KR20120052542A - Mutant of monellin protein, gene coding the same, vector comprising the gene, transformant transformed by the vector, and preparation method of the transformant - Google Patents

Abstract

PURPOSE: A monellin mutant protein is provided to ensure excellent thermal stability and to wide pH resistance and to maintain sweet taste. CONSTITUTION: A monellin mutant protein 24th amion acid(glutamic acid) substituted with alanine, leucine, phenyl alanine, or tryptopane in an amino acid sequence of single chain monellin. The mutant has an amino acid sequence selected from sequence numbers 3-6. A gene encoding the mutant is denoted by sequence number selected from sequence numbers 7-10. A recombinant vector containing the gene encoding the mutant is a recombinant vector for E.coli expression. The recombinant vector is pMOA, pMOL, pMOF or MOW.

Description

Monelin protein variants, genes encoding the variants, recombinant vectors comprising the genes, transformants transformed with the recombinant vectors, and methods for producing the transformants {Mutant of Monellin protein, Gene coding the same, Vector comprising the gene, Transformant transformed by the vector, and Preparation method of the transformant}

The present invention relates to a monelin protein variant, a gene encoding the variant, a recombinant vector comprising the gene, a transformant transformed with the recombinant vector, and a method for preparing the transformant, and more specifically, thermal stability This excellent Monelin protein variant, the gene encoding the variant, a recombinant vector comprising the gene, a transformant transformed with the recombinant vector and the method for producing the transformant.

Monelin is a very sweet protein derived from tropical plants and is known to be about 100,000 times sweeter than sugar on a molar basis. Monelin was first isolated from the West African plant, Dioscoreophyllum cumminsii [Morris and Cagan, Biochem. Biophys. Acta, 1972, 261: 114-122. Monelin consists of the A chain of 45 amino acid residues and the B chain of 50 amino acid residues [Hudson and Biemann, Biochem, Biophys. Res. Comm., 1976, 71: 212-220; Frank and Zuber, Hoppe Seyler's Z Physiol. Chem., 1976, 357: 585-592], and various other physicochemical properties are described in the prior art [Ogata, et al., Nature, 1987, 328: 739-752; Morris et al., J. Biol. Chem., 1973, 248: 534-539; Cagan, Science, 1973, 181: 32-35; Bohak and Li, Biochim. Biophys. Acta, 1976, 427: 153-170.

Regarding this high sweetening protein, US Pat. No. 4,300,576 describes chewing gum coated with monelin, and US Pat. No. 4,412,984 describes flavor enhancing oral compositions containing monelin. However, although powerful as a low-calorie sweetener, Monelin is not commercially available due to its low stability against heat and pH, inability to access plant sources and temperature control for food additives [Dansby, Nature Biotechnology, 1997, 15: 419]. As described above, the Monelin protein has a weak covalent bond between the A-chain of 45 amino acids and the B-chain of 50 amino acids, so that both chains must be introduced into one cell during expression. Production of single-chain monelin in Escherichia coli has been reported [Kim et al., Protein Eng., 1989, 2: 571-575]. Purified single chain monelin has been found to be more thermally stable, resistant to a wide range of pH, and maintain a high sweetness. However, it still has the disadvantage that the heat treatment during the food processing process loses the three-dimensional protein structure and does not give a sweet taste.

One problem to be solved by the present invention is to provide a variant of Monelin protein excellent in thermal stability.

Another problem to be solved by the present invention is to provide a gene encoding a Monelin protein variant with excellent thermal stability.

Another problem to be solved by the present invention is to provide a recombinant vector comprising a gene encoding a Monelin protein variant with excellent thermal stability.

Another problem to be solved by the present invention is to provide a transformant transformed with a recombinant vector comprising a gene encoding a Monelin protein variant with excellent thermal stability.

 Another problem to be solved by the present invention is to provide a method for producing a transformant transformed with a recombinant vector comprising a gene encoding a good heat stability Monelin protein variant.

The present invention provides a Monelin protein variant wherein glutamic acid at position 24 in the amino acid sequence of the single chain monolinin protein consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with one amino acid selected from alanine, leucine, phenylalanine or tryptophan.

The variant may consist of one amino acid sequence selected from SEQ ID NOs: 3 to 6.

The present invention also provides genes encoding the monelin protein variants of the present invention.

The gene may be DNA and / or RNA or the like, and preferably cDNA (complementary DNA).

The gene may be represented by one nucleotide sequence selected from SEQ ID NOs: 7 to 10.

The present invention also provides a recombinant vector comprising the gene of the present invention.

The recombinant vector may be for E. coli expression.

The recombinant vector may have one cleavage map selected from pMOA, pMOL, pMOF or pMOW of FIG.

The present invention also provides a transformant incorporating the recombinant vector of the present invention into a host cell.

The host cell is not limited thereto, but may preferably be Escherichia coli, and the Escherichia coli may be Escherichia coli BL21 (DE3).

The transformant may be one selected from E. coli Escherichia coli BL21 (DE3) transformant EMOA (KCTC18197P), EMOF (KCTC18198P), EMOL (KCTC18199P) or EMOW (KCTC18200P).

In addition, the present invention is a primer comprising a vector comprising a gene encoding a single chain monolinin protein, one primer selected from SEQ ID NO: 13 and 14, SEQ ID NO: 15 and 16, SEQ ID NO: 17 and 18 or SEQ ID NO: 19 and 20 Preparing a recombinant vector of the present invention by performing a clonal polymerase chain reaction (PCR) in pairs; And it provides a method for producing a transformant for producing a monelin protein variant comprising the step of introducing the recombinant vector into the host cell.

Gene encoding the single chain monolin protein may be composed of the nucleotide sequence of SEQ ID NO: 2.

The vector containing the gene encoding the single chain monolinin protein may be for expression in the host cell, the host cell may be E. coli. Preferably, it may be a vector represented by a pMON cleavage map of FIG. 2.

The vector comprising the gene encoding the single chain monolinin protein can be prepared by inserting the gene encoding the single chain monolinin protein into the expression vector for the host cell.

Monelin protein variants of the present invention, the gene encoding the variant, the recombinant vector comprising the gene, the transformants transformed with the recombinant vector and the contents mentioned in the method for producing the transformant are the same unless contradictory Is applied.

The amino acid sequence used in the present invention may be abbreviated as follows.

Glycine Gly, Alanine Ala, Valine Val, Leucine Leu, Isoleucine Ile, Proline Pro, Phenylalanine Phe, Tyrosine Tyr, Tryptophan Trp, Serine Ser, Threonine Thr, Cysteine Cys, Methionine Met, Asparagine Asn, Glutamine Gln, Aspartate Asp, Glutamate Glu, Lysine Lys, Arginine Arg, Histidine His

Monelin protein variants of the present invention is excellent in thermal stability, it is possible to prepare a monelin protein variant excellent in thermal stability by the gene, recombinant vector, transformant, the production method of the present invention.

1 is a diagram showing a primer for amplifying a wild-type single-chain Monelin gene fragment by polymerase chain reaction (PCR).
Figure 2 is a diagram showing the production sequence of the pMON expression vector inserted with the gene encoding the wild-type single-chain Monelin protein.
Figure 3 is a diagram showing the production sequence of the expression vector containing a gene encoding a Monelin protein variant.
Figure 4 is a graph showing the results of analyzing the wavelength curve change of the temperature during the circular dichroism analysis experiments for measuring the stability of the monelin protein variants.
Figure 5 is a graph showing the results of measuring the annealing ratio by temperature at a wavelength of 222nm in the analysis of circular dichroism which is a thermal stability measurement experiment for Monelin protein variants.
Figure 6 is a graph showing the results of measuring the melting temperature of the monelin protein by differential scanning calorimetry.
Figure 7 is a graph showing the results of measuring the melting temperature of one embodiment of the monelin protein variant with a differential scanning calorimetry.

Examples and experimental examples are provided to help understand the present invention. The following Examples and Experimental Examples are provided only to more easily understand the present invention, but the contents of the present invention are not limited by the Examples and Experimental Examples.

Example 1 Preparation of Monelin Protein Variant Coding Gene and Expression Vector

Monelin protein mutant coding gene and expression vector preparation prepared wild-type single-chain Monelin gene-containing expression vector (see Figure 2), using it to produce a Monelin protein variant coding gene and the expression vector comprising the gene. It was prepared by the method (see Fig. 3). Figure 2 schematically shows a method for producing an expression vector containing a wild-type single-chain Monelin gene, and Figure 3 schematically shows a method for producing a Monelin protein variant coding gene and an expression vector comprising the gene using it. . Hereinafter, referring to Figures 2 and 3, the method for producing a Monelin protein variant coding gene and expression vector will be described in detail.

1-1. Preparation of Expression Vectors Containing Wild-type Single Chain Monelin Gene

PLDUTRMon vector inserted with a gene encoding a single chain Monelin protein (SEQ ID NO: 1, cDNA, SEQ ID NO: 2) [K.H. Roh and K.S. Shin, J Plant Biol., 2006, 49 (1): 34-43], were obtained from the Rural Development Administration of Korea (National Institute of Agricultural Science, Seung-Bum Lee Agricultural Research Institute, 224 Suin-ro, Gwon-gu, Suwon-si, Gyeonggi-do). The vector was amplified by polymerase chain reaction (PCR) as a template to obtain a wild type single chain Monelin gene segment. PCR amplification was performed using the primer pairs described in FIG. 1 (forward primer MON-F (SEQ ID NO: 11) and reverse primer MON-R (SEQ ID NO: 12)) under the following conditions. 1 is a diagram showing a primer for amplifying a wild-type single-chain Monelin gene fragment by a polymerase chain reaction (PCR), the underline in Figure 1 is a restriction enzyme recognition sequence portion, respectively.

The conditions of PCR performed to amplify wild type single chain Monelin gene segments are shown in the following table.

[Table 1]

<PCR reaction liquid composition>

TABLE 2

PCR reaction conditions

PET21b (Novagen), which is to be used as a gene fragment and expression vector, was treated with restriction enzymes XhoI (TAKARA, Cat # 1080A, 15units / l) and NdeI (Enzynomics, Cat # R006S, 20units / l) for one hour at 37 degrees Celsius, respectively. This restriction enzyme-treated gene fragment and pET21b were treated with T4 ligase (Promega, Cat # M1801, 1 unit / μl) at 25 degrees Celsius for 1 hour to express an expression vector containing a wild-type single-chain monelin gene (pMON). ) Was produced (see FIG. 2).

The pET21b vector includes a T7 promoter region that regulates the expression of a single-chain Monelin gene, and also has six histidine tags in addition to the T7 promoter region, thereby allowing purification of protein variants produced after expression. Only desired protein variants can be purified quickly and in high yield.

1-2. Preparation of Expression Vectors Containing Monelin Protein Variant Coding Genes

The pMON prepared in 1-1. Was used as a template, and the 24th amino acid residue was converted to alanine, leucine, phenylalanine, and tryptophan from glutamic acid, respectively, and used by a Polygene DNA oligo synthesizer. Thus, the primers of the following table were produced. Using the primers in the table below, the QuickChange (R) Site-Directed Mutagenesis kit (STRATAGENE) according to the manufacturer's instructions for Monelin protein variant coding genes (SEQ ID NO: 7, 8, 9, 10 consisting of each base sequence cDNA) was prepared (see FIG. 3), and transformed into E. coli XL-1 blue competent cells (competent cells) included in the kit. The expression vectors were named pMOA, pMOL, pMOF, and pMOW, respectively.

The expression vector was introduced into Escherichia coli and deposited at the Korea Biotechnology Research Institute (KRIBB) Microbial Resource Center (KCTC) (111, Sciro-ro, Yuseong-gu, Daejeon, Korea) on November 4, 2010. Thus, Accession Nos. KCTC18197P, KCTC18198P, KCTC18199P, and KCTC18200P were respectively assigned to Escherichia coli introduced with pMOA, pMOF, pMOL, and pMOW.

[Table 3]

Underlined sequence portions in the table indicate mutated codon sites.

1-3. Sequencing

In order to confirm that the amino acid mutation as intended, the expression vector prepared in 1-2 above was first isolated purely using GeneAll Exprep ™ Plasmid SV (Plasmid DNA mini-prep kit) according to the manufacturer's instructions. The isolated expression vector was requested to Genotech, and subjected to sequencing using an Applied Biosystems 3730 DNA Analyzer. As a result, the Monelin protein variant coding sequences of pMOA, pMOL, pMOF, and pMOW were identified by encoding the amino acid sequences of SEQ ID NOs: 3, 4, 5, and 6 (SEQ ID NOs: 7, 8, 9, and 10). It became.

Therefore, it was confirmed that the Monelin protein variant coding gene and the expression vector containing the gene was prepared as desired.

Example 2 Transformant Preparation

The expression vectors pMOA, pMOL, pMOF, and pMOW prepared in 1-2 were cultured at 37 ° C. E. coli BL21 (DE3) {purchased from Invitrogen (USA), Catalog No. C6000-06 and ATCC (American Type Culuure Collection) Accession No. BAA-1025} were used to prepare E. coli mutant strains as transformants producing recombinant Monelin protein. By using the strain, it is possible to easily induce protein expression by IPTG (isopropyl-1-thio-β-D-galactopyranoside) and to express the protein in high yield. Genotypes of E. coli BL21 (DE3) are shown in the following table.

[Table 4]

After centrifugation of 100 ml of the E. coli BL21 (DE3) cell suspension (OD 600 = 0.5), the supernatant was removed and the pellet was resuspended in 1/2 volume of 100 mM calcium chloride (CaCl ₂ ). This was again centrifuged to remove the supernatant and resuspended by adding 1/10 volume of 100 mM calcium chloride. Thus, 1 μl of each of the vectors (pMOA, pMOL, pMOF, pMOW) containing the Monelin gene was added to 100 μl of the prepared cells (competent cells) and allowed to stand for 30 minutes on ice. Heat shock was applied at 42 ° C. for 50 seconds to transform the Monelin gene into E. coli cells. Then, the mixture was suspended for 2 minutes on ice, and then 1 ml of LB (Luria-Bertani) medium (tryptone 10 g, yeast 5 g, sodium chloride 5 g, 1 L total) was added and shaken at 37 ° C. for 1 hour. 100 μL of these cells were taken and plated in LB-Ampicillin agar medium and cultured at 37 ° C. to prepare E. coli mutant strains to which the Monelin gene was introduced. The alanine variant was EMOA, the leucine variant was EMOL, and phenylalanine. The (Phenylalanine) variant was named EMOF, and the Tryptophan variant was named EMOW. For control, the same method was performed on pMON to prepare a transformant (named EMON).

The variants were deposited on November 4, 2010, at the Korea Biotechnology Research Institute (KRIBB) Bioresource Management Division (KBRC) Microbial Resource Center (KCTC) (111, Haesong-ro, Yuseong-gu, Daejeon, Korea), respectively. (EMOF), KCTC18199P (EMOL), KCTC18200P (EMOW).

Example 3 Preparation and Confirmation of Monelin Protein Variants

Monelin protein variants were expressed and confirmed from the E. coli strain of E. coli of Example 2.

LB medium containing 10 g tryptone, 5 g calcium chloride, and 5 g yeast extract was used as a medium for culturing the E. coli mutant strain . After inoculating the LB medium with the E. coli mutant strain, the culture was shaken at 37 ° C and cultured at 1/100 volume M9 medium {10X M9 salt per 1L (61.2g Na ₂ HPO ₄ per 1L, KH ₂ PO ₄ 30g, NaCl) 5 g, NH ₄ Cl 10g) 100 ml, 1M MgSO ₄ 2ml, 3g glucose, Gibco MEM vitamin solution 100μl, 100mM CaCl ₂ } and inoculated again at 37 ℃ shaking culture for about 4 hours. When the absorbance of the culture solution was measured to be OD 600 = 0.5, 100 μM of IPTG (isopropyl-1-thio-β-D-galactopyranoside) was added and shaken again for about 20 hours, and the cultured cells were centrifuged. The supernatant was removed by separation.

In order to confirm the expression of Monelin protein from the cultured E. coli strain E. coli , E. coli mutants were lysed using a Bugbuster Protein Extraction Reagent (Novagen), a cell lysis buffer, and then lysed. Centrifugation of the prepared solution at 16,000 g for 30 minutes, loaded onto polyacrylamide gel, Coomassie-blue staining, and the degree of expression of monelin protein and production of protein using E. coli The degree of formation of inclusion bodies having no activity inside one cell was confirmed. The degree of formation of the inclusion body of the monelin protein variants was about 40% of the total expressed amount of the monelin protein, but the amount of the monelin protein dissolved in the cell lysis buffer had a large effect on the high yield purification. It was confirmed that no.

It was confirmed that the Monelin protein variant was expressed by the transformant prepared in Example 2. In addition, it was confirmed that the monelin protein that does not form an inclusion body was expressed, and it was confirmed that the purification was not difficult by using a nickel agarose resin (Ni-NTA agarose resin, Quiagen). Each Monelin protein variant was named as Alanine variant MNEI_E24A, Leucine variant as MNEI_E24L, Phenylalanine variant as MNEI_E24F and Tryptophan variant as MNEI_E24W. For control, EMON was performed in the same manner to express wild type Monelin protein (named MNEI-Wildtype).

Example 4 Preparation of Purified Monelin Protein Variants

Monelin protein variants expressed from E. coli strains cultured in Example 3 were purified.

The E. coli mutant strain E. coli cultured in Example 3 was harvested, dissolved in Bugbuster Protein Extraction Reagent (Novagen), and the monelin protein that formed the inclusion body was difficult to purify. After centrifugation at 16,000 g for 30 minutes to obtain the removed sample, only the supernatant was transferred to a clean tube, and the sample was purified by Ni-NTA agarose resin (Quiagen). Since the gene encoding the histidine protein is contained in the recombinant expression vector pET21b, the recombinant monelin protein also contains six histidine proteins at the C-terminus. Therefore, it can be easily purified by passing through a resin containing nickel or copper. The supernatant sample separated after centrifugation was passed through a nickel resin, and then washed with a buffer wash buffer (pH 8.0, 50 mM NaH ₂ PO ₄ , 300 mM NaCl, 20 mM imidazole) to 8 times the volume of the nickel resin, followed by elution. Monelin protein samples used for circular dichroism (CD) analysis during thermal stability analysis were used with elution buffer (pH 8.0, 50 mM NaH ₂ PO ₄ , 250 mM imidazole), and differential scanning calorimetry (differential scanning) Samples for calorimeter (DSC) analysis were eluted with elution buffer (pH 8.0, 50 mM NaH ₂ PO ₄ , 300 mM NaCl, 250 mM imidazole) and loaded onto polyacrylamide gels for Coomassie-blue staining. The degree of purification of the monelin protein was confirmed. For control, wild type Monelin protein (MNEI-Wildtype) was also purified identically.

As a result, the protein band of about 14 kDa was confirmed, and bradford quantitative analysis confirmed that each monelin protein variant was purified to a concentration of about 10 mg / ml. That is, the monelin protein that can be purified in high yield by the transformant prepared in Example 2 from the fact that the purified protein expressed in the transformant transformed with the monelin gene is almost identical to the molecular weight of the monelin protein. It was confirmed that the variant was expressed.

Experimental Example 1 Sensory Test

The concentrations of salts of sodium chloride (NaCl), imidazole, and the like contained in the protein sample were dialyzed in 20 mM potassium phosphate (KPO ₄ ) buffer by wild-type Monelin protein and Monelin protein variants purified by the method of Example 4. Lowered. This was diluted to 2 mg / ㎖ concentration and heated for 10 minutes at each temperature at 10 ℃ interval in the range of 65 ~ 85 ℃ and cooled at room temperature and then dropped each sample 50 ㎕ on the tongue to perform a sensory test to measure the sweet taste. Ten adult males and females (5 males, 5 females) aged 25-45 years with no abnormality of taste were examined by the difference identification method. Control samples were based on wild type monelin. Since Monelin has a strong scent, avoiding the morning and post-prandial times three times at three pm every two days, the average value is obtained from the table below (Evaluation criteria:-No sweetness, + Some sweet taste, ++ sweet sweet, +++ strong sweet). More + in the table indicates that the sweetness is stronger.

Compared with the wild type Monelin protein, it can be seen that the Monelin protein variants are sweeter when heated to 65 degrees Celsius or more. In the case of MNEI-E24A, MNEI-E24L, and MNEI-E24W, it can be seen that the wild-type Monelin protein maintains sweetness even at a temperature that does not exhibit sweetness. Thus, it can be seen that the protein variants of the present invention exhibit thermal stability and maintain sweetness even at high temperatures.

Experimental Example 2 Monelin Protein Variants Thermostability Measurement

After dialysis in 20mM HEPES buffer (pH8.0) using a dialysis membrane to reduce the concentration of imidazole, NaCl and the like remaining in the monelin protein purified by the method of Example 4 to a minimum, Thermal stability measurements were performed using the final production of Monelin protein samples. Circular dichroism (CD) analysis was first performed for thermal stability analysis, and a differential scanning calorimeter (DSC) analysis of samples showing thermal stability even at high temperatures was performed to obtain more accurate analysis results.

1-1. Circular dichroism analysis

The thermal stability of the purified Monelin protein variants of Example 4 was determined by circular dichroism (CD) analysis using a J-815 spectrometer (JASCO). The wild-type Monelin protein was used by purifying the expression in EMON, a strain transformed with the pMON vector. It was analyzed within the range of 30 ~ 90 ℃ by heating at a interval of 1 ℃ / min within the wavelength 195 ~ 260nm range. The results are shown in FIGS. 4 and 5. Figure 4 is a graph showing the results of analyzing the wavelength curve change of the temperature during the circular dichroism analysis of the heat stability measurement test for Monelin protein variants, Figure 5 is a circular dichroism analysis of the heat stability measurement test for Monelin protein variants This is a graph showing the results of the measurement of the temperature unfolded (Fraction unfolded) at a wavelength of 222nm. As shown in FIGS. 4 and 5, when the α- helix structure and the β- window structure are folded-unfolded, a change is observed in the region around 222 nm. In the result of the Monelin protein variant MNEI_E24A transformed into alanine, The annealing phenomenon could be confirmed at 90 ° C., and the DSC analysis was performed because the accurate melting temperature (T _m ) could not be obtained by CD analysis. Monelin protein mutant MNEI_E24L mutated with leucine (Leucine) results in annealing from 80 ° C. The exact T _m value within 222nm, 30 ~ 90 ° C was analyzed as 81 ° C. In the result of mutant MNEI_E24F mutated with phenylalanine (Phenylalanine), annealing was observed from 70 ° C. The exact T _m value was analyzed as 74.7 ° C. In the results of MNEI_E24W variant of Monelin protein mutant with tryptophan, annealing was observed from 80 ° C. The exact T _m value was analyzed as 76 ° C. MNEI_E24A is more were accurate analysis is required, MNEI_E24L, MNEI_E24F, MNEI_E24W each of which melting temperature via DSC analysis: 6 ℃ compared to the (melting temperature T _m) is 68 ℃ the wild-type (wild type) single-stranded parent nelrin MNEI protein It was confirmed that the thermal stability was improved to 12 ℃.

1-2. Differential Scanning Calorimeter (DSC) Analysis

The differential scanning calorimeter (DSC, VP-DSC, Microcal) was commissioned by the Korea Research Institute of Bioscience and Bio for the Monelin protein variant MNEI_E24A, which was difficult to measure melting temperature (T _m ) by circular dichroism (CD) analysis. The melting temperature was analyzed. The results are shown in FIGS. 6 and 7. Figure 6 is a graph showing the results of measuring the melting temperature of the wild type single-chain Monel MNEI protein by differential scanning calorimetry, Figure 7 is a graph showing the results of measuring the melting temperature of MNEI_E24A. In the figure, Cp is the specific heat, ΔH is the heat of fusion, ΔHv is the heat of vaporization, dsc-cp is the actual measurement, and DSC_CPPEAK1 is the 20mM HEPES buffer. The corrected value is shown. As a result, the T _m value of MNEI_E24A was 82.7 ° C., which was about 14 ° C. higher than the wild type single-chain Monelin MNEI protein.

From the above results, the monelin protein variant of the present invention can be confirmed that the thermal stability is improved compared to the wild type

Korea Biotechnology Research Institute KCTC18197P 20101104 Korea Biotechnology Research Institute KCTC18198P 20101104 Korea Biotechnology Research Institute KCTC18199P 20101104 Korea Biotechnology Research Institute KCTC18200P 20101104

<110> Ensoltek Co., Ltd. <120> Mutant of Monellin protein, Gene coding the same, Vector          comprising the gene, Transformant transformed by the vector, and          Preparation method of the transformant <130> P10-099-ENS <160> 20 <170> Kopatentin 1.71 <210> 1 <211> 97 <212> PRT <213> Artificial Sequence <220> <223> MNEI <400> 1 Met Gly Glu Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu   1 5 10 15 Gly Lys Phe Ala Val Asp Glu Glu Asn Lys Ile Gly Gln Tyr Gly Arg              20 25 30 Leu Thr Phe Asn Lys Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr          35 40 45 Glu Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr      50 55 60 Val Tyr Ala Ser Asp Lys Leu Phe Arg Ala Asp Ile Ser Glu Asp Tyr  65 70 75 80 Lys Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro                  85 90 95 Pro     <210> 2 <211> 291 <212> DNA <213> Artificial Sequence <220> <223> cDNA of MNEI <400> 2 atgggcgagt gggaaatcat cgatattgga ccattcactc aaaacttggg taagttcgct 60 gttgacgaag aaaacaagat tggtcaatat ggtagattga ctttcaacaa ggttattaga 120 ccatgtatga agaagactat ttacgaaaac gaaggattca gagaaattaa ggggtacgaa 180 taccaattgt atgtttacgc ttctgacaag cttttcagag ctgacatttc tgaagactac 240 aagacccgcg gtagaaagtt gttgagattc aacggtccag tcccaccacc a 291 <210> 3 <211> 97 <212> PRT <213> Artificial Sequence <220> <223> MNEI_E24A <400> 3 Met Gly Glu Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu   1 5 10 15 Gly Lys Phe Ala Val Asp Glu Ala Asn Lys Ile Gly Gln Tyr Gly Arg              20 25 30 Leu Thr Phe Asn Lys Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr          35 40 45 Glu Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr      50 55 60 Val Tyr Ala Ser Asp Lys Leu Phe Arg Ala Asp Ile Ser Glu Asp Tyr  65 70 75 80 Lys Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro                  85 90 95 Pro     <210> 4 <211> 97 <212> PRT <213> Artificial Sequence <220> <223> MNEI_E24L <400> 4 Met Gly Glu Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu   1 5 10 15 Gly Lys Phe Ala Val Asp Glu Leu Asn Lys Ile Gly Gln Tyr Gly Arg              20 25 30 Leu Thr Phe Asn Lys Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr          35 40 45 Glu Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr      50 55 60 Val Tyr Ala Ser Asp Lys Leu Phe Arg Ala Asp Ile Ser Glu Asp Tyr  65 70 75 80 Lys Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro                  85 90 95 Pro     <210> 5 <211> 97 <212> PRT <213> Artificial Sequence <220> <223> MNEI_E24F <400> 5 Met Gly Glu Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu   1 5 10 15 Gly Lys Phe Ala Val Asp Glu Phe Asn Lys Ile Gly Gln Tyr Gly Arg              20 25 30 Leu Thr Phe Asn Lys Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr          35 40 45 Glu Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr      50 55 60 Val Tyr Ala Ser Asp Lys Leu Phe Arg Ala Asp Ile Ser Glu Asp Tyr  65 70 75 80 Lys Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro                  85 90 95 Pro     <210> 6 <211> 97 <212> PRT <213> Artificial Sequence <220> <223> MNEI_E24W <400> 6 Met Gly Glu Trp Glu Ile Ile Asp Ile Gly Pro Phe Thr Gln Asn Leu   1 5 10 15 Gly Lys Phe Ala Val Asp Glu Trp Asn Lys Ile Gly Gln Tyr Gly Arg              20 25 30 Leu Thr Phe Asn Lys Val Ile Arg Pro Cys Met Lys Lys Thr Ile Tyr          35 40 45 Glu Asn Glu Gly Phe Arg Glu Ile Lys Gly Tyr Glu Tyr Gln Leu Tyr      50 55 60 Val Tyr Ala Ser Asp Lys Leu Phe Arg Ala Asp Ile Ser Glu Asp Tyr  65 70 75 80 Lys Thr Arg Gly Arg Lys Leu Leu Arg Phe Asn Gly Pro Val Pro Pro                  85 90 95 Pro     <210> 7 <211> 291 <212> DNA <213> Artificial Sequence <220> <223> cDNA of MNEI_E24A <400> 7 atgggcgagt gggaaatcat cgatattgga ccattcactc aaaacttggg taagttcgct 60 gttgacgaag ccaacaagat tggtcaatat ggtagattga ctttcaacaa ggttattaga 120 ccatgtatga agaagactat ttacgaaaac gaaggattca gagaaattaa ggggtacgaa 180 taccaattgt atgtttacgc ttctgacaag cttttcagag ctgacatttc tgaagactac 240 aagacccgcg gtagaaagtt gttgagattc aacggtccag tcccaccacc a 291 <210> 8 <211> 291 <212> DNA <213> Artificial Sequence <220> <223> cDNA of MNEI_E24L <400> 8 atgggcgagt gggaaatcat cgatattgga ccattcactc aaaacttggg taagttcgct 60 gttgacgaat tgaacaagat tggtcaatat ggtagattga ctttcaacaa ggttattaga 120 ccatgtatga agaagactat ttacgaaaac gaaggattca gagaaattaa ggggtacgaa 180 taccaattgt atgtttacgc ttctgacaag cttttcagag ctgacatttc tgaagactac 240 aagacccgcg gtagaaagtt gttgagattc aacggtccag tcccaccacc a 291 <210> 9 <211> 291 <212> DNA <213> Artificial Sequence <220> <223> cDNA of MNEI_E24F <400> 9 atgggcgagt gggaaatcat cgatattgga ccattcactc aaaacttggg taagttcgct 60 gttgacgaat tcaacaagat tggtcaatat ggtagattga ctttcaacaa ggttattaga 120 ccatgtatga agaagactat ttacgaaaac gaaggattca gagaaattaa ggggtacgaa 180 taccaattgt atgtttacgc ttctgacaag cttttcagag ctgacatttc tgaagactac 240 aagacccgcg gtagaaagtt gttgagattc aacggtccag tcccaccacc a 291 <210> 10 <211> 291 <212> DNA <213> Artificial Sequence <220> <223> cDNA of MNEI_E24W <400> 10 atgggcgagt gggaaatcat cgatattgga ccattcactc aaaacttggg taagttcgct 60 gttgacgaat ggaacaagat tggtcaatat ggtagattga ctttcaacaa ggttattaga 120 ccatgtatga agaagactat ttacgaaaac gaaggattca gagaaattaa ggggtacgaa 180 taccaattgt atgtttacgc ttctgacaag cttttcagag ctgacatttc tgaagactac 240 aagacccgcg gtagaaagtt gttgagattc aacggtccag tcccaccacc a 291 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for MNEI <400> 11 ggaattccat atgggcgagt gggaaatcat 30 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for MNEI <400> 12 ccgctcgagt ggtggtggga ctggac 26 <210> 13 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for MNEI_E24A <400> 13 ttcgctgttg acgaagccaa caagattggt caa 33 <210> 14 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for MNEI_E24A <400> 14 ttgaccaatc ttgttggctt cgtcaacagc gaa 33 <210> 15 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for MNEI_E24L <400> 15 tcgctgttga cgaattgaac aagattggtc a 31 <210> 16 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for MNEI_E24L <400> 16 tgaccaatct tgttcaattc gtcaacagcg a 31 <210> 17 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for MNEI_E24F <400> 17 gttcgctgtt gacgaattca acaagattgg tcaat 35 <210> 18 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for MNEI_E24F <400> 18 attgaccaat cttgttgaat tcgtcaacag cgaac 35 <210> 19 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for MNEI_E24W <400> 19 gttcgctgtt gacgaatgga acaagattgg tcaat 35 <210> 20 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for MNEI_E24W <400> 20 attgaccaat cttgttccat tcgtcaacag cgaac 35

Claims (15)

A Monelin protein variant wherein glutamic acid at position 24 in the amino acid sequence of the single chain monelin protein consisting of the amino acid sequence of SEQ ID NO: 1 is substituted with one amino acid selected from alanine, leucine, phenylalanine or tryptophan. The variant of claim 1, wherein the variant consists of one amino acid sequence selected from SEQ ID NOs: 3-6. The gene encoding the Monelin protein variant of any one of claims 1 to 2. The gene according to claim 3, wherein the gene is represented by one nucleotide sequence selected from SEQ ID NOs: 7 to 10. Recombinant vector comprising the gene of claim 3. The recombinant vector according to claim 5, wherein the recombinant vector is for E. coli expression. The recombinant vector according to claim 6, wherein the recombinant vector has one cleavage map selected from pMOA, pMOL, pMOF or pMOW. A transformant, wherein the recombinant vector of claim 5 is introduced into a host cell. The transformant of claim 8, wherein the host cell is Escherichia coli. The transformant of claim 9, wherein the E. coli is Escherichia coli BL21 (DE3). The transformant of claim 10, wherein the transformant is one selected from E. coli Escherichia coli BL21 (DE3) transformant EMOA (KCTC18197P), EMOF (KCTC18198P), EMOL (KCTC18199P), or EMOW (KCTC18200P). Polymerase chain with a primer pair selected from SEQ ID NO: 13 and 14, SEQ ID NO: 15 and 16, SEQ ID NO: 17 and 18, or SEQ ID NO: 19 and 20, as a template, a vector containing a gene encoding a single chain monolinin protein Preparing a recombinant vector of claim 5 by performing a cloning reaction (PCR); And
Method for producing a transformant for producing a monelin protein variant comprising introducing the recombinant vector into the host cell. The method of claim 12, wherein the gene encoding the single chain monolinin protein consists of the nucleotide sequence of SEQ ID NO: 2. 14. The method of claim 12, wherein the vector comprising the gene encoding the single chain monolinin protein is a vector represented by a pMON cleavage map of FIG. 2. The method of claim 12, wherein the vector comprising the gene encoding the single chain monolinin protein is prepared by inserting the gene encoding the single chain monolinin protein into a vector for expression in a host cell.

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