the insolubility of the recombinant protein by increasing the pH by using a salt and NaCO 3 in the microbial disruption solution to provide a recovery rate of 90% or more to the inclusion body, and adding a Chaotropic agent, n-propanol,
  • the protein can be extracted at a high yield compared to the conventional method of extraction and the ion chromatography method is used to remove the impurities and improve the purity of the protein, A mussel adhesive protein having high protein purity and stability can be obtained.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Analytical Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé de production d'une protéine adhésive de moule, qui permet d'obtenir une protéine adhésive de moule à une pureté élevée, un rendement élevé et une stabilité élevée, comprenant les étapes suivantes : expression d'une protéine adhésive de moule par culture d'un micro-organisme ; obtention d'un corps d'inclusion comprenant la protéine adhésive de moule à partir du micro-organisme ; et obtention de la protéine adhésive de moule à l'aide d'au moins un solvant d'extraction constitué d'un un acide organique, d'un alcool, d'un agent chaotropique ou d'un composé de sulfoxyde.
PCT/KR2018/015451 2017-12-06 2018-12-06 Procédé de production d'une protéine adhésive de moule Ceased WO2019112352A2 (fr)

Applications Claiming Priority (2)

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KR10-2017-0166993 2017-12-06
KR20170166993 2017-12-06

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WO2019112352A2 true WO2019112352A2 (fr) 2019-06-13
WO2019112352A3 WO2019112352A3 (fr) 2019-08-01

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119529049A (zh) * 2024-12-04 2025-02-28 合肥贝壳派创新科技有限公司 一种高纯度重组贻贝粘蛋白的制备方法
CN119735467A (zh) * 2024-12-09 2025-04-01 宁夏盛翊生物技术有限责任公司 一种环保型缓释肥及其制备方法
CN120399026A (zh) * 2025-03-20 2025-08-01 上海海洋大学 一种贻贝足丝腺黏蛋白亚基及其制备方法和应用

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KR950009838A (ko) * 1993-09-21 1995-04-24 이헌조 브라운관의 네크 테이핑장치
KR100868047B1 (ko) 2002-08-13 2008-11-10 주식회사 포스코 홍합 접착단백질 Ｍｇｆｐ-5 및 이의 생산방법
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KR101435606B1 (ko) * 2012-06-28 2014-08-29 포항공과대학교 산학협력단 홍합 접착 단백질에의 생체 내 잔기-특이적 ｄｏｐａ 도입 방법
KR101652263B1 (ko) * 2014-06-13 2016-08-30 (주)콜로디스 바이오사이언스 항균 펩티드를 포함하는 접착 단백질 및 이를 포함하는 항균 코팅 조성물
EA035448B1 (ru) * 2014-07-14 2020-06-17 Геннова Биофармасьютикалз Лимитед СПОСОБ ОЧИСТКИ рчГ-КСФ

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119529049A (zh) * 2024-12-04 2025-02-28 合肥贝壳派创新科技有限公司 一种高纯度重组贻贝粘蛋白的制备方法
CN119735467A (zh) * 2024-12-09 2025-04-01 宁夏盛翊生物技术有限责任公司 一种环保型缓释肥及其制备方法
CN120399026A (zh) * 2025-03-20 2025-08-01 上海海洋大学 一种贻贝足丝腺黏蛋白亚基及其制备方法和应用

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KR102870505B1 (ko) 2025-10-15
WO2019112352A3 (fr) 2019-08-01
KR20190067123A (ko) 2019-06-14

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