KR20160072458A - Production Methods of Human Growth Hormone Protein Using Secretion Vector in Pleurotus eryngii - Google Patents

Abstract

The present invention relate to a secretion vector for transforming Pleurotus eryngii Quel(De Candolle ex Fries) and a method for producing human growth hormone protein using the same. More specifically, the present invention provides a secretion vector including an expression construct adequate for mushroom transformation and a method for successfully producing various target proteins such as growth hormone protein from transgenic mushroom using the vector. The recombinant expression vector of the present invention can be applied to a mass-production system of various industrially useful proteins from mushrooms, particularly Pleurotus eryngii Quel(De Candolle ex Fries).

Description

Production Methods of Human Growth Hormone Protein Using Secretion Vector of Saccharomyces cerevisiae {Secretion Vector in Pleurotus eryngii}

The present invention relates to a mushroom transformed by introducing a protein secretion vector and a method for producing human growth hormone 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 human growth hormone protein using the same. As a result, a secretion vector containing an expression construct suitable for transformation of mushroom mushroom was constructed and transformed with mushroom mushroom to successfully produce human growth hormone 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 mushroom was transformed to produce human growth hormone protein successfully.

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

     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 various proteins in mushrooms, particularly mushrooms.

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

Brief Description of Drawings Fig. 1 is a diagram showing a process for producing a vector containing recombinant human growth hormone according to the present invention,
FIG. 2 is an electrophoresis image showing the hGH gene analyzed by PCR using DNA of a vector containing a recombinant human growth hormone according to the present invention as a template,
FIG. 3 is a diagram showing a method of transforming a vector containing recombinant human growth hormone according to the present invention,
FIG. 4 is an electrophoresis image of a hGH gene analyzed by PCR using a chromosomal DNA of a transformed mushroom transformant transformed with a vector containing recombinant human growth hormone according to the present invention as a template.
FIG. 5 is a photograph showing Western blot analysis of expressed hGH protein when cultured Mycelium of mushroom cultured with a vector containing recombinant human growth hormone according to the present invention.

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 Bam HI at 37 ° C for 2 hours, and a DNA fragment of about 10.6 kb in size was extracted from the agarose gel by agarose gel electrophoresis.

<1-2> Preparation of Signal Sequence

Signal sequence was cleaved to [pathogenesis-related protein 1b (pR1b ) signal sequence (96 bp)] [pR1b signal sequence (pEGMT-pR1b)] is subcloned in order to introduce the Nde I and Sma I restriction enzyme, and then, each Was treated with Klenow enzyme to make blunt ends on both ends and then extracted from agarose gel again. 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 hGH gene

In the case of the human growth hormone gene, pGEMT-hGH plasmid in which hGH was cloned was reacted with Eco RI and Hin dIII at 37 ° C for 2 hours, and about 0.57 kb DNA fragment was extracted from the agarose gel by agarose gel electrophoresis The hGH gene DNA to be cloned into the pPEVpR1b secretion vector was used.

<1-4> Preparation of Recombinant Gene Vector

The pPEVpR1b secretion vector prepared in Example <1-1,1-2> and the hGH gene prepared in Example <1-3> were ligated to the pPEVpR1b vector and the hGH gene fragment by the method of Sam Brook, ligation), and the ligation reaction solution was transformed into E. coli XL-1 Blue MRF '( Proc. Natl. Acad. Sci. USA, 1972, 69, 2110-2114). The E. coli XL-1 Blue MRF inoculated on LB medium was cultured at 600 nm with stirring at 200 rpm at 37 DEG C until the absorbance reached about 0.5, and E. coli was recovered by centrifugation at 3500 rpm for 10 minutes, After briefly washing with 0.1 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 200 μl of Escherichia coli suspension, allowed to stand in ice for 30 minutes, thermally shocked at 42 ° C for 1 minute, and left on ice for 2 minutes. 0.5 ml of LB medium was added to the reaction solution, and the mixture was allowed to stand at 37 DEG C for 1 hour. From this solution, 0.1 ml was spread on LB agar medium containing 50 mu g / ml of kanamycin and cultured at 37 for one day So that a colony was generated. The recombinant vector was isolated from the selected colonies by the alkaline digestion method. The clone in which the hGH 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-hGH And Fig. 2).

&Lt; Example 2 > Transformation

<2-1> Transformation of Agrobacterium

In order to introduce pPEVpR1b-hGH prepared in Example 1 into Pleurotus eryngii , transformation with Agrobacterium was used ( Plasmid , 1978, 1, 456-467). Agrobacterium tumefaciens LBA4404 inoculated into YEP medium (1% Bacto tryptone, 1% yeast extract, 0.5% NaCl) was added at 37 ° C to 200 rpm at 600 nm until absorbance reached about 0.5 The cells were incubated with stirring, and the culture was allowed to stand in ice for 10 minutes and then cooled. The bacteria were recovered by centrifugation at 3500 rpm for 10 minutes, suspended in 1 ml of cold 20 mM CaCl ₂ solution, and the recombinant vector pPEVpR1b-hGH solution prepared in the above manner was mixed well with 200 μl of Agrobacterium suspension The mixture was left in liquid nitrogen for rapid freezing, and each frozen mixture was allowed to stand at 37 ° C for 5 minutes to dissolve it. 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, the bacteria were suspended in 0.3 ml of YEP medium, 0.1 ml of the suspension was plated on a YEP agar medium containing 50 μg / ml of kanamycin, Lt; RTI ID = 0.0 &gt; colony. &Lt; / RTI &gt; Each recombinant vector was isolated from the selected colonies by the alkaline digestion method, and the clone in which the hGH gene was cloned was confirmed 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-hGH prepared in Example 2 was inoculated into 5 ml of a minimal medium (0.2% glucose, 0.205% diphosphate phosphate (pH 7.0) containing 50 μg / ml of kanamycin _{dipotassium phosphate; K 2 HPO 4)} , 0.145% mono-potassium phosphate _{(monopotassium phosphate; KH 2 PO 4} ), 0.015% sodium chloride (sodium choloride; NaCl), 0.05 % of magnesium sulfate _{(magnesium sulfate; MgSO 4 7H 2} O), 0.01% calcium chloride hexahydrate (CaCl ₂ 6H ₂ O), 0.00025% ferrous sulfate (FeSO ₄ 7H ₂ O), 0.05% ammonium sulfate (NH ₄ ) ₂ SO _4, ) And incubated at 28 째 C for 2 days while stirring well. 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 uM acetosyringone and 40 mM morpholinoethanesulfonic acid 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 / 3214). 200 占 퐇 of this mixed solution was added to a 3 MM filter paper (Whatman, USA), and an induction medium containing 200 占 아세 of acetosyringone and an induction medium containing no acetocyrinone Were co-cultivated for 2 days at 28 ° C. After each co-cultivation, the filter paper of 3 mm filter paper ( Pleurotus eryngii ) and microorganisms (Agrobacterium tumefaciens ) Were transferred to a PDA (potato dextrose agar) medium containing 200 uM cell troclin to cause Agrobacterium to die. The conidia of each transformed mushroom grown on the PDA medium were suspended in sterilized water (0.4% Potato Starch, 2% Dextrose) containing 200 uM cell trophoblasts and shake-cultured at 28 rpm at 100 rpm. [0091] The mushroom transformant grown in the PDB medium Were recovered by centrifugation and used for various analyzes.

Example 3: Chromosomal DNA analysis of transformed mushroom

The transformed mushroom transformant obtained in Example 2 was cultivated in a PDB medium containing 200 uM cytotoxicity, and 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 The tissue was disrupted by adding 2% hexadecyltrimethylammonium bromide, 1% polyethylene glycol, and 0.2% beta-mercaptoethanol, and heat-treated at 74 ° C for 70 minutes. The solution was extracted with 200 μl of chloroform / isoamyl alcohol (24: 1), left at 0 to 30 minutes, centrifuged at 15,000 rpm for 5 minutes to recover the supernatant, The supernatant was collected and 0.55-fold volume of isopropanol was added, and the mixture was allowed to stand at room temperature for 5 minutes and centrifuged at 15,000 rpm for 10 minutes to recover the precipitate to obtain chromosomal DNA. The whole was dissolved in TE (Tris-EDTA) solution containing 50 의 of 40 / / ml ribonucleic acid hydrolase A (RNase A) and used as template DNA for polymerase chain reaction (PCR) . Using this chromosomal DNA as a template, the hGH gene analysis was confirmed by PCR to confirm the introduction of the hGH gene, and a DNA band of about 0.57 kb in size was identified and it was confirmed whether or not the hGH gene was transformed. Finally, each of the polymerase chain reaction products And the DNA sequence was finally analyzed to confirm that it was transformed into the gene of the correct sequence.

Example 4 Protein Isolation from Transformed Mycelia

The mushroom mycelium transformed with the pPEVpR1b-hGH vector was inoculated in PDB medium and cultured at 25 DEG C for 5 days with shaking culture at 100 rpm. 1 ml of the bacterial culture was inoculated into 250 Erlenmeyer flasks containing 50 ml of Mushroom Complete medium (0.2% Bacto tryptone, 0.2% yeast extract, 2% glucose, 0.05% magnesium sulfate) And cultured at 25 DEG C for 7 days with shaking at 100 rpm. 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. The same volume of 99% ethanol was added to 0.5 ml of the cell extract, followed by mixing well. The mixture was allowed to stand at -20 ° C for 30 minutes and centrifuged at 12,000 rpm for 10 minutes to recover the protein precipitate. Left to dry. The dried protein extracts were suspended in 20 μl of distilled water and suspended in 20 μl of 2 × SDS-PAGE buffer (0.1 M Tris-HCl (pH 6.8), 0.2 M dithiothreitol (DTT ), 4% SDS, 20% glycerol, 0.2% bromophenol blue) and heated at 100 for 5 minutes to confirm by SDS-PAGE ( Nature, 1970, 227, 680- 685). The separated gel was stained in a Coomassie brilliant blue solution for 1 hour and transferred to decolorization solution (30% methanol, 10% acetic acid) for decolorization. In addition, the protein developed by SDS-PAGE was transferred to a PVDF (polyvinylidene fluoride) membrane, and the hGH protein expressed in the cultured mycelia of mycelia was transformed into a known antibody blot using anti-hGH antibody produced in rabbit Respectively. As a result, it was confirmed that hGH protein having a molecular weight of about 21 kDa was expressed in the cultured Mycelium of mushroom mycelia transformed with pPEVpR1b-hGH of the present invention (see FIG. 5).

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) a nucleotide sequence encoding a target protein located between (b) and (c) above.
An Agrobacterium sp. Microorganism transformed with the recombinant vector of claim 1.
A mushroom transformed with the recombinant vector of claim 1.
The method according to claim 1, wherein the target protein is human growth hormone.
The transformed mushroom according to claim 3, wherein the mushroom is a fresh mushroom.

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