KR20150044195A - Production Methods of Foreign Protein Using Secretion Vector in Pleurotus eryngii - Google Patents

Abstract

The present invention relates to a secretion vector used for transformation of king trumpet mushrooms (Pleurotus eryngii) and to a target protein preparation method thereof. According to the present invention, the secretion vector includes expression constructs appropriate for the transformation of mushrooms. The method can be also used to successfully produce various target proteins such as growth hormone proteins from the transgenic mushrooms thereof. The recombination expression vector enables various industrially useful proteins to be mass-produced from mushrooms, and especially from the king trumpet mushrooms.

Description

{Production Methods of Foreign Protein Using Secretion Vector in Pleurotus eryngii}

The present invention relates to a mushroom transformed by introducing a protein secretion vector and a method for producing a target protein using the mushroom.

Useful protein materials are used not only for the treatment of human body but also for industrial use, and their use is getting wider, and their importance is becoming increasingly important worldwide. However, these useful proteins, especially those derived from animals, are produced in a very small amount in the living body. Therefore, in order to use proteins for therapeutic purposes, mass production is necessary. In the case of proteins separated from blood, And the possibility of residual cancer inducing factors. In order to solve the above-mentioned problems, the present invention can be solved by using a gene recombination technique which mass-produces a gene of a useful protein in a protein expression host cell such as a microorganism. BACKGROUND ART [0002] Genetic recombination technology is a technology for synthesizing a desired protein by introducing a gene having an important function for industrial and medical functions (enzyme, hormone, growth factor, etc.) into a cell such as microorganism or yeast, It is a technology that enables mass production. Since recombinant protein products were first sold by Genentech in 1982 to E. coli, insulin was approved by the FDA. According to the Industrial Technology Development Project of the Ministry of Commerce, Industry and Energy, about 130 kinds of protein products were sold in 2005 It is a high-value-added industry with a global market size estimated at about $ 50 billion, and in 2011 it is expected to reach $ 110 billion, with more than 100 multinational corporations participating worldwide. Therefore, protein production using genetic recombination technology is rapidly expanding into the protein market. Recombinant proteins are produced using various expression systems depending on the purpose of use, the type of protein, and the production scale. Typically used cells are microorganisms such as Escherichia coli or yeast, or cells such as insect cells, animal cells, and plant cells . In particular, protein expression using E. coli is easy to genetically manipulate, has a high growth rate of cells, is easy to cultivate, can be produced in a relatively large amount, and can be synthesized in an economical manner. However, when it is intended to produce proteins derived from eukaryotic cells such as glycoprotein, E. coli can not synthesize glycosylated proteins, and many proteins are insoluble proteins in the form of inclusion bodies which do not fold properly In order to produce biologically active products, refolding process is required and production of secreted proteins is difficult. Early recombinant protein drugs were mainly produced in E. coli, but as technology developed, the proportion of recombinant proteins produced in E. coli decreased in the 2000s, while production in yeast, insects, plant and animal cells is increasing. In the case of animal cells or insect cells, most of the protein derived from human body can be produced as a fully active protein, but it has a problem that the culture time is long, the culture condition is difficult, and the cost of the culture medium is high. The recombinant proteins produced using plants have chemical modifying functions such as saccharification and phosphorylation, which are essential for the biological activity of useful proteins derived from human body. In addition, it has the advantage of producing biologically stable proteins. It can be produced economically inexpensively by using sugar and salt as main components. However, it has disadvantages such as slow growth rate and low yield ( Biotechnol . Bioeng., 1997, 56, 473-484). Due to various problems of the recombinant protein production system, there is a need for a new recombinant protein production system.

Mushrooms are taxonomically belonging to the fungi (Eumycetes) of higher fungi, most of which belong to the family Mycobacterium. The mushroom is distributed in southern Europe, North Africa, East Asia, South Russia, etc. It has been widely used for edible purposes. Its scientific name is Pleurotus eryngii , and is a species of Basidiomycetes belonging to the genus Bacillus subtilis, the subgenus Bacillus subsp. The common name is King Oyster Mushroom or Boletus of the Steppes, and it has been known in Korea as large oyster mushroom or king oyster mushroom. This mushroom is a parasitic organism that attaches to the stump or stem of a dead tree and is able to grow in some plant tissues and has been reported as a pathogen causing illnesses in the roots of herbaceous plants such as mountain type,

On the other hand, Agrobacterium tumefaciens, a gram-negative bacterium, is known as a phytopathogenic bacterium that causes crown gall in plants. Agrobacterium tumefaciens has a Ti plasmid DNA that can interfere with plant cell chromosomal DNA and is widely used to introduce a desired gene into a plant. The Ti plasmid contains a number of vir genes necessary for the transport of Ti DNA and its insertion into plant cell chromosomal DNA and is induced by phenolic compounds such as acetosyringone, And transferred to the chromosomal DNA of the host cell in the form of single-stranded DNA ( Plant Physiol , 2000, 124, 13631371). Transformation of plants mediated by Agrobacterium has long been used to introduce genes into plants, has also been used to introduce mutations into plants, and is also used as a transformation method for white rot fungi. It has also been used as a successful transformation method for a variety of fungi such as Aspergillus, Fusarium and Trichoderma (Nature Biotechn., 1998, 16, 839- 842; J. Appl. Microbiol, 2002, 92, 189-195; Symbiosis. J, 2006, 41, 71-79). However, up to now, the transgenic transformation system that introduces genes that produce useful proteins in mushrooms has been limited in the attempted studies, and thus industrialization research on the production of useful recombinant proteins has not been done yet.

Growth hormone, one of the useful proteins, is synthesized as a protein consisting of 217 amino acids in the anterior pituitary gland, and then the leader peptide of the N-terminal part is removed, resulting in 191 (N-terminal alanine) or 190 (N-terminal phenylalanine) is converted into a protein having a molecular weight of about 22,000 daltons and has biological activity (Pro. Natl. Acad. Sci. 1977, 74, 2432-2436). Growth hormone secretion regulation is stimulated by growth hormone releasing hormone (GHRH) secreted from the hypothalamus and secreted by somatostatin. The secreted growth hormone is involved in the regulation of the metabolism of proteins, carbohydrates, fats and minerals by inducing the production of insulin-like growth factor-1 (IGF-1) in the liver, As a protein (Nature, 1982, 300, 611-615), are used to promote growth of animals in the treatment and livestock industry of dwarfism-deficient patients with growth hormone deficiency.

The present invention provides a method for transformation of mushroom mushroom using Agrobacterium to obtain a transformed white rot fungus capable of producing a useful recombinant protein, and a mushroom transformant obtained by this method. And thus the present invention has been completed.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to develop a secretory vector for transformation of mushroom mushroom and a method for producing a target protein using the same. As a result, a secretion vector containing an expression construct suitable for transformation of mushroom mushroom was constructed and transformed into mushroom mushroom using this to successfully produce the desired protein, thereby completing the present invention.

In order to accomplish the above object, a feature of the present invention is to provide a mushroom vector for transformation of mushroom.

As a result, a vector containing an expression construct suitable for transformation of mushroom mushroom was constructed, and the target protein was successfully produced by transforming mushroom using the vector.

In the recombinant vector for transformation of mushrooms of the present invention, CaMV 35S promoter, signal sequence (pathogenesis-related protein 1b), and bovine growth hormone gene which is a useful gene of the present invention are used as a promoter.

     The present invention provides a secretory vector containing an expression construct suitable for mushroom transformation and a method for successfully producing a target protein such as a growth hormone protein from the transformed mushroom using the secretion vector.

Based on these results, recombinant expression vectors are well suited for transformation of mushrooms and allow the expression and acquisition of a variety of proteins in mushrooms, particularly mushrooms.

In addition, it can be used industrially by establishing protein expression system capable of mass production.

Brief Description of the Drawings Fig. 1 is a diagram showing a process for producing a vector containing a recombinant bovine growth hormone according to the present invention,
FIG. 2 is a graph showing the results of electrophoresis of bGH gene analyzed by PCR using DNA of a vector containing recombinant bovine growth hormone as a template according to the present invention,
FIG. 3 is a diagram illustrating a method of transforming a vector containing recombinant bovine growth hormone according to the present invention,
FIG. 4 is an electrophoresis image showing a bGH gene analyzed by PCR using a chromosomal DNA of a mushroom transformant transformed with a vector containing a recombinant bovine growth hormone according to the present invention as a template.

     Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

≪ Example 1 > Preparation of vector for producing recombinant protein

<1-1> Preparation of vector

The pPEV vector isolated from the Escherichia coli culture by alkaline lysis method was reacted with BamHI at 37 for 2 hours and a DNA fragment of about 10.6 kb size was extracted from the agarose gel by agarose gel electrophoresis. The linearized pPEV vector was treated with Klenow enzyme to make both ends of the vector blunt end and then extracted again from the agarose gel.

<1-2> Preparation of Signal Sequence

      The pR1b signal sequence (pEGMT-pR1b), which was subcloned to introduce the signal sequence [pathogenesis-related protein 1b (pR1b) signal sequence (96 bp)], was digested with NdeI and SmaI restriction enzymes, The Klenow enzyme was treated to make blunt ends at both ends, and then extracted from the agarose gel. The signal sequence and vector fragments were ligated using ligation enzymes. The E. coli XL1-blue MRF 'strain was transformed to screen the pPEVpR1b secretion vector (10.7 kb).

&Lt; 1-3 > Production of bGH gene

For the bovine growth hormone gene, pGEMT-bGH plasmid in which bGH was cloned was reacted with EcoRI and HindIII at 37 for 2 hours, and about 0.57 kb DNA fragment was extracted from the agarose gel by agarose gel electrophoresis. The separated bGH gene fragment was treated with Celanov enzyme, and the both ends were blunt ended. Then, the bGH gene was extracted from the agarose gel and used as the bGH gene DNA to be cloned into the pPEVpR1b secretion vector.

<1-4> Preparation of Recombinant Gene Vector

The pPEVpR1b secretion vector having a blunt end prepared in Example <1-1, 1-1> and the bGH gene prepared in Example <1-3> were transformed into pPEVpR1b vector And bGH gene fragments were ligated and the ligation reaction solution was transformed into E. coli XL-1 Blue MRF '(Proc. Natl. Acad. Sci. USA, 1972, 69, 2110-2114 ). E. coli XL-1 Blue MRF 'inoculated on LB medium was cultured at 600 nm with stirring at 37 to 200 rpm at an absorbance of about 0.5, and the E. coli was recovered by centrifugation at 3500 rpm for 10 minutes to obtain a cold 0.1 After washing with M CaCl ₂ solution, the obtained E. coli was suspended in 1/10 volume of cold 0.1 M CaCl ₂ solution and left on ice for 30 minutes. The CaCl ₂ -treated E. coli was recovered by centrifugation and suspended in 1/2 volume of cold 0.1 M CaCl ₂ solution. The ligation reaction mixture prepared in the above manner was mixed well in Escherichia coli suspension 200, left for 30 minutes on ice, subjected to thermal shock for 42 minutes for 1 minute, and left on ice for 2 minutes. 0.5 LB medium was added to the reaction solution, and the mixture was allowed to stand at 37 for 1 hour. From this solution, 0.1 was plated on LB agar medium containing 50 / kanamycin and cultured at 37 for one day to obtain a colony Respectively. The recombinant vector was isolated from the selected colonies by the alkaline digestion method. The clone in which the bGH gene was cloned was confirmed to have a DNA band of about 0.57 kb using PCR, and the recombinant vector thus constructed was named pPEVpR1b-bGH And Fig. 2).

< Example  2> Transformation

<2-1> Transformation of Agrobacterium

In order to introduce pPEVpR1b-bGH prepared in Example 1 into Pleurotus eryngii, transformation with Agrobacterium was used (Plasmid, 1978, 1, 456-467). Agrobacterium tumefaciens LBA4404 inoculated on YEP medium (1% Bacto tryptone, 1% yeast extract, 0.5% NaCl) was stirred at 37 to 200 rpm until the absorbance reached about 0.5 at 600 nm , And the culture solution was allowed to stand in ice for 10 minutes and then cooled. The bacteria were recovered by centrifugation at 3500 rpm for 10 minutes and suspended well in 1 cold 20 mM CaCl ₂ solution. The recombinant vector pPEV-bGH solution prepared in the above manner was mixed well in Agrobacterium suspension 200, The mixed solution was left in liquid nitrogen for quick freezing, and each frozen mixed solution was left to stand at 37 for 5 minutes to dissolve. Then, 10 times as much volume of YEP medium as the mixed solution was added, and the bacteria were cultured while being stirred at 28 for 2 hours. The bacteria were recovered by centrifugation at 3500 rpm for 10 minutes, and the bacteria were suspended in a YEP medium of 0.3, 0.1 of which was plated on YEP agar medium containing 50 / kanamycin and cultured at 37 for one day to obtain colonies colony. Each recombinant vector was isolated from the selected colonies by the alkaline digestion method, and the clone in which the bGH gene was cloned was identified by the method described in Example 1-3 (see Fig. 2). The gene inserted into the recombinant vector by DNA sequencing Was finally confirmed.

&Lt; 2-2 > Transformation of New mushroom by Agrobacterium tomefaciens

Transformation of the mushroom by Agrobacterium tumefaciens was slightly modified by Michielse et al. (Nat. Protoc, 2008, 3, 16711678). Briefly, Agrobacterium LBA4404 transformed with pPEV-bGH prepared in Example 2 was inoculated into 5 minimal medium (0.2% glucose, 0.205% dipotassium phosphate (pH 8.0) containing 5% kanamycin, K ₂ HPO _{4 )} , 0.145% monopotassium phosphate (KH ₂ PO _{4 )} , 0.015% sodium choloride (NaCl), 0.05% magnesium sulfate (MgSO ₄ 7H ₂ O) Calcium sulfate hexahydrate (CaCl ₂ 6H ₂ O), 0.00025% ferrous sulfate (FeSO ₄ 7H ₂ O), 0.05% ammonium sulfate (NH ₄ ) ₂ SO _{4 )} 28 for 2 days. Agrobacterium cultures were grown in an induction medium (10 mM glucose, 0.205% diphotassium phosphate, 0.145% monopotassium phosphate, 0.015% sodium chloride, 0.05% magnesium sulfate, 0.01% calcium (2-morpholinoethanesulfonic acid (MES)) in the presence of 0.00025% ferrous sulfate, 0.05% ammonium sulfate, 0.5% glycerol, 200 M acetosyringone, Biol. Organ, 1995, 14, 3206-8). The cells were then incubated for 3 h at the same conditions and then mixed with the same volume of conidial suspension (10 ⁶ conidia / This mixed solution 200 was added to a 3-mm filter paper (Watts, USA), and an induction medium containing 200 M of acetosyringone and an induction medium containing no acetosyringone The filter paper was soaked and co-cultivated for 28 days. After each co-cultivation, the mushroom fungus (Pleurotus eryngii) grown on the 3 MM filter paper and the microbial fungi (Agrobacterium tumefaciens ) Was transferred to a PDA (potato dextrose agar) medium containing 200 M cell troclin to kill Agrobacterium. The conidia of each transformed mushroom grown on the PDA medium was suspended in sterilized water, and 200 M cells (0.4% Potato Starch, 2% Dextrose) containing shrimp, and shake-cultured at 28 to 100 rpm. [0060] The transformed mushroom transformants grown in the PDB medium were centrifuged Collected, and used for various analyzes.

< Example  3> transformed Mushroom  chromosome DNA  analysis

The transformed mushroom transformant obtained in Example 2 was cultured in a PDB medium containing 200 M cells, and the transformed cells were transformed with hexadecyltrimethylammonium bromide (CTAB) method (FOCUS, 1990, 12, 13-15). Briefly, 0.1 g of the mushroom mycelia obtained in Example 2 was dissolved in 0.5 ml of DNA extraction buffer (0.1 M Tris-Cl (pH 9), 20 mM ethylenediaminetetraacetic acid (EDTA), 1.4 M sodium chloride ), 2% hexadecyltrimethylammonium bromide, 1% polyethylene glycol, 0.2% beta-mercaptoethanol) was added and the tissue was disrupted and heat-treated at 74 for 70 minutes. The above solution was extracted with 200 chloroform / isoamyl alcohol (24: 1), left at 0 to 30 minutes, centrifuged at 15,000 rpm for 5 minutes to recover the supernatant, The process was repeated once more. The supernatant was recovered and 0.55 volume of isopropanol was added. The mixture was allowed to stand at room temperature for 5 minutes and then centrifuged at 15,000 rpm for 10 minutes to recover the precipitate to obtain chromosomal DNA. The DNA precipitate was dissolved in TE (Tris-EDTA) solution containing 50/40 of ribonucleic acid hydrolase A (RNase A) and used as template DNA for polymerase chain reaction (PCR). Using this chromosomal DNA as a template, the bGH gene analysis was confirmed by PCR to confirm the introduction of the bGH gene. The DNA band of about 0.57 kb was confirmed, and the bGH gene was transformed. Finally, each of the polymerase chain reaction products Cloning was performed to analyze the DNA sequence and finally it was confirmed that the gene was transformed into the correct sequence gene.

< Example  4> transformed Mushroom mushroom  Protein Isolation from Mycelium

The mushroom mycelium transformed with the pPEVpR1b-bGH vector was inoculated in PDB medium and cultured for 25 to 5 days at 100 rpm with shaking culture. The bacterial culture 1 was inoculated into 250 Erlenmeyer flasks containing 50 Mushroom Complete medium (0.2% Bacto tryptone, 0.2% yeast extract, 2% glucose, 0.05% magnesium sulfate) And shake cultured at 100 rpm for 7 days. Mycelia were recovered at each time zone, and the recovered mycelium was suspended in an ultrasonic disruption buffer (10 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 5 mM beta-mercaptoethanol) per 3 g of mycelium, The mycelia of P. japonica were disrupted with a homogenizer and an ultrasonic crusher. The mycelium was disrupted and centrifuged at 15,000 rpm for 5 minutes. The supernatant was recovered and used as a protein expression assay sample.

Claims (5)

A recombinant vector for mushroom transformation comprising:
(a) CaMV 35S promoter (cauliflower mosaic virus 35S promoter);
(b) CaMV 35S 5-untranslated sequence (UTS);
(c) CaMV 35S 3-noninterpected region;
(d) pR1b signal sequence (pathogenesis-related protein 1b, 96 bp)
(e) a nopaline synthase terminator; And (f) repeating steps (b) and (c)
A nucleotide sequence coding for the desired protein located between.
An Agrobacterium sp. Microorganism transformed with the recombinant vector of claim 1.
A mushroom transformed with the recombinant vector of claim 1.
2. The method of claim 1, wherein the target protein is bovine growth hormone.
The transformed mushroom according to claim 3, wherein the mushroom is a fresh mushroom.

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