JPWO2008133141A1 - Osmotin recombinant protein, method for producing the same, and use thereof - Google Patents

Abstract

An osmotin recombinant protein is produced using a non-plant transformant into which a polynucleotide encoding osmotin or a variant thereof is introduced so that it can be expressed. Thus, the present invention provides an osmotin recombinant protein having a physiological activity that can be obtained easily, inexpensively and in large quantities, a method for producing the same, and use thereof.

Description

  The present invention relates to an osmotin recombinant protein, a method for producing the same, and use thereof, and in particular, an osmotin recombinant protein having physiological activity that can be obtained simply, inexpensively and in large quantities, and a method for producing the same, and the method thereof It is about use.

  The leading cause of death among Japanese is cardiovascular diseases such as myocardial infarction and cerebral infarction. The major cause of cardiovascular disease is thought to be metabolic syndrome that overlaps obesity, diabetes, hyperlipidemia, and hypertension. Therefore, establishment of a method for preventing and treating metabolic syndrome is a very important issue.

  Metabolic syndrome is a disease concept defined to narrow down high-risk groups for the primary purpose of preventing cardiovascular disease. Specifically, it is a pathological condition in which visceral fat accumulation is combined with insulin resistance (glucose tolerance abnormality), arteriosclerosis-induced lipoprotein abnormality, and high blood pressure.

  In 2002, WHO and NECP (USA) announced a policy of emphasizing metabolic syndrome as a health measure and published diagnostic criteria for metabolic syndrome. The diagnostic criteria include multiple risk factors. Even in the Japanese group, it has been reported that in the group in which three or more of these risk factors are combined, the cardiovascular disease risk rate reaches 30 times or more compared to the target group.

  Metabolic syndrome has been conventionally referred to as syndrome X, death quartet, insulin resistance syndrome, visceral fat syndrome and the like. However, in 2005, the Japan Society of Internal Medicine and 7 related academic societies reported that “in the multiple risk factor syndrome with multiple visceral fat accumulation and insulin resistance and glucose metabolism abnormality, lipid metabolism abnormality and hypertension, It was defined as “a pathological condition that is prone to hardening”.

  There are many unexplained causes of the development of metabolic syndrome, but it is thought that the stress that occurs in the body is part of it. For example, it is estimated that “oxidative stress” and “carbonyl stress” are involved in the onset mechanism of diabetic complications. Oxidative stress refers to damage to biological parts such as proteins and DNA caused by highly reactive reactive oxygen species. Carbonyl stress is a phenomenon in which a carbonyl compound such as glucose attacks a biomolecule such as a protein. This reaction is called the Maillard reaction. Although it has been estimated that the “wound” received by these living body parts leads to the onset, the detailed mechanism is still unclear.

  Chronic hyperglycemia causes excessive generation of reactive oxygen species (ROS) and acts as a powerful oxidative stress via glycation reaction, mitochondrial electron transport system, and hexosamine pathway. However, since more than half of the current diabetics do not have any treatment at all, these pregnancies are more commonly used by people with lifestyle habits such as overeating, obesity, and lack of exercise. It is essential to take measures to reduce oxidative stress and protect the living body in daily life.

  As an example, diabetes has been described, but oxidative stress has been reported not only to metabolic syndrome but also to cancer, inflammation, allergic diseases, Alzheimer's disease, age-related macular degeneration, skin aging, and the like. Therefore, reduction of oxidative stress can be expected to have effects such as prevention and improvement of various diseases caused by oxidative stress, and anti-aging.

  When visceral fat (intraperitoneal fat) accumulates due to satiety and overnutrition due to lack of exercise, fat cells cause abnormal secretion of various physiologically active substances, particularly adipocytokines. As a result, abnormal sugar metabolism, abnormal lipid metabolism, hypertension, and cardiovascular disease are caused.

  Adipocytokine is a general term for physiologically active substances produced and secreted in adipocytes. Various adipocytokines are produced and secreted from adipocytes. These adipocytokines are roughly classified into good adipocytokines that improve the pathological condition and bad adipocytokines that worsen the pathological condition.

  Adiponectin (also known as Acrp30) is a good adipocytokine with anti-diabetic and anti-arteriosclerotic effects. Adiponectin is a product of the gene apM1 (adipose most abundant gene transcript 1) expressed specifically in adipose tissue and is a secreted protein consisting of 224 amino acids.

  Adiponectin is followed by a signal sequence commonly found in secreted proteins, and the N-terminal part consists of a collagen-like fibrous domain and the C-terminal part consists of a complement C1q-like globular domain. Adiponectin forms multimers around the collagen-like domain in plasma. Further, it has been reported that a full-length adiponectin produces a globular adiponectin having only a C-terminal complement C1q-like globular domain and exhibits higher activity than a full-length adiponectin even in a small amount. Further, in Non-Patent Document 1, the amount of adiponectin in blood decreases due to obesity, insulin resistance, and type II diabetes. However, in an experiment using mice, administration of adiponectin decreases the blood glucose level, resulting in insulin resistance. Is described as being improved. It has also been suggested that adiponectin can be a drug for weight control.

  Non-Patent Document 2 describes that there is an adiponectin receptor (AdipoR) to which adiponetin binds, and that AdipoR mediates the regulation of energy state, fatty acid oxidation, and glucose transport of organisms by adiponetin. Yes. It has also been suggested that AdipoR agonists can be anti-diabetic drugs or anti-atherologic drugs.

  Non-Patent Document 3 discloses that adiponectin phosphorylates 5′-AMP-activated protein kinase (hereinafter also referred to as “AMPK”) via AdipoR, based on a study using C2C12 which is a skeletal muscle model cell. It is described that it exhibits various physiological actions.

  By the way, in Non-Patent Document 4, tobacco osmotin is an antibacterial protein of tobacco belonging to the PR-5 family, and induces apoptosis in yeast by suppressing cellular stress response via the RAS2 / cAMP pathway. Is described. Moreover, it is described that tobacco osmotin shows a three-dimensional homology with globular adiponectin. Furthermore, it is described that the tobacco osmotin receptor PHO36 is a yeast AdipoR homolog.

  In addition, Non-Patent Document 4 describes that osmotin can phosphorylate AMPK in the same manner as adiponectin, based on studies using skeletal muscle model cells, C2C12. Moreover, it has been shown that the phosphorylation activity of AMPK by osmotin decreases with decreasing expression of AdipoR. Therefore, it is suggested that osmotin may phosphorylate AMPK via AdipoR.

  As described above, various studies on the physiological activity of osmotin have been conducted. Conventionally, for example, tobacco osmotin is extracted from plant tobacco and purified. However, in tobacco, the expression level of tobacco osmotin is low and its yield is very low.

Therefore, various attempts have been made to obtain more tobacco osmotin. For example, tobacco osmotin is known to accumulate in vacuoles due to osmotic stress. Therefore, there are a method of extracting and purifying tobacco cultured cells in which expression of tobacco osmotin has been induced by salt stress treatment, and a method of extracting and purifying tobacco osmotin recombinant protein from a transformed tobacco produced using recombinant DNA technology. It has been reported. Furthermore, Non-Patent Documents 5 and 6 disclose methods for extracting and purifying tobacco osmotin recombinant protein from transformed tobacco that secretes the expressed tobacco osmotin to the extracellular matrix.
Lodish et al, Proc. Natl. Acad. Sci. USA 98, 2005-2010, 2001. Yamauchi et al, Nature 423, 762-769, 2003. Yamauchi et al, Nature Medicine 8, 1288-1295, 2002. Narasimhan et al, Mol. Cell 17, 171-180, 2005. Maggio et al, Plant Mol. Biol. Rep. 14, 249-260, 1996. Liu et al, Plant Sci. 121, 123-131, 1996. Xu Hu and ASN Reddy, Plant Mol. Biol. 34; 949-959, 1997

  As described above, a substance that activates AdipoR, that is, an AdipoR agonist, is considered to be effective for prevention and treatment of metabolic syndrome. Osmotin is considered to be one of AdipoR agonists and can be used for prevention and treatment of metabolic syndrome.

  When osmotin is used for the prevention and treatment of metabolic syndrome, it is desired to mass-produce osmotin. However, as described above, only a very small amount of osmotin can be obtained from a wild-type tobacco plant. Moreover, even if the conventional large-scale expression system for tobacco osmotin recombinant protein as disclosed in Non-Patent Document 5 is used, only a few mg of tobacco osmotin recombinant protein can be obtained from 1 kg of the leaves of the transformed tobacco. Absent. Thus, in the conventional technology, the yield of tobacco osmotin is very low regardless of which method is used. Therefore, in order to obtain a large amount of tobacco osmotin or a recombinant protein thereof, tobacco or transformed tobacco needs to be cultivated on a large area. That is, there arises a problem that tobacco osmotin cannot be produced with high productivity in terms of cultivation place and cultivation equipment. In addition, extraction and purification of tobacco osmotin or a recombinant protein thereof requires a great deal of labor because it is necessary to extract and purify from a large amount of tobacco or transformed tobacco.

  As another protein of the PR-5 family to which osmotin belongs, a recombinant protein expression system using Escherichia coli has been reported (see Non-Patent Document 7). However, although osmotin has been attempted to be expressed in heterologous organisms such as bacteria and yeast using DNA recombination technology, it has not yet been obtained as an osmotin recombinant protein (see Non-Patent Document 5). .

  The present invention has been made in view of the above problems, and an object of the present invention is to provide an osmotin recombinant protein having physiological activity that can be easily obtained at low cost and in large quantities, a method for producing the same, and use thereof. There is.

  As a result of intensive studies in view of the above problems, the present inventors have formed an inclusion body containing an osmotin recombinant protein in a non-plant transformant into which a polynucleotide encoding osmotin or a variant thereof has been introduced. Originally, it is possible to easily and inexpensively obtain a large amount of osmotin recombinant protein having physiological activity by extracting osmotin recombinant protein from the inclusion body and refolding the osmotin recombinant protein. The present invention has been completed. That is, the present invention includes the following inventions.

  (1) A method for producing an osmotin recombinant protein, comprising producing an osmotin recombinant protein using a non-plant transformant into which a polynucleotide encoding osmotin or a variant thereof is introduced so as to be expressed.

  (2) The method for producing an osmotin recombinant protein according to (1), comprising a step of detecting phosphorylation activity of 5'-AMP-activated protein kinase of the osmotin recombinant protein.

  (3) The method for producing an osmotin recombinant protein according to (1) or (2), wherein the host of the non-plant transformant is Escherichia coli, yeast, fungi, insect, or mammal.

  (4) In the non-plant transformant, an inclusion body containing an osmotin recombinant protein is formed, the inclusion body is extracted, and the inclusion body is dissolved in a solubilized solution, whereby the osmotin contained in the inclusion body The method for producing an osmotin recombinant protein according to any one of (1) to (3), wherein the recombinant protein is solubilized and the solubilized osmotin recombinant protein is refolded.

(5) The polynucleotide according to any one of (1) to (4), wherein the polynucleotide is any one polynucleotide selected from the group consisting of the following (a) to (l): A method for producing an osmotin recombinant protein.
(A) a polynucleotide comprising the base sequence shown in SEQ ID NO: 1 (b) a base in which one or several bases are substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 1 A polynucleotide comprising the sequence and encoding a polypeptide having the activity of phosphorylating 5'-AMP-activated protein kinase (c) a polynucleotide and a string comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 1 A polynucleotide that encodes a polypeptide that hybridizes under mild conditions and phosphorylates 5′-AMP-activated protein kinase (d) from position 1 to position 678 of the base sequence represented by SEQ ID NO: 1 A polynucleotide comprising the base sequence consisting of the positions (e) from position 1 to position 678 of the base sequence shown in SEQ ID NO: 1 A polypeptide having a base sequence in which one or several bases are substituted, deleted, inserted and / or added, and having an activity of phosphorylating 5′-AMP-activated protein kinase The polynucleotide to be encoded (f) hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence consisting of positions 1 to 678 of the base sequence represented by SEQ ID NO: 1, and 5 A polynucleotide encoding a polypeptide having an activity to phosphorylate AMP-activated protein kinase (g) a polynucleotide comprising a nucleotide sequence consisting of positions 4 to 738 of the nucleotide sequence represented by SEQ ID NO: 1 (h ) In the base sequence consisting of positions 4 to 738 of the base sequence shown in SEQ ID NO: 1, one or several bases are A polynucleotide (i) encoding a polypeptide consisting of a base sequence that has been substituted, deleted, inserted, and / or added, and having the activity of phosphorylating 5′-AMP-activated protein kinase, as shown in SEQ ID NO: 1. It has the activity of hybridizing under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence consisting of positions 4 to 738 of the nucleotide sequence and phosphorylating 5'-AMP activated protein kinase. A polynucleotide encoding the polypeptide (j) a polynucleotide comprising the base sequence consisting of positions 4 to 678 of the base sequence shown in SEQ ID NO: 1 (k) the fourth position of the base sequence shown in SEQ ID NO: 1 ~ In the base sequence consisting of position 678, one or several bases substituted, deleted, inserted and / or added. And a polynucleotide encoding a polypeptide having an activity to phosphorylate 5′-AMP-activated protein kinase (l) a nucleotide sequence comprising positions 4 to 678 of the nucleotide sequence represented by SEQ ID NO: 1 A polynucleotide encoding a polypeptide that hybridizes with a polynucleotide comprising a complementary base sequence under stringent conditions and has an activity of phosphorylating 5′-AMP-activated protein kinase. (6) The osmotin group according to any one of (1) to (4), which is a polynucleotide derived from any one plant selected from the group consisting of potato, tomato, pepper, pepper, and tobacco A method for producing a replacement protein.

  (7) The osmotin recombinant protein according to (4), wherein the solubilized osmotin recombinant protein is refolded at 2 ° C. to 40 ° C. in a solution containing reduced glutathione and oxidized glutathione. A method for producing a recombinant protein.

  (8) The method for producing an osmotin recombinant protein according to (4) or (7), wherein the solubilized solution contains N-lauroyl sarcosine.

  (9) The host of the non-plant transformant is E. coli, and the osmotin recombinant protein is produced by inducing expression of the polynucleotide at 20 ° C. to 30 ° C. (1) The manufacturing method of the osmotin recombinant protein in any one of-(8).

  (10) An osmotin recombinant protein produced by the method for producing an osmotin recombinant protein according to any one of (1) to (9).

  (11) The osmotin recombinant protein according to (10), which has an activity of phosphorylating 5'-AMP-activated protein kinase.

  (12) A composition comprising the osmotin recombinant protein according to (10) or (11).

  (13) The composition according to (12), which is used for prevention or treatment of lifestyle-related diseases.

  (14) The lifestyle-related disease is at least one disease selected from the group consisting of type II diabetes, insulin resistance, hyperlipidemia, hypertension, arteriosclerosis, and heart disease ( The composition according to 13).

  (15) The composition according to (12), which is used for prevention or treatment of a disease caused by oxidative stress.

  (16) The composition according to (15), wherein the disease caused by oxidative stress is at least one disease selected from the group consisting of cancer, Alzheimer's disease, age-related macular degeneration, and skin aging. object.

(17) A screening method for an osmotin recombinant protein having an activity of phosphorylating 5′-AMP-activated protein kinase,
Extracting an osmotin recombinant protein from a non-plant transformant in which a polynucleotide encoding osmotin or a variant thereof is introduced so as to be expressed,
A method for screening an osmotin recombinant protein, comprising detecting the activity of phosphorylating 5′-AMP-activated protein kinase of the osmotin recombinant protein.

  (18) The method for screening an osmotin recombinant protein according to (17), wherein the host of the non-plant transformant is Escherichia coli, yeast, fungi, insect, or mammal.

(19) The osmotin recombinant protein is
In the non-plant transformant, an inclusion body containing an osmotin recombinant protein is formed,
Extracting the inclusion body and dissolving the inclusion body in a solubilizing solution solubilizes the osmotin recombinant protein contained in the inclusion body,
The method for screening an osmotin recombinant protein according to (17) or (18), which is obtained by refolding the solubilized osmotin recombinant protein.

(20) The polynucleotide according to any one of (17) to (19), wherein the polynucleotide is any one polynucleotide selected from the group consisting of the following (a) to (l): A screening method for the described osmotin recombinant protein.
(A) a polynucleotide comprising the base sequence shown in SEQ ID NO: 1 (b) a base in which one or several bases are substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 1 A polynucleotide comprising the sequence and encoding a polypeptide having the activity of phosphorylating 5'-AMP-activated protein kinase (c) a polynucleotide and a string comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 1 A polynucleotide that encodes a polypeptide that hybridizes under mild conditions and phosphorylates 5′-AMP-activated protein kinase (d) from position 1 to position 678 of the base sequence represented by SEQ ID NO: 1 A polynucleotide comprising the base sequence consisting of the positions (e) from position 1 to position 678 of the base sequence shown in SEQ ID NO: 1 A polypeptide having a base sequence in which one or several bases are substituted, deleted, inserted and / or added, and having an activity of phosphorylating 5′-AMP-activated protein kinase The polynucleotide to be encoded (f) hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence consisting of positions 1 to 678 of the base sequence represented by SEQ ID NO: 1, and 5 A polynucleotide encoding a polypeptide having an activity to phosphorylate AMP-activated protein kinase (g) a polynucleotide comprising a nucleotide sequence consisting of positions 4 to 738 of the nucleotide sequence represented by SEQ ID NO: 1 (h ) In the base sequence consisting of positions 4 to 738 of the base sequence shown in SEQ ID NO: 1, one or several bases are A polynucleotide (i) encoding a polypeptide consisting of a base sequence that has been substituted, deleted, inserted, and / or added, and having the activity of phosphorylating 5′-AMP-activated protein kinase, as shown in SEQ ID NO: 1. It has the activity of hybridizing under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence consisting of positions 4 to 738 of the nucleotide sequence and phosphorylating 5'-AMP activated protein kinase. A polynucleotide encoding the polypeptide (j) a polynucleotide comprising the base sequence consisting of positions 4 to 678 of the base sequence shown in SEQ ID NO: 1 (k) the fourth position of the base sequence shown in SEQ ID NO: 1 ~ In the base sequence consisting of position 678, one or several bases substituted, deleted, inserted and / or added. And a polynucleotide encoding a polypeptide having an activity to phosphorylate 5′-AMP-activated protein kinase (l) a nucleotide sequence comprising positions 4 to 678 of the nucleotide sequence represented by SEQ ID NO: 1 A polynucleotide encoding a polypeptide that hybridizes with a polynucleotide comprising a complementary base sequence under stringent conditions and has an activity of phosphorylating 5′-AMP-activated protein kinase. (21) The polynucleotide according to any one of (17) to (19), wherein the polynucleotide is derived from any one plant selected from the group consisting of potato, tomato, pepper, pepper, and tobacco Screening method for osmotin recombinant protein.

  (22) The osmotin according to (19), wherein the solubilized osmotin recombinant protein is refolded at 2 ° C. to 40 ° C. in a solution containing reduced glutathione and oxidized glutathione. Recombinant protein screening method.

  (23) The method for screening an osmotin recombinant protein according to (19) or (22), wherein the solubilized solution contains N-lauroyl sarcosine.

(24) The host of the non-plant transformant is E. coli,
The osmotin recombinant protein according to any one of (17) to (23), wherein the osmotin recombinant protein is produced by inducing expression of the polynucleotide at 20 ° C to 30 ° C. Screening method.

  Other objects, features, and advantages of the present invention will be fully understood from the following description. The advantages of the present invention will become apparent from the following description with reference to the accompanying drawings.

FIG. 1 is a diagram showing a method for constructing an expression vector for tobacco osmotin recombinant protein in Examples. FIG. 2 is a diagram showing the results of SDS-PAGE of tobacco osmotin recombinant protein according to the example. FIG. 3 is a diagram showing the results of Western blotting using a lysate of C2C12 cells to which the tobacco osmotin recombinant protein according to the example was added. FIG. 4 is a diagram showing the results of measuring the AMPK phosphorylation activity of C2C12 cells using the tobacco osmotin recombinant protein according to the example. FIG. 5: is a figure which shows the result of SDS-PAGE of the tobacco osmotin recombinant protein concerning an Example. FIG. 6 is a diagram showing the results of Western blotting using a lysate of C2C12 cells to which the tobacco osmotin recombinant protein according to the example was added. FIG. 7 is a diagram showing the results of measuring the AMPK phosphorylation activity of C2C12 cells using the tobacco osmotin recombinant protein according to the example. FIG. 8 is a diagram showing the evaluation results of the lipid metabolism abnormality improving effect of the tobacco osmotin recombinant protein according to the example. FIG. 9 is a diagram showing the evaluation results of the effect of improving the sugar metabolism abnormality of the tobacco osmotin recombinant protein according to the example. FIG. 10 is a diagram showing the evaluation results of the oxidative stress reduction effect of the tobacco osmotin recombinant protein according to the example.

  An embodiment of the present invention will be described as follows, but the present invention is not limited to this.

<I. Osmotin recombinant protein>
The osmotin recombinant protein according to the present invention (hereinafter also simply referred to as “recombinant protein according to the present invention”) is an osmotin (hereinafter also referred to as “osmotin” or “Osm”) that is naturally induced by osmotic stress. A recombinant protein having an activity (hereinafter also referred to as “AMPK phosphorylation activity”) that phosphorylates 5′-AMP activated protein kinase (hereinafter also referred to as “AMPK”). In the present specification, “protein AMPK phosphorylation activity” refers to phosphorylation in the total AMPK (hereinafter also referred to as “total-AMPK” or “t-AMPK”) when the protein is allowed to act on AMPK. The proportion of AMPK received (hereinafter also referred to as “p-AMPK”) is intended. That is, the higher the proportion of p-AMPK in t-AMPK, the higher the AMPK phosphorylation activity of the protein. The AMPK phosphorylation activity can be measured by the method described in this specification. As used herein, the term “protein” is used interchangeably with “polypeptide” or “peptide”.

  The AMPK phosphorylation activity is one of the physiological activities possessed by Osm. Therefore, the recombinant protein according to the present invention may have physiological activities other than AMPK phosphorylation activity. Examples of physiological activities other than AMPK phosphorylation activity include oxidative stress reduction activity.

The oxidative stress reducing activity specifically refers to Superoxide disumutase (hereinafter also referred to as “SOD”) expression enhancing activity. Superoxide (O ₂ ^-) is granulocytes and monocytes is one of the white blood cells which is a kind of essential free radicals phagocytosis and bactericidal action indicate to exogenous infection, while the Ya various biological response It is considered as a causative substance of oxidative stress because when excessively produced by external stress, it damages DNA. SOD is an enzyme that catalyzes the reaction of producing hydrogen peroxide and oxygen from superoxide and hydrogen ions, and is considered as a mechanism for erasing the only superoxide in the living body. Therefore, a substance having SOD expression enhancing activity is effective in eliminating oxidative stress in the living body and preventing and ameliorating various diseases caused by oxidative stress.

  The recombinant protein according to the present invention is preferably produced by a host other than plants. Specifically, for example, it is preferable to produce Escherichia coli, yeast, fungi, insects, mammals and the like as a host, and it is more preferable to produce Escherichia coli as a host. More specifically, it is preferably produced by the method for producing an osmotin recombinant protein according to the present invention described later.

  As described above, the recombinant protein according to the present invention has AMPK phosphorylation activity. The AMPK phosphorylation activity can be measured by the method described in Examples described later. The recombinant protein according to the present invention preferably has AMPK phosphorylation activity equivalent to or higher than that of naturally-occurring osmotin.

  Here, the structure of the recombinant protein according to the present invention will be specifically described. Examples of the recombinant protein according to the present invention are registered in GenBank as accession numbers CAA43854 (derived from tobacco), AAU95241 (derived from potato), AAC64171 (derived from tomato), CAC34055 (derived from pepper), or AAP86781 (derived from red pepper). And a polypeptide consisting of the amino acid sequence. In addition, the recombinant protein according to the present invention may be a complex polypeptide containing a structure other than a polypeptide. As used herein, examples of the “structure other than the polypeptide” include sugar chains and isoprenoid groups, but are not particularly limited. Furthermore, the recombinant protein according to the present invention may contain an additional polypeptide at the N-terminus or C-terminus. Examples of the additional polypeptide include epitope-tagged polypeptides such as His, Myc, and Flag, and a glutathione-S-transferase (GST) tag. If necessary, affinity purification of the recombinant protein according to the present invention may be performed using these additional polypeptides.

  In one embodiment, the recombinant protein according to the present invention may be a mutant of Osm. That is, for example, it may be a variant of a polypeptide consisting of an amino acid sequence registered in GenBank as accession numbers CAA43854, AAU95241, AAC64171, CAC34055, or AAP86781. Specifically, for example, a polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence registered in GenBank as accession number CAA43854 Can be mentioned. In addition, when using regarding protein or polypeptide in this specification, the term "variant" intends the polypeptide which has AMPK phosphorylation activity.

  The variants also include deletions, insertions, inversions, repeats, and type substitutions (eg, replacement of a hydrophilic residue with another residue, but usually a strongly hydrophilic residue is strongly hydrophobic) Mutants containing residues that do not substitute) are included. In particular, “neutral” amino acid substitutions in a polypeptide generally have little effect on the activity of the polypeptide.

  Preferred mutants include mutants having conservative or non-conservative amino acid substitutions, deletions, or additions. Such a variant also does not change the AMPK phosphorylation activity of the recombinant protein according to the present invention. Representative conservative substitutions include substitution of one amino acid with another in the aliphatic amino acids Ala, Val, Leu, and Ile; exchange of hydroxyl residues Ser and Thr, acidic residues Asp and Glu Exchange of amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe and Tyr.

  It is well known in the art that some amino acids in the amino acid sequence that make up a polypeptide can be easily modified without significantly affecting the structure or function of the polypeptide.

  One skilled in the art can readily mutate one or several amino acids in the amino acid sequence of a polypeptide using well-known techniques. For example, according to a known point mutation introduction method, an arbitrary base of a polynucleotide encoding a polypeptide can be mutated. In addition, a deletion mutant or an addition mutant can be prepared by designing a primer corresponding to an arbitrary site of a polynucleotide encoding a polypeptide. Furthermore, if the method described in this specification is used, it can be easily determined whether the produced mutant has a desired AMPK phosphorylation activity.

  Moreover, the recombinant protein concerning this invention can be polypeptide which consists of an amino acid sequence which consists of 1st position-225th of the amino acid sequence registered into GenBank as accession number CAA43854 as one Embodiment. A polypeptide consisting of 20 amino acid residues on the C-terminal side in Osm is a signal sequence for transport to the vacuole after protein synthesis, and it has been reported that deletion does not affect the activity of Osm. (See Non-Patent Document 6). Therefore, since the above polypeptide also has AMPK phosphorylation activity, it is included in the recombinant protein according to the present invention.

  In another embodiment, the recombinant protein according to the present invention includes, as another embodiment, a polypeptide comprising an amino acid sequence consisting of positions 2 to 245 of the amino acid sequence registered in GenBank as accession number CAA43854; It may be a polypeptide consisting of an amino acid sequence consisting of positions 2 to 225 of the amino acid sequence registered under the sequence number CAA43854.

  Since the recombinant protein according to the present invention has the above physical properties and structure, it can be used for the treatment and improvement of diseases caused by activation or inhibition of the pathway through AMPK. In addition, since the recombinant protein according to the present invention has the above physical properties and structure, it can be used for the treatment and improvement of diseases caused by the occurrence of oxidative stress.

<II. Method for producing osmotin recombinant protein>
The method for producing an osmotin recombinant protein according to the present invention (hereinafter also simply referred to as “the production method according to the present invention”) is a non-plant transformant in which a polynucleotide encoding Osm or a variant thereof is introduced so as to be expressed. Is used to produce an osmotin recombinant protein having AMPK phosphorylation activity (hereinafter also referred to as “Osm recombinant protein”). According to the production method of the present invention, the above-described recombinant protein according to the present invention can be efficiently produced.

  As one embodiment, the production method according to the present invention can be specifically an embodiment in which an Osm recombinant protein is extracted from the non-plant transformant using a conventionally known method. In this embodiment, more specifically, the cells or tissues of the non-plant transformant are obtained from the non-plant transformants by a conventionally known cell or tissue disruption method in a conventionally known protein extract. By destroying and obtaining a soluble fraction by centrifugation or the like, an Osm recombinant protein can be obtained. Moreover, you may refine | purify Osm recombinant protein from the said soluble fraction using a conventionally well-known method.

  In another embodiment, the production method according to the present invention includes a step of forming an inclusion body containing an Osm recombinant protein in the non-plant transformant (hereinafter also referred to as “inclusion body formation step”), Extracting inclusion bodies and dissolving the inclusion bodies in a solubilization solution to solubilize the Osm recombinant protein (hereinafter also referred to as “solubilization step”), and the solubilized Osm recombination The structure may include a step of refolding the protein (hereinafter also referred to as “refolding step”).

  In any of the above embodiments, the production method according to the present invention further includes a step of producing the non-plant transformant (hereinafter, also referred to as “transformant production step”), and the refolding step. The step of detecting the AMPK phosphorylation activity of the obtained Osm recombinant protein (hereinafter also referred to as “activity detection step”) may be included. The transformant production step and activity detection step may include only one or both. In the present specification, “non-plant transformant” means a transformant having an organism other than plants as a host.

  Hereinafter, embodiments including the above-described transformant preparation step, inclusion body formation step, solubilization step, refolding step, and activity detection step will be described in detail.

(I-1) Transformant production process In the said transformant production process, the non-plant transformant in which the polynucleotide which codes Osm or its variant was introduce | transduced was produced. Specifically, a polynucleotide encoding Osm or a variant thereof is introduced into a host excluding plants so that it can be expressed. In the present specification, “polynucleotide is introduced so as to allow expression” means that the polynucleotide is introduced into a subject (host) so as to be expressed by a known genetic engineering technique (gene manipulation technique). In addition, the term “transformant” is intended to include not only cells, tissues and organs but also individual organisms.

  The host excluding the plant is not particularly limited as long as it is an organism excluding the plant. Specific examples include Escherichia coli, yeast, fungi, insects, and mammals, but the present invention is not limited to these. Among the above-exemplified hosts, E. coli is preferably used as a host excluding the above-mentioned plants, and among them, a strain having a genotype that expresses a transcription factor that activates a high expression promoter such as T7 promoter or lac promoter is used. Is more preferable. Examples of such strains include BL21 strain, Origami strain, Rosetta strain, Rosetta-gami strain and the like. Moreover, it is more preferable that the E. coli used as the host is a protease-deficient strain. Therefore, as a particularly preferred Escherichia coli, for example, BL21 (DE3) strain can be mentioned. According to such a host, an Osm recombinant protein can be produced more efficiently.

  In one embodiment, the polynucleotide is a polynucleotide encoding Osm. Specifically, for example, a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 can be mentioned. The polynucleotide is a polynucleotide having a base sequence registered under GenBank as accession number X61679, and encodes osmotin derived from tobacco (Nicotiana tabacum). In addition, the above polynucleotides include GenBank with accession numbers AY73731 (polynucleotide encoding osmotin derived from potato), AF093743 (polynucleotide encoding osmotin derived from tomato), AJ297410 (polynucleotide encoding osmotin derived from pepper) ) And AY262059 (polynucleotide encoding osmotin derived from red pepper). Furthermore, the polypeptides encoded by these polynucleotides have AMPK phosphorylation activity. As used herein, the term “polynucleotide” is used interchangeably with “gene”, “nucleic acid” or “nucleic acid molecule” and is intended to be a polymer of nucleotides. As used herein, the term “base sequence” is used interchangeably with “nucleic acid sequence” or “nucleotide sequence” and refers to the sequence of deoxyribonucleotides (abbreviated A, G, C, and T). As shown.

  In one embodiment, the polynucleotide may be a polynucleotide encoding a variant of Osm. Such a polynucleotide can also be referred to as a variant of a polynucleotide encoding Osm. As used herein, the term “variant” when used with respect to DNA or polynucleotide is intended to be a polynucleotide encoding a polypeptide having AMPK phosphorylation activity.

  Specifically, in one embodiment of the polynucleotide variant encoding the Osm, one or several bases are substituted, deleted, inserted, and / or substituted in the base sequence represented by SEQ ID NO: 1. It may be a polynucleotide encoding a polypeptide consisting of an added base sequence and having AMPK phosphorylation activity.

  One skilled in the art can readily mutate one or several bases in a polynucleotide base sequence using well-known techniques. For example, according to a known point mutation introduction method, any base of the polynucleotide can be mutated. In addition, a deletion mutant or an addition mutant can be prepared by designing a primer corresponding to an arbitrary site of the polynucleotide.

  In one embodiment of the variant, a polypeptide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 1 and has AMPK phosphorylation activity is provided. It can be an encoding polynucleotide.

  Hybridization can be performed by well-known methods such as those described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989). Usually, the higher the temperature and the lower the salt concentration, the higher the stringency (harder to hybridize), and a more homologous polynucleotide can be obtained. The appropriate hybridization temperature varies depending on the base sequence and the length of the base sequence. For example, when a DNA fragment consisting of 18 bases encoding 6 amino acids is used as a probe, a temperature of 50 ° C. or lower is preferable.

  As used herein, the term “stringent hybridization conditions” refers to hybridization solution (50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate. (Containing pH 7.6), 5 × Denhart solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA) overnight at 42 ° C., then 0.1 × at about 65 ° C. It is intended to wash the filter in SSC. Depending on the polynucleotide that hybridizes to a “portion” of the polynucleotide, at least about 15 nucleotides (nt) of the reference polynucleotide, and more preferably at least about 20 nt, even more preferably at least about 30 nt, and even more preferably about Polynucleotides (either DNA or RNA) that hybridize to polynucleotides longer than 30 nt are contemplated. A polynucleotide (oligonucleotide) that hybridizes to a “part” of such a polynucleotide is also useful as a primer used to isolate a polynucleotide, as used in the Examples.

  The “polynucleotide comprising the base sequence represented by SEQ ID NO: 1” is intended to be a polynucleotide comprising the sequence represented by each deoxynucleotide A, G, C and / or T of SEQ ID NO: 1.

  The polynucleotide may be present in the form of RNA (eg, mRNA) or in the form of DNA (eg, cDNA or genomic DNA). DNA can be double-stranded or single-stranded.

  As used herein, the term “oligonucleotide” is intended to be a combination of several to several tens of nucleotides, and is used interchangeably with “polynucleotide”. Oligonucleotides are called dinucleotides (dimers) and trinucleotides (trimers) as short ones, and are represented by the number of nucleotides polymerized such as 30 mers or 100 mers as long ones. Oligonucleotides can be produced as fragments of longer polynucleotides or chemically synthesized.

  In another embodiment, the polynucleotide comprises, as another embodiment, a polynucleotide consisting of a base sequence consisting of positions 1 to 678 of the base sequence shown in SEQ ID NO: 1; first position of the base sequence shown in SEQ ID NO: 1. A polynucleotide encoding a polypeptide having a base sequence in which one or several bases are substituted, deleted, inserted and / or added in the base sequence consisting of position 678 and having AMPK phosphorylation activity A polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence consisting of positions 1 to 678 of the nucleotide sequence represented by SEQ ID NO: 1 and has AMPK phosphorylation activity; It can be a polynucleotide encoding a peptide. According to such a polynucleotide, a polypeptide comprising an amino acid sequence in which 20 amino acid residues are deleted from the C-terminal of the amino acid sequence registered in GenBank as accession number CAA43854 can be produced.

  Furthermore, in another embodiment, the polynucleotide is a polynucleotide comprising a base sequence consisting of positions 4 to 738 of the base sequence shown in SEQ ID NO: 1; fourth position of the base sequence shown in SEQ ID NO: 1 A polynucleotide encoding a polypeptide having a base sequence in which one or several bases are substituted, deleted, inserted, and / or added in the base sequence consisting of position 738 and having AMPK phosphorylation activity A polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence consisting of positions 4 to 738 of the nucleotide sequence represented by SEQ ID NO: 1 and has AMPK phosphorylation activity; It can be a polynucleotide encoding a peptide. According to such a polynucleotide, a polypeptide comprising an amino acid sequence in which the N-terminal methionine residue of the amino acid sequence registered in GenBank as accession number CAA43854 is deleted can be produced.

  In addition, the polynucleotide is a polynucleotide consisting of a base sequence consisting of positions 4 to 678 of the base sequence shown in SEQ ID NO: 1; from the 4th position to 678th position of the base sequence shown in SEQ ID NO: 1 A polynucleotide comprising a nucleotide sequence in which one or several bases are substituted, deleted, inserted and / or added, and which encodes a polypeptide having AMPK phosphorylation activity; A polypeptide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence consisting of positions 4 to 678 of the nucleotide sequence shown and that encodes a polypeptide having AMPK phosphorylation activity Can be a nucleotide. According to such a polynucleotide, a polypeptide comprising an amino acid sequence in which the N-terminal methionine residue of the amino acid sequence registered in GenBank as accession number CAA43854 and 20 amino acid residues from the C-terminal are deleted. Can be manufactured. Thus, in the base sequence shown in SEQ ID NO: 1, translation and expression efficiency may be improved by using a polynucleotide in which the first to third positions, which are initiation codons, are deleted. Therefore, according to the said structure, the production efficiency of Osm recombinant protein can be improved.

  The polynucleotide can also be fused to the polynucleotide encoding the tag tag (tag sequence or marker sequence) described above on the 5 'or 3' side. Specifically, for example, a polynucleotide encoding an epitope tag polypeptide such as His, Myc, Flag or the like can be fused.

  The polynucleotide may include a sequence such as a sequence of an untranslated region (UTR) or a vector sequence (including an expression vector sequence).

  Although it does not specifically limit as a supply source for acquiring the said polynucleotide, It is preferable that it is a biological material. As used herein, the term “biological material” intends a biological sample (a tissue sample or cell sample obtained from an organism). The polynucleotide can also be obtained by chemical synthesis.

  Furthermore, when the polynucleotide is a mutant, as described above, it is well known that a desired mutant can be obtained from nature other than by artificially introducing a mutation.

  In the transformant production step, the method for introducing the polynucleotide into the host excluding the plant so that it can be expressed is not particularly limited, and may be introduced by a conventionally known method depending on the host. Good. For example, the polynucleotide can be incorporated into a recombinant expression vector and then introduced into a host capable of expression by a known method. The method for introducing the expression vector into a host cell, that is, the transformation method is not particularly limited, and electroporation method, calcium phosphate method, liposome method, DEAE dextran method, microinjection method, cationic lipid-mediated transfection, electro Conventionally known methods such as poration, transduction and infection can be suitably used.

  The recombinant expression vector is preferably a plasmid, but the present invention is not limited to this, and any other expression vector may be used as long as it can introduce the target polynucleotide into a host so that it can be expressed. Can be used. The recombinant expression vector preferably incorporates a promoter that functions in the host so as to express a foreign polynucleotide. Moreover, it is preferable that the said promoter is what improves the expression level of Osm recombinant protein. Further, the recombinant expression vector may contain a tag sequence for attaching a tag to a polypeptide that is a translation product of a foreign polynucleotide. By having such a tag sequence, it is possible to purify the polypeptide relatively easily.

  Preferred recombinant expression vectors for use in bacteria include, for example, pET vector, pQE70, pQE60, pQE-9, pBS vector, Phagescript vector, Bluescript vector, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233- 3, pDR540, and pRIT5. Among these, a plasmid vector having a T7 promoter or a lac promoter is preferable.

  Examples of preferable eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, pSG, pSVK3, pBPV, pMSG, and pSVL.

  The method for incorporating the polynucleotide into the recombinant vector is not particularly limited, and may be incorporated by a conventionally known method. Such techniques are well known to those skilled in the art.

(I-2) Inclusion body formation step In the inclusion body formation step, an inclusion body containing an Osm recombinant protein is formed in a non-plant transformant in which a polynucleotide encoding Osm or a variant thereof is introduced. Let In the present specification, “inclusion body” is used interchangeably with “inclusion body”.

  The method for forming the inclusion body is not particularly limited, and the non-plant transformant may be cultured under conditions where the polynucleotide is expressed. In the present invention, it is preferable to induce the expression of the polynucleotide using a conventionally known gene expression induction system to produce an Osm recombinant protein to form the inclusion body. Examples of the gene expression induction system include an expression induction system by temperature shift, an isopropyl-β-D-galactopyranoside (IPTG) expression induction system, and an auxin (IAA) expression induction system. These gene expression induction systems may be used alone or in combination.

  As an embodiment of the non-plant transformant, when using an E. coli transformant into which a polynucleotide encoding osmotin or a variant thereof is introduced so as to be expressed, for example, using an IPTG gene induction system, It is preferable to induce the expression of the polynucleotide at ˜30 ° C. By inducing the expression of the polynucleotide in the temperature range, the Escherichia coli transformant can efficiently produce the Osm recombinant protein and form the inclusion body.

(I-3) Solubilization step In the solubilization step, first, the inclusion body formed in the inclusion body formation step is extracted from the non-plant transformant. The method for extracting the inclusion body is not particularly limited. For example, since the inclusion body is insoluble in water, the cells or tissues of the non-plant transformant are disrupted in an appropriate aqueous buffer to obtain an extract, and then the extract is centrifuged. It can be obtained by separating and collecting the insoluble fraction. The method for destroying the cells or tissues of the non-plant transformant is not particularly limited. For example, destruction by physical lysis such as sonication or French press, or lysis with a surfactant or lysozyme. can do. More specifically, after suspending in a buffer containing a surfactant such as Triton X-100, physically pulverizing with ultrasonic waves or a French press, lysozyme and nuclease are added, and the enzyme is incubated at 30 ° C to 37 ° C. By making it react, the said inclusion body can be extracted.

  The buffer solution is not particularly limited, and examples thereof include a Tris-HCl buffer and a phosphate buffer (10 mM to 50 mM / neutral pH). Further, the buffer solution may contain about 1% to 2% of Triton X-100. Thereby, the protein which is an impurity derived from the membrane fraction of the non-plant transformant can be removed.

  Furthermore, commercially available reagents such as BugBuster MasterMix (Novagen) may be used to destroy cells or tissues of the non-plant transformant. For example, according to BugBuster MasterMix (Novagen), lysozyme that dissolves bacterial cells and nuclease for removing genomic DNA derived from E. coli are included, so when the host of the non-plant transformant is E. coli, The inclusion bodies can be recovered by breaking the cells without disruption.

  Next, in the solubilization step, the extracted inclusion body is dissolved in a solubilization solution. Thereby, the Osm recombinant protein contained in the inclusion body is solubilized.

  The solubilizing solution is not particularly limited and may be any solubilizing solution that can solubilize the inclusion body. Specific examples of the solubilizing solution include a buffer solution containing a surfactant and / or a denaturing agent. Examples of the surfactant include nonionic surfactants such as polyoxyethylene octyl phenyl ether, polyoxyethylene sorbitan monolaurate, and polyoxyethylene sorbitan monooleate; dodecyltrimethylammonium bromide, dodecyltrimethylammonium chloride And cationic surfactants such as cetyltrimethylammonium bromide; anionic surfactants such as sodium dodecyl sulfate (SDS), N-lauroylsarcosine, deoxycholic acid, sodium cholate; amphoteric surfactants such as phosphatidylethanolamine Although an agent can be mentioned, this invention is not limited to these. Among them, it is preferable to use sodium N-lauroyl sarcosine. Examples of the modifying agent include urea, thiourea, guanidine hydrochloride and the like. These surfactants and modifiers may be used alone or in combination.

  The concentration of the surfactant and protein denaturant in the solubilized solution is not particularly limited, and can be set as appropriate according to the type of surfactant and protein denaturant used. Specifically, for example, when N-lauroyl sarcosine is contained in the solubilized solution, the concentration is preferably 0.1% (w / v) to 5% (w / v), and 2% (W / v) is more preferable. If it is the said range, the said inclusion body can be dissolved effectively.

  The solubilizing solution may contain a reducing agent. Examples of the reducing agent include dithiothreitol, dithioerythritol, glutathione, cysteine, cystamine, and 2-mercaptoethanol, but are not particularly limited. Moreover, these reducing agents may be used alone or in combination.

  The buffer solution contained in the solubilized solution is not particularly limited, and examples thereof include a Tris buffer solution, a phosphate buffer solution, a HEPES buffer solution, and a CAPS buffer solution. The solubilized solution preferably has a pH of 5.5 to 12.5. Among them, it is preferable to use an alkaline buffer solution, more specifically, a buffer solution having a pH of 8.0 to 12.5. According to the solubilized solution in which the above exemplified surfactant is contained in such a buffer solution, the inclusion body can be solubilized more efficiently.

Further, the solubilizing solution may contain 0.1M to 0.5M salt. Examples of the salt include NaCl, KCl, CaCl _{2 and the} like, but are not limited thereto.

  Moreover, the conditions which process the said inclusion body with the said solubilization solution are not specifically limited, What is necessary is just to process on the conditions which can solubilize the said inclusion body. That is, the treatment temperature, the treatment time, and the like may be appropriately set so that the inclusion body is solubilized by the solubilizing solution according to the composition and pH of the solubilizing solution. Specifically, when the pH of the solubilized solution is near neutral, in other words, when the pH is 7 to 8, the treatment temperature is preferably 4 ° C to 37 ° C, and preferably 4 ° C. Is more preferable. The treatment time is preferably 30 minutes to 24 hours, and more preferably 18 hours. On the other hand, when the pH of the solubilized solution is alkaline, in other words, when the pH is 9 to 12, the treatment temperature is preferably 4 ° C to 37 ° C, and is room temperature (15 ° C to 30 ° C). It is more preferable. The treatment time is preferably 15 minutes to 2 hours. According to said process conditions, the said inclusion body can be dissolved efficiently.

  In the solubilization step, the solubilized Osm recombinant protein may be purified from a solution containing the solubilized inclusion body, in other words, from a solution containing the solubilized Osm recombinant protein. The method for purifying the solubilized Osm recombinant protein is not particularly limited. Specifically, for example, from a solution containing the solubilized inclusion body, the cells are removed from the cells or tissues by a well-known method (for example, a method of recovering the soluble fraction by centrifuging after disrupting the cells or tissues). After preparing the extract, well-known methods (for example, ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography) are prepared from this cell extract. Chromatography, hydroxyapatite chromatography, and lectin chromatography). In addition, since the solution containing the solubilized inclusion body contains a surfactant and / or a denaturing agent, a usable method and conditions can be selected even in the presence of the surfactant and / or the denaturing agent. preferable.

(I-4) Refolding step In the refolding step, the Osm recombinant protein solubilized in the solubilization step is refolded. Specifically, after the solubilization step, the Osm recombinant protein is dissolved in the solubilization solution. In the refolding step, using the solution containing the solubilized Osm recombinant protein, the Osm recombinant protein is refolded into an Osm recombinant protein having AMPK phosphorylation activity. The refolding method is not particularly limited, and a conventionally known method can be used. Specifically, for example, it can be performed by a dialysis method or a dilution method.

  The reaction solution used for refolding (hereinafter also referred to as “refolding solution”) is not particularly limited, but promotes or assists formation of a three-dimensional structure, that is, formation of an intermolecular or intramolecular disulfide bond. It preferably contains an S—S bond formation promoting / adjuvant. Specific examples of the SS bond formation promoting / adjuvant include, for example, reduced glutathione (hereinafter also referred to as “GSH”), oxidized glutathione (hereinafter also referred to as “GSSG”), dithiothreitol ( (Hereinafter also referred to as “DTT”) or the like can be used. These may be used alone or in combination. The concentration of the SS bond formation promotion / adjuvant in the refolding solution is not particularly limited, and may be set according to the type of the SS bond formation promotion / adjuvant used. For example, when GSH and GSSG are used as the SS bond formation promoting / adjuvant, the GSH concentration is preferably 1 mM to 5 mM, and the GSSG concentration is preferably 0.1 mM to 0.5 mM. . Moreover, when using DTT, it is preferable that DTT density | concentration shall be 0.1 mM-10 mM.

The pH of the refolding solution is preferably 5-8. Furthermore, the refolding solution may contain 0.1 M to 0.5 M salt. Examples of the salt include NaCl, KCl, CaCl ₂ , and MgCl _2, but the present invention is not limited to this.

  The temperature at the time of refolding may be any temperature at which the Osm recombinant protein can be refolded into a polypeptide having AMPK phosphorylation activity. Specifically, it is preferably 2 ° C to 40 ° C, more preferably 4 ° C to 37 ° C, and further preferably 20 ° C to 30 ° C. By performing refolding in the above temperature range, it can be refolded so as to be an Osm recombinant protein having high AMPK phosphorylation activity.

  In the refolding step, the Osm recombinant protein may be purified after the Osm recombinant protein is refolded. A method for purifying the Osm recombinant protein is not particularly limited. For example, from a refolding solution containing the refolded Osm recombinant protein, well-known methods (eg, ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction) Chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography).

(I-5) Activity detection step In the activity detection step, the AMPK phosphorylation activity of the Osm recombinant protein refolded in the refolding step is detected. The method for detecting AMPK phosphorylation activity is not particularly limited. Specifically, for example, after adding / administering the Osm recombinant protein to a cell (cultured cell) or an animal individual and stimulating the cell, By detecting the ratio of p-AMPK to t-AMPK in the organ, AMPK phosphorylation activity can be detected. More specifically, for example, t-AMPK is detected by using an anti-αAMPK antibody, p-AMPK is detected by using an anti-p-AMPK antibody, and the ratio between the two is used to obtain AMPK phosphorylation. Oxidation activity can be detected.

  A method for detecting t-AMPK and p-AMPK using the anti-αAMPK antibody and the anti-p-AMPK antibody is not particularly limited. For example, a method of performing Western blotting of a cell or tissue sample using these antibodies Alternatively, it can be detected by an ELISA method in which an anti-αAMPK antibody and / or a p-AMPK antibody is immobilized.

  As described above, cells or individual animals can be used to detect the AMPK phosphorylation activity, but cultured cells are preferably used. According to the cultured cells, the AMPK phosphorylation activity of the Osm recombinant protein can be detected simply and rapidly.

  The cells and animal individuals may be cells and animal individuals expressing an adiponectin receptor (hereinafter also referred to as “AdipoR”). Specific examples of cells expressing AdipoR include C2C12 cells that are mouse skeletal muscle models and Fao cells that are mouse liver-derived cells. Since these cells express AdipoR, they can be suitably used in the present invention. A cell line in which AdipoR is forcibly expressed can also be used as the cell expressing AdipoR.

  As described above, according to the production method of the present invention, an Osm recombinant protein having physiological activity can be produced without the need for special equipment or equipment. That is, according to the production method of the present invention, it is possible to produce an Osm recombinant protein that is inexpensive, simple, and has a large amount of physiological activity. Specifically, for example, when an Escherichia coli transformant is used as the non-plant transformant, an Osm recombinant protein having physiological activity can be produced in a yield of about 10 mg from 1 L of the culture solution. On the other hand, the yield of the Osm recombinant protein from tobacco transformants as disclosed in Non-Patent Document 5 is 2 mg to 3 mg from 1 kg of leaves of tobacco transformants. Therefore, according to the production method of the present invention, a significantly higher yield of Osm recombinant protein can be obtained than in the prior art. Furthermore, according to the production method of the present invention, since there are no restrictions on the host to be used, various conditions such as use, production scale, equipment, and cost can be accommodated.

  Moreover, if it is set as the structure including the said activity detection process, the mutant which has the activity which phosphorylates 5'-AMP activation protein kinase will be easily screened from the mutant of osmotin, and this Osm recombinant protein will be acquired. be able to. That is, the present invention includes screening for an osmotin recombinant protein having an activity of phosphorylating 5′-AMP-activated protein kinase, including at least the inclusion body formation step, the solubilization step, the refolding step, and the activity detection step. Methods are included. According to the screening method of the present invention, an osmotin recombinant protein having an activity of phosphorylating 5'-AMP-activated protein kinase can be easily screened without requiring special equipment or technology.

<III. Composition containing osmotin recombinant protein>
The composition according to the present invention contains the above-described recombinant protein according to the present invention. The recombinant protein according to the present invention has AMPK phosphorylation activity. Therefore, the composition according to the present invention can be used as a composition for preventing or treating a disease caused by activation or inhibition of a pathway mediated by AMPK.

  Examples of the disease include lifestyle-related diseases. More specifically, type II diabetes, insulin resistance, hyperlipidemia, hypertension, arteriosclerosis, and heart disease can be mentioned. The composition concerning this invention may be applied with respect to the patient who develops the said disease independently, and may be applied with respect to the patient who has a plurality of diseases.

  In addition, as will be further described, as demonstrated in the examples described later, in one aspect, the recombinant protein according to the present invention has a glucose metabolism abnormality improving effect and a lipid metabolism abnormality improving effect. Those skilled in the art will recognize that improvement in abnormal glucose metabolism or abnormal lipid metabolism may lead to improvement or prevention of symptoms of lifestyle-related diseases such as type II diabetes, insulin resistance, hyperlipidemia, arteriosclerosis, and heart disease. Easy to understand.

  In other words, the composition according to the present invention can be used as a “lifestyle-related disease improving agent”. In this specification, the term “lifestyle-related disease-improving agent” intends a pharmaceutical, food, or cosmetic composition having an effect of alleviating or eliminating the symptoms of the disease by ingestion.

  The recombinant protein according to the present invention has oxidative stress reducing activity. Therefore, the composition according to the present invention can be used for prevention or treatment of diseases caused by oxidative stress.

  Examples of the disease include cancer, inflammation, allergic disease, Alzheimer's disease, age-related macular degeneration, and skin aging. The composition concerning this invention may be applied with respect to the patient who develops the said disease independently, and may be applied with respect to the patient who has a plurality of diseases.

  In other words, the composition according to the present invention can be used as an “oxidative stress reducing agent”. In this specification, the term “oxidative stress reducing agent” intends a pharmaceutical, food, or cosmetic composition having an effect of alleviating or eliminating the symptoms of the disease by ingestion.

  When the composition according to the present invention is used as a therapeutic agent for clinical application, the administration conditions can be determined using a model animal system that develops a disease to be treated. That is, administration conditions including dosage, administration interval, and administration route can be examined using the model animal, and conditions for obtaining appropriate preventive or therapeutic effects can be determined.

  Further, the composition according to the present invention can be combined with a desired pharmaceutically acceptable carrier. Examples of the carrier include sterilized water, physiological saline, buffer, vegetable oil, emulsifier, suspension, salt, stabilizer, preservative, surfactant, sustained release agent, other proteins (BSA, etc.), transfection Examples include reagents (including lipofection reagents and liposomes). Furthermore, usable carriers include glucose, lactose, gum arabic, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea and the like. Further, the composition according to the present invention may contain any substance other than the above exemplified carriers as long as it is within a pharmaceutically acceptable range.

However, the content of the recombinant protein according to the present invention and components other than the recombinant protein are not particularly limited. What is necessary is just to set according to a use application, an administration method, etc. Also,
When the composition according to the present invention is formulated, the dosage form is not particularly limited. For example, it may be a solution (injection), powder, microcapsule, tablet or the like. The method for administering the composition according to the present invention to the administration subject is not particularly limited, and can be administered systemically or locally. Specifically, for example, it can be administered transcutaneously, intranasally, transbronchially, intramuscularly, intraperitoneally, intravenously, intraarticularly, subcutaneously, intrathecally, intraventricularly, or orally. . Of these, intravenous administration or oral administration is preferred, and oral administration is more preferred. In addition, when there is a side effect due to systemic administration or a decrease in the effect, local administration is preferred. For example, it is considered that the composition according to the present invention can be effectively treated by drug delivery targeting a disease site by combining the composition with a sustained-release agent. The dosage and administration method vary depending on the tissue transferability of the active ingredient of the therapeutic agent, the purpose of treatment, the weight and age of the patient, symptoms, etc., but can be appropriately selected by those skilled in the art.

  Moreover, in the composition concerning this invention, content of the recombinant protein concerning this invention is not specifically limited, What is necessary is just to set according to a use use, an administration method, etc. In general, the content of the recombinant protein according to the present invention is preferably 0.1 to 90% by weight of the total composition. Further, the dose of the composition according to the present invention to the administration subject is not particularly limited, but generally, in parenteral administration, 0.0001 mg to 1000 mg, preferably 0. 001 mg to 300 mg, more preferably 0.01 mg to 100 mg. However, these dosages should be adjusted accordingly depending on the disease state, weight, and individual patient response to treatment, the type of composition being administered, and the mode of administration, the stage of the disease process or the interval between doses. Is preferred. That is, the dose may be smaller than the minimum dose described above, or may be administered in excess of the maximum dose described above in order to obtain a therapeutic effect. Administration can be performed once to several times and can be administered 1 to 5 times per day.

  Moreover, the subject which administers the composition concerning this invention is not specifically limited. Examples include humans and non-human mammals such as mice, rats, rabbits, monkeys, and other vertebrates. Application to non-human mammals is also useful as a model for developing preventive or therapeutic methods for diseases caused by activation or inhibition of AMPK-mediated pathways. Thereby, for example, a new treatment protocol for preventing lifestyle-related diseases can be developed.

  Moreover, the composition according to the present invention can be used not only as a pharmaceutical product but also as a food or cosmetic composition. Use as such a food or cosmetic composition is effective in preventing and improving diseases caused by activation or inhibition of pathways mediated by AMPK, such as lifestyle-related diseases.

  It should be noted that those skilled in the art who have contacted the present specification will be aware of diseases resulting from activation or inhibition of the pathway through AMPK, such as type II diabetes, insulin resistance, hyperlipidemia, arteriosclerosis, and heart disease. The protein according to the present invention is administered for the prevention or treatment of lifestyle-related diseases, or abnormal sugar metabolism or abnormal lipid metabolism, and the use of the protein according to the present invention in the manufacture of a medicament for the prevention or treatment. Are easily understood to be within the scope of the present invention.

  Furthermore, since the composition according to the present invention has an effect of reducing oxidative stress, it can be used not only as a pharmaceutical product but also as a food or cosmetic composition. Use as such a food or cosmetic composition is effective in preventing or improving diseases caused by oxidative stress.

  It should be noted that those skilled in the art who have come into contact with the present specification are in the present invention for the prevention or treatment of diseases caused by oxidative stress, such as cancer, inflammation, allergic diseases, Alzheimer's disease, age-related macular degeneration, and skin aging. It will be readily understood that it is within the scope of the present invention to administer such proteins and to use the proteins according to the present invention in the manufacture of a medicament for the prevention or treatment described above.

<III-A. Food composition>
The form of the food composition containing the composition according to the present invention is not particularly limited. Therefore, the food of the present invention is a tablet, a pill, a granule, a powder, a powder, a capsule, in the form of the above-mentioned composition, or an excipient or additive commonly used in foods. , Wettable powders, emulsions, solutions, extracts, elixirs and the like can be formulated by known methods. Preferred dosage forms are solid dosage forms such as tablets, pills, granules, powders, powders and capsules, and more preferred are granules, or compressed dosage forms such as tablets and pills.

  Examples of excipients commonly used in food include, for example, binders (for example, syrup, gum arabic, sucrose, lactose, powdered reduced maltose, cellulose sugar, mannitol, maltitol, dextran, starches, gelatin, sorbit, tragacanth. , Polyvinylpyrrolidone, etc.), lubricant (eg, sucrose fatty acid ester, glycerin fatty acid ester, magnesium stearate, calcium stearate, talc, polyethylene glycol), disintegrant (eg, potato starch), wetting agent (eg, lauryl sulfate) Sodium), an anti-caking agent (for example, silica) and the like. Examples of additives include fragrances, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, preservatives, and the like.

  In addition, in addition to supplements having the above-mentioned preparation form (including health foods (specific health foods, foods for nutritional function) and so-called health foods such as nutritional supplements), the foods targeted by the present invention include the above Foods prepared using antioxidants as one of the raw materials for producing foods and drinks (in addition to general foods (including health foods), health function foods (including foods for specified health use and nutritional function foods)) included.

  The type of food is not particularly limited, and drinks (milk drinks, lactic acid bacteria drinks, soft drinks with fruit juice, carbonated drinks, fruit juice drinks, vegetable drinks, vegetable and fruit drinks, alcoholic drinks, coffee drinks, powdered drinks, sports drinks , Supplement beverages, tea beverages, green tea, beverages with jelly, tea beverages such as blended tea), confectionery (puddings such as custard pudding, milk pudding, pudding with fruit juice, desserts such as jelly, bavaroa and yogurt; milk Ice cream, ice cream with fruit juice, soft ice cream, ice candy and other frozen desserts; chewing gums, bubble gums and other gums (plate gums, sugar-coated grain gums); marble chocolates and other coated chocolates, strawberry chocolates, blueberry chocolates And added flavor such as melon chocolate Chocolates such as chocolate; hard candy (including bonbon, butterball, marble, etc.), soft candy (including caramel, nougat, gummy candy, marshmallow, etc.), caramels such as drop, toffee, etc .; hard biscuits, cookies, oak Baked confectionery such as rice crackers), breads, soups (consomme soup, potage soup, etc.), processed fish products (fish meat ham, fish sausage, fish meat surimi, salmon, bamboo rings, hampen, fried satsuma, Date roll, whale bacon ), Processed meat products (ham, sausage, grilled pork, etc.), noodles (udon, cold wheat, somen noodles, buckwheat, Chinese soba noodles, spaghetti, macaroni, rice noodles, salmon and wonton), sauces (separate dressing, non-oil dressing, ketchup) , Sauce, sauce) The like may be exemplified delicatessen. Among these, beverages are more preferable from the viewpoint of continuous intake.

  The food composition of the present invention involves active oxygen generated more than necessary by ultraviolet rays, in vivo phagocytes, radiation, chemical substances, tobacco, stress, etc. due to the oxidative stress reducing action of the osmotin recombinant protein, which is an active ingredient. It can be effectively used for the prevention or improvement of various diseases, such as cancer, inflammation, allergic diseases, Alzheimer's disease, age-related macular degeneration, and skin aging.

  For this reason, the food of the present invention includes, in addition to general foods, foods that have the effect of activating AMPK or reducing oxidative stress and are used for specific health uses. Examples of such foods include foods for specified health use or health foods equivalent thereto. In addition, food for specified health use (including food for specified health use with conditions) is a food that can be labeled for function or health use approved or approved by the Ministry of Health, Labor and Welfare in its packaging container. In the present invention, a specific health purpose is to treat and ameliorate diseases caused by AMPK activation, specifically AMPK-mediated pathway activation or inhibition, or oxidative stress reduction, specifically oxidative stress. Examples of indications include "antimetabolic syndrome action", "antioxidant action", "radical scavenging", "reactive oxygen scavenging", "preventing oxidation", and "suppressing aging" Or prevent ”. The food for specified health use is a preferred embodiment of the food as referred to in the present invention because it can be differentiated from a general food that does not allow function display in that such function display is possible.

<III-B. Cosmetic composition>
The form of the cosmetic composition containing the composition according to the present invention is not particularly limited. Therefore, it may take any state such as solid, liquid, paste, jelly, and powder. In order to form such a state, it can be solidified using, for example, a gelling agent, or can be dispersed using a liquid. Moreover, a solvent can be added to make a solution, or it can be spray-dried to form a powder.

  Further, the dosage form of the cosmetic composition containing the composition according to the present invention according to the present invention is not limited, and ampoules, capsules, powders, granules, pills, tablets, solids, liquids, gels, bubbles, emulsions, Various forms such as creams, ointments, sheets, mousses and bath preparations can be used.

  The cosmetic composition containing the composition according to the present invention according to the present invention is preferably applied to humans, but should be applied to animals other than humans as long as each effect can be expected. You can also.

  In the cosmetic composition containing the composition according to the present invention, a whitening agent, a moisturizing agent, an ultraviolet absorber, an antibacterial agent, and various other components may be further added according to the purpose of use. Can do. The example of an additive is shown below.

(Whitening agent)
The whitening agent may be a melanin production inhibitor and / or a tyrosinase activity inhibitor.

  For example, L-ascorbic acid and derivatives thereof, hydroquinone and derivatives thereof, lucinol, edetic acid and derivatives thereof, and placenta extract, t-AMCHA, acerola extract, age extract, ellagic acid or derivatives thereof, fire retardant Extract, chamomile extract, chamomile flower extract, kiwi extract, glutathione, tocotrienol, ferulic acid, raspberry ketone, lucinol, walnut extract, pyridoxine dipalmitate, sulfur, kojic acid or derivatives thereof, glucosamine or derivatives thereof, hydroxycinnamic acid or derivatives thereof , Glutathione, Arnica extract, Ogon extract, Sohakuhi extract, Psycho extract, Bowfu extract, Manntake mycelium culture or extract thereof, Linden extract, Peach leaf extract, Ages extract, Examples include kujin extract, gill extract, toki extract, yakuinin extract, oyster leaf extract, daiou extract, button pi extract, hamamelis extract, maroni extract, hypericum extract, oil-soluble licorice extract and the like.

  These whitening agents can be used alone or in combination of two or more.

(Anti-aging agent)
The anti-aging agent may be an anti-inflammatory agent and / or an antioxidant.

  For example, anti-inflammatory agents and antioxidants are used as anti-aging agents. Anti-inflammatory agents include glycyrrhizic acid and its salts, glycyrrhetinic acid and its salts, isopropylaminocaproic acid and its salts, allantoin, lysozyme chloride, guaiazulene, salicylic acid. Examples of methyl, γ-oryzanol, and antioxidants include carotenoids such as α-carotene, β-carotene, γ-carotene, lycopene, cryptoxanthine, lutein, zeaxanthin, isozeaxanthin, rhodoxanthine, capsanthin, and crocetin; 1,4-diazacyclo Octane, 2,5-dimethylfuran, 2-methylfuran, 2,5-diphenylfuran, 1,3-diphenylisobenzofuran, α-tocopherol, β-tocopherol, γ-tocopherol, d-tocopherol, histidine, tryptophan, Thionine, alanine or alkyl ester thereof; dibutylhydroxytoluene, butylhydroxyanisole, ascorbic acid, tannic acid, epicatechin, epicacatechin, epicatechin gallate, tannins such as rucaine, flavonoids such as rutin, and other gallic acids Propyl, astaxanthin, carotene, tocopherol, ascorbic acid, tetrasodium edetate, erythorbic acid, tocopherol acetate, retinol acetate, dibitylhydroxytoluene, ascorbyl stearate, ascorbyl palmitate, methylsilanol dioletocopherol silicic acid mixture, nicotinic acid Benzyl, photosensitizer 401, aspartic acid, adenosine triphosphate 2Na, aminobutyric acid, fennel extract, Dutch carassier Kiss, caffeine, chlorella extract, saffron extract, ginger extract, soybean extract, tiso extract, folic acid, retinol, retinol palmitate, inositol, turmeric extract, oryzanol, carotene, carrot extract, wheat ointment extract, sensyu extract, chimney extract, Toki extract, toki root extract, dokudami extract, tocopherol, tocopherol nicotinate, button extract, ergocalciferol, pyridoxine dicaprylate, batyl alcohol, glycyrrhetinyl stearate, glycyrrhizic acid, glycyrrhetinic acid, diphenhydramine hydrochloride, lysozyme chloride, aminocaproic acid, Ganoderma Extract, Yokuinin, Merirot Extract, Button Extract, Toki Extract, Toki Root Extract, Senkyu Extract, Gen Shoko extract, Allantoin, Arnica extract, Arnica flower extract, Ogon extract, Ouren extract, Odorikosou extract, Catfish ear extract, Chamomile extract, Calamine, Pepper extract, Licorice extract, Guaiazulene, Gardenia extract, Kumazasa extract, Glycyrrhizic acid 2K, Glycyrrhetic acid Stearyl, Gentian extract, Black tea extract, Comfrey extract, Comfrey leaf extract, Tocopherol acetate, Methyl salicylate, Zinc oxide, Sicon extract, Murasaki root extract, Perilla extract, Perilla leaf extract, Periwinkle extract, Peonies extract, Honeysuckle extract, Sage extract, Kizuta extract, Elderberry extract, Achillea millefolium extract, Assembly extract, Sakuhakuhi extract, Tokisenka extract, Piri Xing HCl, loquat leaf extract, Tilia cordata extract, peach leaf or fruit extract, cornflower extract, saxifrage extract, mugwort extract, lettuce extract, Roman chamomile extract, burnet extract and Kakaroru, polyamines and the like.

  These anti-aging agents can be used individually by 1 type or in combination of 2 or more types.

(Humectant)
The humectant may be a hydrophilic humectant and / or a lipophilic humectant. Examples of the hydrophilic humectant include amino acids, peptides, proteins, higher alcohols and derivatives thereof, and lipophilic humectants such as phospholipids, sugars. Lipids, steroids and the like may be used.

  As moisturizer, sodium pyridonecarboxylate, glycol, glycerin, glucose, maltose, maltitol, sucrose, fructose, xylitol, sorbitol, maltotriose, threitol, erythritol, amylolytic sugar-reducing alcohol, sorbitol, polyhydric alcohols Is ethylene glycol, 1,4-butylene glycol, diglycerin, triglycerin, tetraglycerin, 1,3-butylene glycol, propylene glycol, dipropylene glycol, polyethylene glycol, 1,3-propanediol, other serine, glycine, Threonine, alanine, collagen, hydrolyzed collagen, hydronectin, fibronectin, keratin, elastin, royal jelly, chondroitin sulfate hepa Glycerophospholipid, glyceroglycolipid, sphingophospholipid, sphingoglycolipid, linoleic acid or its esters, eicosapentaenoic acid or its esters, pectin, bifidobacteria fermentation product, lactic acid fermentation product, yeast extract, litchi mycelium Culture or extract thereof, wheat germ oil, avocado oil, rice germ oil, jojoba oil, soybean phospholipid, γ-oryzanol, bellows oyster extract, yokuinin extract, siamese extract, typhoid extract, diatomaceous earth extract, kidachi aloe extract, burdock extract, mannenrou Extract, Arnica extract, wheat bran, pyrrolidone carboxylic acid, tomato extract, camellia oil, soybean phospholipid, hyaluronic acid, threonine, ammonium glycolate, methylsilanol alginate, yocuinine, toki extract, toki root extract , Soybean extract, Asparagus extract, DNA-Na, PCA-Na, RNA-Na, Ashitaba extract, Aspartic acid, Amatya extract, Alanine, Arginine, Alginic acid Na, Altea extract, Aloe vera extract, Oyster extract, Barley extract, Oyster leaf , Hydrolyzed keratin, hydrolyzed collagen, hydrolyzed conchiolin, hydrolyzed eggshell membrane, hydrolyzed egg white, hydrolyzed silk, hydrolyzed soy protein, brown algae extract, karin extract, raspberry extract, xylitol, chitosan, cucumber extract, cucumber fruit extract , Guava confectionary extract, quince seed extract, glycine, glycerin, glucose, grapefruit extract, grapefruit fruit extract, clematis extract, burdock extract, rice fermentation broth, chondroitin sulfate N , Fish Collagenino, Hawthorn Extract, Shiitake Extract, Giant Extract, Glycerin, Cystine, Cysteine, Horsetail Extract, Xenopus Extract, Serine, Sorbitol, Soy Protein, Tomato Extract, Lactic Acid Na, Lactobacillus, Soybean Ferment Extract, Novara Extract, Corn Oil Honey, hyaluronic acid Na, fukuno extract, betaine, loofah extract, maltitol, maltose, mannitol, lily extract, lactoferrin, lysine, apple extract, lotus extract, royal jelly, lanolin fatty acid polyethylene glycol 1000, lanolin fatty acid isopropyl, isopropyl myristate, myristin Isobutyl acid, hexyl decanol, myristyl lactate, lanolin fatty acid, tricapryl glyceryl, oleyl alcohol, oleic acid Chilled decyl, decyl oleate, reduced lanolin, octyl decanol, almond oil, avocado oil, olive oil, oleic acid, orange rough oil, cocoa butter, carrot extract, sesame oil, sasanqua oil, safflower oil, dihydrocholesterol, squalane, stearin Acid cholesteryl, ceramide 2, evening primrose oil, sunflower oil, sunflower oil, cefnide 3, sunflower seed oil hybrid sunflower oil, phytosphingosine, grape seed oil, jojoba oil, jojoba seed oil, mineral oil, mink oil, macadamia nut oil, meadowweed Examples include oil, yuri oil, eucalyptus leaf oil, lanolin, linoleic acid, rosehip oil, petrolatum and polyglutamic acid.

  These humectants can be used alone or in combination of two or more.

(UV absorber)
As UV absorbers, use benzoic acid UV absorbers, anthranilic acid UV absorbers, salicylic acid UV absorbers, cinnamic acid UV absorbers, triazine UV absorbers, benzophenone UV absorbers, etc. Also good.

  For example, as an ultraviolet absorber, amyl benzoate, octyl paraaminobenzoate, ethylene glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicylate, butylphenyl salicylate, homomentyl salicylate, benzyl cinnamate, paramethoxycinnamate 2 -Ethoxyethyl, octyl paramethoxycinnamate, glyceryl mono-2-ethylhexanoate diparamethoxycinnamate, isopropyl paramethoxycinnamate, diisopropyl-diisopropylcinnamate ester mixture, urocanic acid, ethyl urocanate, hydroxymethoxy Benzophenone, hydroxymethoxybenzophenonesulfonic acid and its salts, dihydroxymethoxybenzophenone, dihydroxymethoxybenzophenone disulfonic acid Sodium, dihydroxybenzophenone, tetrahydroxybenzophenone, 4-tert-butyl-4′-methoxy-dibenzoylmethane, 2,4,6-trianilino-p- (carbo-2′-ethylhexyl-1′-oxy) -1, 3,5-triazine, 2- (2-hydroxy-5-methylphenyl) benzotriazole, 2-ethylhexyl paradimethylaminobenzoate, ethyl paraaminobenzoate, toptylmethoxydipenzoylmethane, oxybenzone-1, ethyl guaiazulenesulfonate Examples of zinc oxide, sinoxate, and anthranilic acid-based UV absorbers include homomenthyl-N-acetylanthranylate.

  These ultraviolet absorbers can be used alone or in combination of two or more.

(Antimicrobial agent)
The antibacterial agent may be an organic antibacterial agent and / or an inorganic antibacterial agent.

  For example, as an antibacterial agent, buckwheat extract, halocarban, chlorophenesine, lysozyme chloride, alkyldiaminoethylglycine hydrochloride, isopropylmethylphenol, thymol, hexachlorophene, berberine, thioxolone, salicylic acid and their derivatives, benzoic acid, benzoic acid Sodium, paraoxybenzoic acid ester, parachlorometacresol, benzalkonium chloride, phenoxyethanol, isopropylmethylphenol, carboxylic acid, sorbic acid, potassium sorbate, hexachlorophene, chlorhexidine chloride, trichlorocarbanilide, photosensitizer, bis (2- Pyridylthio-1-oxide) zinc, thianthol, hinokitiol, triclosan, trichlorohydroxydiphenyl ether, chlorhexidine gluconic acid , Phenoxyethanol, resorcin, azulene, salicylic acid, zinc pyrithione, sodium mononitroguayacol, fennel extract, pepper extract, cetylpyridinium chloride, etc. benzethonium chloride and undecylenic acid derivatives.

  These antibacterial agents can be used alone or in combination of two or more.

  The cosmetic composition containing the composition according to the present invention may contain a surfactant. For example, as the surfactant, anionic surfactant (carboxylate, sulfonate, sulfate ester salt, phosphate ester salt), cationic surfactant (amine salt, quaternary ammonium salt), amphoteric surfactant ( Carboxylic acid type amphoteric surfactant, sulfate ester type amphoteric surfactant, sulfonic acid type amphoteric surfactant, phosphate ester type amphoteric surfactant), nonionic surfactant (ether type nonionic surfactant, ether ester) Type nonionic surfactant, ester type nonionic surfactant, block polymer type nonionic surfactant, nitrogen-containing type nonionic surfactant), other surfactants (natural surfactants, protein hydrolysates) Derivatives, polymeric surfactants, surfactants containing titanium or silicon, fluorocarbon surfactants, and the like.

  The cosmetic composition containing the composition according to the present invention may be used by dissolving in a nonionic surfactant, a lower alcohol, a polyhydric alcohol, or a natural oil such as olive oil, squalane, or a fatty acid.

  Nonionic surfactants include, for example, sorbitan fatty acid esters (for example, sorbitan monooleate, sorbitan monoisostearate, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan sesquioleate, sorbitan trioleate). Glycerol, penta-2-ethylhexyl diglycerol sorbitan, tetra-2-ethylhexyl diglycerol sorbitan, etc.); glycerin polyglycerin fatty acids (eg mono cottonseed oil fatty acid glycerin, monoerucic acid glycerin, sesquioleate glycerin, glyceryl monostearate) , Α, α′-oleic acid pyroglutamate glycerin, monostearate glycerin malate, etc.); propylene glycol fatty acid esters (for example, monos Propylene glycol tea acrylate, etc.); hardened castor oil derivative; glycerin alkyl ether and the like.

  Examples of the POE-based hydrophilic nonionic surfactant include POE-sorbitan fatty acid esters (for example, POE-sorbitan monooleate, POE-sorbitan monostearate, POE-sorbitan monooleate, POE-sorbitan tetraoleate). POE sorbite fatty acid esters (eg, POE-sorbite monolaurate, POE-sorbite monooleate, POE-sorbite pentaoleate, POE-sorbite monostearate, etc.); POE-glycerin fatty acid esters (eg, POE-glycerol monostearate, POE-glycerin monoisostearate, POE-monooleate such as POE-glycerol triisostearate, etc.); POE-fatty acid esters (for example, POE-distearate) POE-alkyl ethers (for example, POE-lauryl ether, POE-oleyl ether, POE-stearyl ether, POE-behenyl ether, POE-2-octyldodecyl ether), POE-monodiolate, ethylene glycol distearate, etc. POE-cholestanol ether, etc.); Pluronic type (for example, Pluronic, etc.); POE • POP-alkyl ethers (for example, POE • POP-cetyl ether, POE • POP-2-decyltetradecyl ether, POE • POP−) Monobutyl ether, POE / POP-hydrogenated lanolin, POE / POP-glycerin ether, etc.); tetra-POE / tetra-POP-ethylenediamine condensates (for example, Tetronic, etc.); POE-castor oil-cured castor Oil derivatives (eg POE-castor oil, POE-hardened castor oil, POE-hardened castor oil monoisostearate, POE-hardened castor oil triisostearate, POE-hardened castor oil monopyroglutamic acid monoisostearic acid diester, POE -Hardened castor oil maleic acid, etc.); POE-beeswax lanolin derivatives (eg POE-sorbite beeswax etc.); alkanolamides (eg coconut oil fatty acid diethanolamide, lauric acid monoethanolamide, fatty acid isopropanolamide etc.); POE- Examples thereof include propylene glycol fatty acid ester; POE-alkylamine; POE-fatty acid amide; sucrose fatty acid ester; alkylethoxydimethylamine oxide; trioleyl phosphate.

  Examples of the lower alcohol include ethanol, propanol, isopropanol, isobutyl alcohol, t-butyl alcohol and the like.

  Examples of the polyhydric alcohol include divalent alcohols (for example, ethylene glycol, propylene glycol, trimethylene glycol, 1,2-butylene glycol, 1,3-butylene glycol, tetramethylene glycol, 2,3-butylene glycol, Pentamethylene glycol, 2-butene-1,4-diol, hexylene glycol, octylene glycol, etc.); trivalent alcohol (eg, glycerin, trimethylolpropane, etc.); tetravalent alcohol (eg, 1, 2, 6) Pentaerythritol such as hexanetriol, etc .; pentavalent alcohol (eg, xylitol, etc.); hexavalent alcohol (eg, sorbitol, mannitol, etc.); polyhydric alcohol polymer (eg, diethylene glycol, dipropylene glycol, Tylene glycol, polypropylene glycol, tetraethylene glycol, diglycerin, polyethylene glycol, triglycerin, tetraglycerin, polyglycerin, etc.); divalent alcohol alkyl ethers (for example, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol) Monobutyl ether, ethylene glycol monophenyl ether, ethylene glycol monohexyl ether, ethylene glycol mono 2-methylhexyl ether, ethylene glycol isoamyl ether, ethylene glycol benzyl ether, ethylene glycol isopropyl ether, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, ethylene glycol Dibutyl Dihydric alcohol alkyl ethers (for example, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, diethylene glycol butyl ether, diethylene glycol methyl ethyl ether, triethylene glycol monomethyl ether, triethylene glycol) Monoethyl ether, propylene glycol monomethyl ether, propylene glycol monoethyl ether, propylene glycol monobutyl ether, propylene glycol isopropyl ether, dipropylene glycol methyl ether, dipropylene glycol ethyl ether, dipropylene glycol Dibutyl alcohol ether ester (for example, ethylene glycol monomethyl ether acetate, ethylene glycol monoethyl ether acetate, ethylene glycol monobutyl ether acetate, ethylene glycol monophenyl ether acetate, ethylene glycol diazebate, ethylene glycol disuccine) , Diethylene glycol monoethyl ether acetate, diethylene glycol monobutyl ether acetate, propylene glycol monomethyl ether acetate, propylene glycol monoethyl ether acetate, propylene glycol monopropyl ether acetate, propylene glycol monophenyl ether acetate, etc.); glycerin monoalkyl ether (example) Sugar alcohol (eg, sorbitol, maltitol, maltotriose, mannitol, sucrose, erythritol, glucose, fructose, starch-degrading sugar, maltose, xylitol, starch degradation) Sugar-reduced alcohol, etc.); Glysolid; Tetrahydrofurfuryl alcohol; POE-Tetrahydrofurfuryl alcohol; POP-Butyl ether; POP / POE-Butyl ether; Tripolyoxypropylene glycerin ether; POP-glycerin ether; POP-glycerin ether phosphate POP · POE-pentane erythritol ether, polyglycerin and the like.

  Oils include avocado oil, olive oil, sesame oil, camellia oil, evening primrose oil, turtle oil, macadamian nut oil, corn oil, mink oil, rapeseed oil, egg yolk oil, persic oil, wheat germ oil, sasanca oil, castor oil , Linseed oil, safflower oil, cottonseed oil, eno oil, soybean oil, peanut oil, tea seed oil, kaya oil, rice bran oil, kiri oil, jojoba oil, cacao butter, palm oil, horse oil, palm oil, palm kernel oil Animal and vegetable oils such as beef tallow, sheep fat, pork tallow, lanolin, whale wax, beeswax, carnauba wax, molasses, candelilla wax, squalane and their hardened oils. There are mineral oils such as liquid paraffin and petrolatum, and synthetic triglycerins such as glycerin tripalmitate.

  Examples of higher fatty acids include lauric acid, myristic acid, palmitic acid, oleic acid, linoleic acid, linolenic acid, stearic acid, behenic acid, 12-hydroxystearic acid, isostearic acid, undecylic acid, tallic acid, eicosapentaenoic acid, docosahexaene. There are acids. Examples of higher alcohols include lauryl alcohol, cetyl alcohol, stearyl alcohol, behenyl alcohol, myristyl alcohol, oleyl alcohol, cetostearyl alcohol, jojoba alcohol, lanolin alcohol, batyl alcohol, 2-decyltetrathececinol, cholesterol, phytosterol, and isostearyl. There is alcohol. Synthetic esters include, for example, cetyl octanoate, octyldodecyl myristate, isopropyl myristate, myristyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, decyl orenate, dimethyl octanoate, cetyl lactate, myristyl lactate, etc. There is. Examples of silicone include linear polysiloxanes such as dimethylpolysiloxane and methylphenylpolysiloxane, cyclic polysiloxanes such as decamethylcyclopolysiloxane, and three-dimensional network structures such as silicone resins.

  The present invention is not limited to the configurations exemplified above, and various modifications are possible within the scope shown in the claims, and the technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention.

  The present invention will be described more specifically based on Examples and Comparative Examples and FIGS. 1 to 9, but the present invention is not limited thereto. Those skilled in the art can make various changes, modifications, and alterations without departing from the scope of the present invention.

[Preparation Example 1: Isolation of tobacco osmotin gene from tobacco BY-2 cultured cells]
The tobacco osmotin gene was isolated from tobacco BY-2 cultured cells by the flow shown in FIG. Hereinafter, the method for isolating the tobacco osmotin gene will be described in detail.

(1) Preparation of template DNA sample Total RNA was extracted from tobacco BY-2 cultured cells using RNeasy Plant RNA Kit (QIAGEN). 3 μg of the extracted total RNA was subjected to DNase removal treatment using RQ1 RNase-Free DNase (Promega), reverse transcription reaction was performed using TaqMan Reverse Transcription Reagent (Applied Biosystems), and cDNA (hereinafter also referred to as “Sample 1”). ) Was synthesized.

(2) Synthesis of Oligonucleotide Complementary to Tobacco Osmotin Gene Using a DNA synthesizer type 392 manufactured by PerkinElmer, the oligonucleotide consisting of the nucleotide sequences shown in SEQ ID NOs: 2 and 3 (hereinafter, respectively) , Also referred to as “oligo 1” and “oligo 2”). The synthesis was carried out according to the manual, and the deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, it was commissioned to a DNA synthesis consignment company (Japan Bioservice Co., Ltd., Operon Co., Ltd., Sigma Genosis Co., Ltd., etc.). Oligo 1 is a forward primer consisting of a sequence homologous to the 1st to 20th bases of the base sequence registered in GenBank as accession number X61679 (base sequence shown in SEQ ID NO: 1). Reference numeral 2 denotes a reverse primer comprising a sequence complementary to the bases at positions 718 to 738 of the base sequence registered in GenBank as accession number X61679 (base sequence shown in SEQ ID NO: 1).

(3) Amplification of tobacco osmotin gene by PCR method 2 μl of the solution containing the above sample 1, 25 pmoles of oligo 1 and oligo 2 and 10 × PCRmix, 0.2 mM dNTPs, 1 mM MgSO ₄ , KOD-plus DNA polymerase with milliQ To make the total volume 50 μl. The PCR reaction was first carried out at 94 ° C. for 1 minute, followed by a cycle of 94 ° C. for 15 seconds denaturation step, 55 ° C. for 30 seconds annealing and 68 ° C. for 1 minute DNA extension reaction step, This was carried out by performing a total of 30 cycles.

(4) Cloning of tobacco osmotin gene The PCR reaction solution containing the tobacco osmotin gene amplified above is isolated by 2% agarose gel electrophoresis, purified by QIAquick Gel Extraction Kit (QIAGEN), and contains only the tobacco osmotin gene. A solution containing DNA (PCR product; hereinafter also referred to as “sample 2”) was prepared. Sample 2 was inserted into a plasmid vector using TArget Clone-plus (Toyobo). E. coli DH5α strain was transformed to prepare a solution containing plasmid DNA (hereinafter also referred to as “sample 3”) into which the full-length tobacco osmotin gene was inserted.

[Preparation Example 2: Production of recombinant expression vector containing tobacco osmotin gene]
Using the tobacco osmotin gene isolated in Preparation Example 1, a recombinant expression vector was constructed according to the flow shown in FIG. Hereinafter, a method for constructing the recombinant vector will be described in detail.

(1) Synthesis of Oligo for Adding Restriction Enzyme Site to Tobacco Osmotin Gene Oligonucleotide comprising the nucleotide sequences shown in SEQ ID NOs: 3 and 4 by the phosphoramidite method using Perkin Elmer DNA synthesizer type 392 (Hereinafter also referred to as “oligo 3” and “oligo 4”, respectively) were synthesized. The synthesis was carried out according to the manual, and the deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, it was commissioned to a DNA synthesis consignment company (Japan Bioservice Co., Ltd., Operon Co., Ltd., Sigma Genosis Co., Ltd., etc.). Oligo 3 is an EcoRI site on the 5 ′ end side of the sequence homologous to the 4th to 19th bases of the base sequence registered in GenBank as accession number X61679 (base sequence shown in SEQ ID NO: 1). Is a forward primer consisting of a base sequence to which oligo 4 is added at positions 662 to 675 of the base sequence registered in GenBank as accession number X61679 (base sequence shown in SEQ ID NO: 1). This reverse primer consists of a base sequence in which a SacI site is added to the 5 ′ end side of a complementary sequence.

(2) Addition of restriction enzyme site to tobacco osmotin gene 2 μl of the solution containing the above sample 3, 25 pmoles of oligo 3 and oligo 4, and 10 × PCRmix, 0.2 mM dNTPs, 1 mM MgSO ₄ , KOD-plus DNA polymerase MilliQ was added to make a total volume of 50 μl.

  The PCR reaction was first carried out at 94 ° C. for 1 minute, followed by a cycle of 94 ° C. for 15 seconds denaturation step, 55 ° C. for 30 seconds annealing and 68 ° C. for 1 minute DNA extension reaction step, This was carried out by performing a total of 30 cycles. The PCR reaction solution amplified above is isolated by 2% agarose gel electrophoresis, purified by QIAquick Gel Extraction Kit (QIAGEN), and contains only the tobacco osmotin gene to which a restriction enzyme site is added (EcoRI / SacI site) A solution containing the tobacco osmotin gene to which “A” was added; hereinafter also referred to as “sample 4”) was prepared.

(3) Construction of tobacco osmotin gene protein expression vector The sample 4 was treated with restriction enzymes EcoRI (Takara Shuzo) and SacI (Takara Shuzo), and the DNA solution containing the sample 4 subjected to the restriction enzyme treatment was subjected to 1% agarose gel electrophoresis. A solution containing DNA (osmotin-EcoRI / SacI; hereinafter also referred to as “sample 5”) containing tobacco osmotin gene cleaved with a restriction enzyme, isolated by QIAquick Gel Extraction Kit (QIAGEN) Prepared. The reagent composition and reaction conditions for the restriction enzyme treatment were in accordance with the recommended reagent conditions. Similarly, the pET28a vector was treated with restriction enzymes EcoRI and SacI, and the DNA containing the pET28a vector cleaved by the restriction enzyme from the DNA solution containing the pET28a vector (pET28-EcoRI / SacI; hereinafter referred to as “sample 6”) Solution) was prepared.

  Sample 5 and sample 6 were mixed at a molar ratio of 5: 1, DNA Ligation Kit Ver.1 (Takara Shuzo) was added, and a ligation reaction was performed at 16 ° C. for 18 hours. 1 μl of the obtained reaction solution was mixed with 50 μl of E. coli DH5α strain (Takara Shuzo), and transformed according to a conventional method. Plasmid DNA was extracted from the colonies that appeared, and a solution containing plasmid DNA containing the tobacco osmotin gene incorporated into pET28a (osmotin-pET28; hereinafter also referred to as “sample 7”) was prepared.

[Example 1: Mass expression of recombinant protein of tobacco osmotin using Escherichia coli]
(1) Preparation of transformant Escherichia coli and induction of expression of tobacco osmotin recombinant protein 1 μl of the solution containing Sample 7 prepared in Preparation Example 2 and 20 μl of Escherichia coli BL21 (DE3) strain were mixed and transformed according to a conventional method. . The fungus was picked from the appearing colonies, inoculated into 5 ml of LB-Kam (kanamycin) liquid medium, and cultured at 37 ° C. for 16 hours. On the next day, 5 ml of the culture solution was added to 500 ml of TB-Kam liquid medium, cultured at 37 ° C. for 3 hours, IPTG 1 mM was added, and further cultured at 25 ° C. for 6 hours.

(2) Preparation and solubilization of inclusion bodies The cells were collected from the TB-Kam liquid medium cultured as described above, and the resulting E. coli pellet was suspended in 20 ml of BugBuster MasterMix (Novagen). After incubating at room temperature for 20 minutes, centrifugation was performed at 5800 × g for 20 minutes, and the supernatant was removed to prepare inclusion bodies. The prepared inclusion bodies were suspended in solubilization buffer 1 (2% N-lauroyl sarcosine, 50 mM Tris-HCl, 0.3 M NaCl: pH 8.0), solubilized by incubation at 4 ° C. for 18 hours, and tobacco osmotin A lysate containing the recombinant protein was prepared.

(3) Refolding The lysate prepared above was regenerated with buffer (0.5 mM oxidized glutathione, 5 mM reduced glutathione, 50 mM Tris-HCl, 0.3 M NaCl: pH 8.0) at 25 ° C. or 4 ° C. for 6 hours. Dialyzed. After completion of dialysis, the lysate was recovered, desalted with PD-10 Column (GE Healthcare), and replaced with PBS (pH 7.4). Desalted and PBS-substituted lysate was treated with reduced Remuri and subjected to SDS-PAGE using a 2-15% gradient gel. When the gel was stained with Coomassie after SDS-PAGE, a single band was detected at a position of about 26 kD as shown in FIG.

[Example 2: Measurement of AMPK phosphorylation activity of tobacco osmotin recombinant protein]
(1) Induction of differentiation of C2C12 cells C2C12 cells, which are mouse skeletal muscle cells, are seeded in a 12-well plate, cultured to 100% confluent in DMEM medium containing 10% fetal bovine serum, and then 2.5% horses. The culture medium was replaced with DMEM medium containing serum and cultured for 7 days to induce differentiation.

(2) Addition of tobacco osmotin recombinant protein The C2C12 cells induced to differentiate as described above were replaced with serum-free medium, cultured at 37 ° C. for 7 hours, and then AICAR so that the concentration was 0.5 mM / well in the medium. (1-bD-ribofuranoside: TRC) was added and further cultured at 37 ° C. for 1 hour. One hour after the addition of AICAR, tobacco osmotin recombinant protein refolded at 25 ° C. or 4 ° C. was added to the medium in the same manner as AICAR, and cultured at 37 ° C. for 7 minutes, and then C2C12 cells were recovered. The collected cells were frozen with liquid nitrogen and stored at −80 ° C. until measurement.

(3) Western blotting To the C2C12 cells collected above, 100 μl of cell lysis buffer (50 mM HEPES, 2 mM sodium orthovanadate, 10 mM pyrophosphate, 10 mM sodium fluoride, 2 mM EDTA, 2 mM EGTA, 1 mM PMSF) was added to each well. The cells were collected, sonicated, and centrifuged at 10,000 × g for 15 minutes, and the supernatant was collected. The collected supernatant was subjected to reduction-remlem treatment, and 10 μg per sample was used for SDS-PAGE on 7.5% acrylamide gel. After completion of SDS-PAGE, the sample was transferred to a PVDF membrane according to a conventional method.

  The PVDF membrane to which the protein was transferred was subjected to a blocking reaction for 1 hour at room temperature using a 5% skim milk solution. After blocking, a primary antibody reaction is performed for 1 hour at room temperature using an anti-αAMPK polyclonal antibody (1/1000 Total AMPK: Cell Signaling) and a phosphorylated AMPK monoclonal antibody (1/1000 p-AMPK: Cell Signaling). It was. After the completion of the primary antibody reaction, a secondary antibody reaction was performed at room temperature for 45 minutes using HRP-Goat Anti-Rabbit IgG (ZYMED). After completion of the secondary antibody reaction, detection was performed with ECL Detection Reagent (GE Healthcare). The result is shown in FIG.

(4) Activity evaluation The band detected by said Western blotting (refer FIG. 3) was image-analyzed in Scion Images, and the ratio of p-AMPK with respect to Total AMPK was computed. As a result, as shown in FIG. 4, it can be confirmed that the ratio of phosphorylated AMPK is increased depending on the concentration of the recombinant tobacco osmotin protein compared to the blank added with only serum-free medium. It was. Moreover, as shown in FIG. 4, it was confirmed that the tobacco osmotin recombinant protein refolded at 25 ° C. rather than 4 ° C. showed stronger AMPK phosphorylation activity.

[Example 3: Examination of expression induction condition of tobacco osmotin recombinant protein]
The tobacco osmotin gene isolated in Preparation Example 1 was introduced into a pET32 expression vector so that expression was possible, and pET32 containing the tobacco osmotin gene was transformed into E. coli BL21 (DE3) strain by a conventional method. When the thus obtained transformant was cultured at 37 ° C. after addition of 1 mM IPTG, almost no expression of recombinant tobacco osmotin protein was observed. On the other hand, when the culture temperature after addition of IPTG was lowered to 30 ° C. and 25 ° C., an increase in the expression level of tobacco osmotin recombinant protein was observed as the culture temperature decreased.

  In addition, when the culture temperature was changed under the same conditions of the IPTG concentration (in other words, the intensity of expression induction) and the culture time, a tendency was observed that the cell growth was better at a lower culture temperature. From this, it was speculated that by culturing at a low temperature after addition of IPTG, the antibacterial activity of tobacco osmotin is partially suppressed, allowing Escherichia coli to grow and the expression level to increase.

[Comparative Example 1]
Sample 7 prepared in Preparation Example 2 and E. coli BL21 (DE3) pLysS strain were mixed and transformed by a conventional method. Using the transformant thus obtained, expression of tobacco osmotin recombinant protein was induced in the same manner as in Example 1, but tobacco osmotin recombinant protein was not expressed at all.

[Comparative Example 2]
Instead of the solubilization buffer 1, the inclusion bodies of Example 1 were solubilized buffer 2 (2M urea, 50 mM Tris-HCl, 0.3 M NaCl: pH 8.0) or solubilization buffer 3 (4 M urea, 50 mM Tris- When suspended in HCl, 0.3 M NaCl: pH 8.0) and incubated at 37 ° C. for 1 hour, the inclusion bodies could not be sufficiently solubilized.

[Example 4: Mass expression of recombinant protein of tobacco osmotin using Escherichia coli]
(1) Preparation of transformant Escherichia coli and induction of expression of tobacco osmotin recombinant protein 1 μl of the solution containing Sample 7 prepared in Preparation Example 2 and 20 μl of Escherichia coli BL21 Star (DE3) strain were mixed and transformed according to a conventional method. did. The fungus was picked from the appearing colonies, inoculated into 5 ml of LB-Kam (kanamycin) liquid medium, and cultured at 37 ° C. for 16 hours. On the next day, 5 ml of the culture solution was added to 500 ml of TB-Kam liquid medium, cultured for 4 hours at 37 ° C., 0.1 mM IPTG was added, and further cultured at 25 ° C. for 4 hours.

(2) Recovery of tobacco osmotin recombinant protein from soluble fraction The cells were collected from the TB-Kam liquid medium cultured as described above, and the resulting E. coli pellet was suspended in 20 ml of BugBuster MasterMix (Novagen). After incubating at room temperature for 20 minutes, lysate (soluble fraction) was prepared by centrifuging at 5800 × g for 20 minutes and collecting the supernatant. The prepared lysate was mixed with Ni-NTA agarose beads equilibrated with a purification buffer (50 mM Tris-HCl, 0.3 M NaCl: pH 8.0) and then incubated at 4 ° C. for 1 hour to be contained in the lysate. Tobacco osmotin recombinant protein adsorbed on beads. After completion of the incubation, centrifugation was performed at 1500 × g for 5 minutes, and the supernatant was removed to collect the beads. The beads were washed with a washing buffer (20 mM imidazole, 20% glycerol, 50 mM Tris-HCl, 0.3 M NaCl: pH 8.0), and elution buffer (250 mM imidazole, 50 mM Tris-HCl, 0.3 M NaCl: pH 8.0). And purified tobacco osmotin recombinant protein was recovered.

(3) Removal of E. coli-derived chaperonin The tobacco osmotin recombinant protein recovered above was desalted and replaced with 50 mM Tris-HCl (pH 8.0) using PD-10 Column (GE Healthcare). . Desalted and buffer-substituted tobacco osmotin recombinant protein is made to a final concentration of 50 μg / ml using reaction buffer (50 mM Tris-HCl, 10 mM ATP, 10 mM MgSO ₄ , 5 mg / ml casein: pH 8.0). And then incubated at 37 ° C. for 30 minutes to remove E. coli-derived chaperonin bound to the tobacco osmotin recombinant protein.

  To the tobacco osmotin recombinant protein solution from which chaperonin was removed, NaCl was added to a final concentration of 300 mM, and then centrifuged at 5800 × g for 20 minutes to collect the supernatant.

  The collected supernatant was mixed with Ni-NTA agarose beads equilibrated with a purification buffer (50 mM Tris-HCl, 0.3 M NaCl: pH 8.0), and then incubated at 4 ° C. for 1 hour. The tobacco osmotin recombinant protein contained was adsorbed to the beads. After completion of the incubation, centrifugation was performed at 1500 × g for 5 minutes, and the supernatant was removed to collect the beads. The collected beads were washed buffer (20 mM imidazole, 20% glycerol, 50 mM Tris-HCl, 0.3 M NaCl: pH 8.0), and LPS removal buffer (1.25% Triton X-114, 50 mM Tris-HCl, 0. After washing with 3M NaCl: pH 8.0, elution was carried out with an elution buffer (250 mM imidazole, 50 mM Tris-HCl, 0.3 M NaCl: pH 8.0) to recover tobacco osmotin recombinant protein.

  The recovered tobacco osmotin recombinant protein was dialyzed against PBS (pH 7.4) at 4 ° C. for 16 hours, and desalted and replaced with PBS. Tobacco osmotin recombinant protein substituted with PBS was treated with reduced Remuri and subjected to SDS-PAGE using a 2-15% gradient gel. When the gel was stained with Coomassie after SDS-PAGE, a band was detected at a position of about 26 kD as shown in FIG.

[Example 5: Measurement of AMPK phosphorylation activity of tobacco osmotin recombinant protein]
(1) Induction of C2C12 cell differentiation C2C12 cells, which are mouse skeletal muscle cells, are seeded in a 12-well plate, cultured in DMEM medium containing 10% fetal bovine serum until 100% confluent, and then the medium is added to 2.5. The medium was replaced with DMEM medium containing% horse serum and cultured for 7 days to induce differentiation.

(2) Addition of Tobacco Osmotin Recombinant Protein For the above-mentioned differentiation-induced C2C12 cells, the medium was changed to a serum-free medium and cultured at 37 ° C. for 7 hours, and then 2 mM / well in the medium. AICAR (1-bD-ribofuranoside: TRC) was added, and further cultured at 37 ° C. for 30 minutes. One hour after the addition of AICAR, tobacco osmotin recombinant protein purified from the soluble fraction and freed of chaperonin was added to the medium in the same manner as AICAR, and cultured at 37 ° C. for 7 minutes, and then C2C12 cells were recovered. The collected cells were frozen in liquid nitrogen and stored at −80 ° C. until the measurement described later.

(3) Western blotting To the C2C12 cells recovered above, 100 μl of cell lysis buffer (50 mM HEPES, 2 mM sodium orthovanadate, 10 mM pyrophosphate, 10 mM sodium fluoride, 2 mM EDTA, 2 mM EGTA, 1 mM PMSF) was added to each well. The cells were collected, and after sonication, centrifuged at 10,000 × g for 15 minutes, and the supernatant was collected. The collected supernatant was subjected to reduction Lemmri treatment, and SDS-PAGE was performed on 7.5% acrylamide gel using 10 μg per sample. After completion of SDS-PAGE, it was transferred to a PVDF membrane according to a conventional method.

  The PVDF membrane to which the protein was transferred was subjected to a blocking reaction for 1 hour at room temperature using a 5% skim milk solution. After blocking, a primary antibody reaction is performed for 1 hour at room temperature using an anti-αAMPK polyclonal antibody (1/1000 Total AMPK: Cell Signaling) and a phosphorylated AMPK monoclonal antibody (1/1000 p-AMPK: Cell Signaling). It was. After the completion of the primary antibody reaction, a secondary antibody reaction was performed at room temperature for 45 minutes using HRP-Goat Anti-Rabbit IgG (ZYMED). After completion of the secondary antibody reaction, detection was performed with ECL Detection Reagent (GE Healthcare). The result is shown in FIG.

(4) Activity evaluation The band (refer FIG. 6) detected by said western blotting was image-analyzed in Scion Images, and the ratio of p-AMPK with respect to Total AMPK was computed. As a result, as shown in FIG. 7, it was confirmed that the ratio of phosphorylated AMPK was increased by the addition of the tobacco osmotin recombinant protein, as compared with the blank to which only the serum-free medium was added.

[Example 6: Lipid metabolism abnormality improving effect of tobacco osmotin recombinant protein]
(1) Animal Male + Lepr ^db / + Lepr ^db mouse (db mouse) (aged 7-10) was purchased from Japan Charles River.

(2) Measurement of lipid metabolism abnormality improvement effect by intraperitoneal administration Tobacco osmotin recombinant protein (Osm) suspended in PBS is 0.05 mg / kg per body weight for a group of db mice (Osm administration group). Was administered intraperitoneally. As a control, an equal amount of PBS was intraperitoneally administered to another group of db mice (PBS administration group). Blood was collected before administration (0 h) and 9 hours after administration (9 h), and the concentration of free fatty acid (FFA) in the obtained plasma was measured. The result is shown in FIG. In the Osm administration group, the FFA concentration was significantly reduced as compared with the PBS administration group, and the effect of improving lipid metabolism abnormality was recognized in the tobacco osmotin recombinant protein.

[Example 7: Glucose metabolism abnormality improving effect of tobacco osmotin recombinant protein]
(1) Animal Male + Lepr ^db / + Lepr ^db mouse (db mouse) (age 7-10) was purchased from Japan Charles River.

(2) Measurement of glucose metabolism abnormality improving effect by oral glucose tolerance test An osmotic pump (Alzet) was surgically inserted into the back of a db mouse. The osmotic pump was set so that tobacco osmotin recombinant protein (Osm) suspended in PBS was continuously administered to a group of db mice (Osm administration group) at 0.05 mg / kg per body weight per day. did. As a control, it was set so that an equal amount of PBS was continuously administered to another group of db mice (PBS administration group). On the 10th day from the start of administration, an oral glucose tolerance test was performed, and the glucose metabolism capacity of the Osm administration group and the PBS administration group was measured.

(3) Evaluation of glucose metabolism abnormality improvement effect In an oral glucose tolerance test, blood glucose levels at 0 minutes, 15 minutes, 30 minutes, 60 minutes, and 120 minutes after glucose loading were measured to evaluate the effect of improving sugar metabolism abnormality. The result is shown in FIG. In the graph showing fluctuations in blood glucose level, the blood glucose level is significantly lower in the Osm administration group than in the PBS administration group at 15 minutes, 30 minutes, and 60 minutes, and the tobacco osmotin recombinant protein has an effect of improving glucose metabolism abnormality. It was.

[Example 8: Effect of reducing tobacco osmotin recombinant protein on oxidative stress]
(1) Animal Male + Lepr ^db / + Lepr ^db mouse (db mouse) (age 7-10) was purchased from Japan Charles River.

(2) Continuous administration of tobacco osmotin recombinant protein and collection of adipose tissue Four mice were divided into two groups so that the blood glucose level and body weight of db mice were equal. An osmotic pump (Alzet) was surgically inserted under the back of a db mouse. The osmotic pump was set so that tobacco osmotin recombinant protein (Osm) suspended in PBS was continuously administered to a group of db mice (Osm administration group) at 0.05 mg / kg per body weight per day. did. As a control, it was set so that an equal amount of PBS was continuously administered to another group of db mice (PBS administration group). On day 12 from the start of administration, dissection was performed, and adipose tissue around the testis was collected.

(3) Preparation of total RNA and cDNA from testicular adipose tissue Total RNA was prepared from collected mouse testicular adipose tissue. Specifically, the peritesticular adipose tissue after 12 days of tobacco osmotin recombinant protein administration with an osmotic pump was removed, TRIZOL (Gibco-BRL) was added, and each RNA was crushed with a homogenizer. Was prepared. The total RNA was prepared using TRIZOL according to the attached protocol. The obtained total RNA was dissolved in DEPC-treated water (manufactured by Nacalai Tesque). 3 μg of the prepared total RNA was subjected to DNase removal treatment using RQ1 RNase-Free DNase (Promega), reverse transcription reaction was performed using TaqMan Reverse Transcription Reagent (Applied Biosystems), and cDNA (hereinafter also referred to as “Sample 8”). ) Was synthesized.

(4) Variation analysis of gene expression by real-time PCR Sample 8 is sampled in an equal amount, and the mixed cDNA is serially diluted with DEPC water 1-fold, 3-fold, 9-fold, and 27-fold to prepare a calibration curve. A standard sample was used. The remaining sample 8 was diluted 3-fold with DEPC water to obtain a measurement sample. Standard samples and measurement samples were subjected to real-time PCR using TaqMan Gene Expression Master Mix (ABI) and TaqMan Gene Expression Assays (ABI). Note that. The operation was performed according to the protocol attached to the kit.

(5) Gene analysis The results of the above-mentioned real-time PCR are shown as a result of relative gene expression fluctuations using SDS2.2 (ABI) and the gene expression fluctuations in each measurement sample represented by a standard curve prepared with standard samples. 10 shows. When the gene expression of the enzyme Super Oxide Dismutase (SOD) having the action of eliminating oxidative stress was corrected by the expression level of 36B4 gene used as an internal standard, a significant increase in SOD gene expression was observed in the Osm administration group. That is, it was found that Osm administration reduces oxidative stress due to adipose tissue.

  Note that the present invention is not limited to the configurations described above, and various modifications are possible within the scope of the claims, and technical means disclosed in different embodiments and examples respectively. Embodiments and examples obtained by appropriately combining them are also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

  In the method for producing an osmotin recombinant protein according to the present invention, an osmotin recombinant protein is produced using a non-plant transformant into which a polynucleotide encoding osmotin or a variant thereof has been introduced. Therefore, the osmotin recombinant protein can be obtained easily, inexpensively and in large quantities.

  The osmotin recombinant protein screening method according to the present invention comprises extracting an osmotin recombinant protein from a non-plant transformant into which a polynucleotide encoding osmotin or a variant thereof is introduced so that the osmotin can be expressed. The activity of phosphorylating the 5′-AMP activated protein kinase of the replacement protein is detected. Therefore, there is an effect of easily screening an osmotin recombinant protein having an activity of phosphorylating 5'-AMP-activated protein kinase.

  The specific embodiments or examples made in the detailed description section of the invention are merely to clarify the technical contents of the present invention, and are limited to such specific examples and are interpreted in a narrow sense. It should be understood that various modifications may be made within the spirit of the invention and the scope of the following claims.

  As described above, the present invention provides an Osm recombinant protein having physiological activity and a method for producing the Osm recombinant protein simply, inexpensively and in large quantities. Therefore, it can be widely used in the fields utilizing the physiological activity of osmotin, specifically in the pharmaceutical field, the medical field, the food field, the cosmetic field, and the like.

Claims (24)

  A method for producing an osmotin recombinant protein, comprising producing an osmotin recombinant protein using a non-plant transformant in which a polynucleotide encoding osmotin or a variant thereof is introduced so as to allow expression.   The method for producing an osmotin recombinant protein according to claim 1, comprising a step of detecting phosphorylation activity of 5'-AMP-activated protein kinase of the osmotin recombinant protein.   The method for producing an osmotin recombinant protein according to claim 1 or 2, wherein the host of the non-plant transformant is Escherichia coli, yeast, fungi, insect, or mammal. In the non-plant transformant, an inclusion body containing an osmotin recombinant protein is formed,
Extracting the inclusion body and dissolving the inclusion body in a solubilizing solution solubilizes the osmotin recombinant protein contained in the inclusion body,
The method for producing an osmotin recombinant protein according to any one of claims 1 to 3, wherein the solubilized osmotin recombinant protein is refolded. The osmotin recombination according to any one of claims 1 to 4, wherein the polynucleotide is any one polynucleotide selected from the group consisting of the following (a) to (l): A method for producing a protein.
(A) a polynucleotide comprising the base sequence shown in SEQ ID NO: 1 (b) a base in which one or several bases are substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 1 A polynucleotide comprising the sequence and encoding a polypeptide having the activity of phosphorylating 5'-AMP-activated protein kinase (c) a polynucleotide and a string comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 1 A polynucleotide that encodes a polypeptide that hybridizes under mild conditions and phosphorylates 5′-AMP-activated protein kinase (d) from position 1 to position 678 of the base sequence represented by SEQ ID NO: 1 A polynucleotide comprising the base sequence consisting of the positions (e) from position 1 to position 678 of the base sequence shown in SEQ ID NO: 1 A polypeptide having a base sequence in which one or several bases are substituted, deleted, inserted and / or added, and having an activity of phosphorylating 5′-AMP-activated protein kinase The polynucleotide to be encoded (f) hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence consisting of positions 1 to 678 of the base sequence represented by SEQ ID NO: 1, and 5 A polynucleotide encoding a polypeptide having an activity to phosphorylate AMP-activated protein kinase (g) a polynucleotide comprising a nucleotide sequence consisting of positions 4 to 738 of the nucleotide sequence represented by SEQ ID NO: 1 (h ) In the base sequence consisting of positions 4 to 738 of the base sequence shown in SEQ ID NO: 1, one or several bases are A polynucleotide (i) encoding a polypeptide consisting of a base sequence that has been substituted, deleted, inserted, and / or added, and having the activity of phosphorylating 5′-AMP-activated protein kinase, as shown in SEQ ID NO: 1. It has the activity of hybridizing under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence consisting of positions 4 to 738 of the nucleotide sequence and phosphorylating 5'-AMP activated protein kinase. A polynucleotide encoding the polypeptide (j) a polynucleotide comprising the base sequence consisting of positions 4 to 678 of the base sequence shown in SEQ ID NO: 1 (k) the fourth position of the base sequence shown in SEQ ID NO: 1 ~ In the base sequence consisting of position 678, one or several bases substituted, deleted, inserted and / or added. And a polynucleotide encoding a polypeptide having an activity to phosphorylate 5′-AMP-activated protein kinase (l) a nucleotide sequence comprising positions 4 to 678 of the nucleotide sequence represented by SEQ ID NO: 1 A polynucleotide encoding a polypeptide that hybridizes with a polynucleotide comprising a complementary base sequence under stringent conditions and has an activity of phosphorylating 5'-AMP-activated protein kinase   5. The polynucleotide according to claim 1, wherein the polynucleotide is a polynucleotide derived from any one plant selected from the group consisting of potato, tomato, pepper, pepper, and tobacco. A method for producing the described osmotin recombinant protein.   The osmotin recombinant protein according to claim 4, wherein the solubilized osmotin recombinant protein is refolded at 2 ° C to 40 ° C in a solution containing reduced glutathione and oxidized glutathione. Manufacturing method.   The method for producing an osmotin recombinant protein according to claim 4 or 7, wherein the solubilized solution contains N-lauroyl sarcosine. The host of the non-plant transformant is E. coli,
The method for producing an osmotin recombinant protein according to any one of claims 1 to 8, wherein the osmotin recombinant protein is produced by inducing expression of the polynucleotide at 20 ° C to 30 ° C. .   The osmotin recombinant protein manufactured with the manufacturing method of the osmotin recombinant protein of any one of Claims 1-9.   The osmotin recombinant protein according to claim 10, which has an activity of phosphorylating 5'-AMP-activated protein kinase.   A composition comprising the osmotin recombinant protein according to claim 10 or 11.   The composition according to claim 12, which is used for prevention or treatment of lifestyle-related diseases.   14. The lifestyle-related disease is at least one disease selected from the group consisting of type II diabetes, insulin resistance, hyperlipidemia, hypertension, arteriosclerosis, and heart disease. The composition as described.   The composition according to claim 12, which is used for prevention or treatment of a disease caused by oxidative stress.   The disease caused by the oxidative stress is at least one disease selected from the group consisting of cancer, inflammation, allergic disease, Alzheimer's disease, age-related macular degeneration and skin aging. The composition as described. A screening method for an osmotin recombinant protein having an activity of phosphorylating 5′-AMP-activated protein kinase,
Extracting an osmotin recombinant protein from a non-plant transformant in which a polynucleotide encoding osmotin or a variant thereof is introduced so as to be expressed,
A method for screening an osmotin recombinant protein, comprising detecting the activity of phosphorylating 5′-AMP-activated protein kinase of the osmotin recombinant protein.   The method for screening an osmotin recombinant protein according to claim 17, wherein the host of the non-plant transformant is Escherichia coli, yeast, fungi, insect, or mammal. The osmotin recombinant protein is
In the non-plant transformant, an inclusion body containing an osmotin recombinant protein is formed,
Extracting the inclusion body and dissolving the inclusion body in a solubilizing solution solubilizes the osmotin recombinant protein contained in the inclusion body,
The method for screening an osmotin recombinant protein according to claim 17 or 18, wherein the solubilized osmotin recombinant protein is obtained by refolding. The osmotin recombination according to any one of claims 17 to 19, wherein the polynucleotide is any one polynucleotide selected from the group consisting of the following (a) to (l): Protein screening method.
(A) a polynucleotide comprising the base sequence shown in SEQ ID NO: 1 (b) a base in which one or several bases are substituted, deleted, inserted and / or added in the base sequence shown in SEQ ID NO: 1 A polynucleotide comprising the sequence and encoding a polypeptide having the activity of phosphorylating 5'-AMP-activated protein kinase (c) a polynucleotide and a string comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 1 A polynucleotide that encodes a polypeptide that hybridizes under mild conditions and phosphorylates 5′-AMP-activated protein kinase (d) from position 1 to position 678 of the base sequence represented by SEQ ID NO: 1 A polynucleotide comprising the base sequence consisting of the positions (e) from position 1 to position 678 of the base sequence shown in SEQ ID NO: 1 A polypeptide having a base sequence in which one or several bases are substituted, deleted, inserted and / or added, and having an activity of phosphorylating 5′-AMP-activated protein kinase The polynucleotide to be encoded (f) hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence consisting of positions 1 to 678 of the base sequence represented by SEQ ID NO: 1, and 5 A polynucleotide encoding a polypeptide having an activity to phosphorylate AMP-activated protein kinase (g) a polynucleotide comprising a nucleotide sequence consisting of positions 4 to 738 of the nucleotide sequence represented by SEQ ID NO: 1 (h ) In the base sequence consisting of positions 4 to 738 of the base sequence shown in SEQ ID NO: 1, one or several bases are A polynucleotide (i) encoding a polypeptide consisting of a base sequence that has been substituted, deleted, inserted, and / or added, and having the activity of phosphorylating 5′-AMP-activated protein kinase, as shown in SEQ ID NO: 1. It has the activity of hybridizing under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence consisting of positions 4 to 738 of the nucleotide sequence and phosphorylating 5'-AMP activated protein kinase. A polynucleotide encoding the polypeptide (j) a polynucleotide comprising the base sequence consisting of positions 4 to 678 of the base sequence shown in SEQ ID NO: 1 (k) the fourth position of the base sequence shown in SEQ ID NO: 1 ~ In the base sequence consisting of position 678, one or several bases substituted, deleted, inserted and / or added. And a polynucleotide encoding a polypeptide having an activity to phosphorylate 5′-AMP-activated protein kinase (l) a nucleotide sequence comprising positions 4 to 678 of the nucleotide sequence represented by SEQ ID NO: 1 A polynucleotide encoding a polypeptide that hybridizes with a polynucleotide comprising a complementary base sequence under stringent conditions and has an activity of phosphorylating 5'-AMP-activated protein kinase   The polynucleotide according to any one of claims 17 to 19, wherein the polynucleotide is derived from any one plant selected from the group consisting of potato, tomato, pepper, pepper, and tobacco. A screening method for the described osmotin recombinant protein.   The osmotin recombinant protein according to claim 19, wherein the solubilized osmotin recombinant protein is refolded at 2 to 40 ° C in a solution containing reduced glutathione and oxidized glutathione. Screening method.   The method for screening an osmotin recombinant protein according to claim 19 or 22, wherein the solubilized solution contains N-lauroyl sarcosine. The host of the non-plant transformant is E. coli,
The method for screening an osmotin recombinant protein according to any one of claims 17 to 23, wherein the osmotin recombinant protein is produced by inducing expression of the polynucleotide at 20 ° C to 30 ° C. .