KR20100000771A - Ice-binding protein gene derived from chaetoceros neogracile, recombinant vector comprising the gene, and protein encoded by the gene - Google Patents

Abstract

PURPOSE: A gene encoding ice-binding protein is provided to massively express ice-binding protein. CONSTITUTION: An ice-binding protein(IBP) has gene sequence of sequence number 1 or 2. The gene is derived from Chaetoceros neogracile. A recombinant vector contains the gene of sequence number 1 or 2. The recombinant vector contains ori site, ampicillin resistant site, multiple cloning site, 6XHis-Tag site and Trc promoter. The ice-binding protein is a mature IBP comprising an amino acid of sequence number 3 or a pre-mature IBP comprising an amino acid of sequence number 4.

Description

Ice-binding protein gene derived from Chaetoceros neogracile, recombinant vector comprising the gene, and protein encoded by the gene }

The present invention relates to a gene encoding an ice-binding protein, and more particularly, a gene represented by the nucleotide sequence of SEQ ID NO: 1 derived from Chitoceros neogracile , wherein It relates to a recombinant vector comprising a gene, and a protein encoded by the gene.

Water, which accounts for more than 70% of the world, is made up of extreme ecosystems that are maintained at temperatures near or below freezing. Extreme habitats include deep waters, alpine and polar environments. The main stresses in the polar environment include unchanging low and near freezing temperatures, physical disturbances in the glacier and regular extreme seasons.

Creatures living in polar environments have several ways of survival to adapt to low temperatures and have evolved this survival mechanism evolutionarily. All living creatures in the polar region, from higher animals like birds to bacteria, have a mechanism of protection against low temperatures, which allows them to survive in extreme environments.

There are many different mechanisms for low temperature adaptation. Among them, many microalgae or bacteria living in seawater and fresh water lower the freezing point around the cell by secreting extracellular matrix and increasing the salt concentration around the cell.This method reduces the freezing point around the cell to prevent freezing of the cell. . In addition, a mechanism is known to secrete extracellular proteins, which are known to directly adhere to ice crystals and prevent them from growing. In particular, proteins secreted from polar diatoms attach to the surface of the ice, forming holes in the surface when the ice crystals grow or changing the shape of the ice crystals (Buckley 1951 Buckley, HE 1951. Crystal Growth. Wiley, New York, 319 pp. After the observation of.), The properties of the protein exhibiting the effect of ice-binding activity have been studied. Ice-binding protein (IBP) has since been found in fish, insects, plants, fungi and microorganisms, and has an anti-freeze effect and the ability to inhibit ice recrystallization. Can act as ice nucleators (Janech MG., A. Krell, T. Mock, JS. Kang and JA. Raymond. 2006. Ice-binding proteins from sea ice diatom ( Bacillariophyceae ). J. Phycol. 42 , 410-416).

Anti-freezing protein (AFP) derived from some fish is also an extracellular secreted protein and is known to require glycosylation, a post-translational modification process (Raymond et al. al. 2007). Such proteins maintain ion concentrations between cell membranes (Negulescu et al. 1992, Fish Antifreeze proteins block Ca entry into rabbit parietal cells.Am J Physiol 263, C1310-C1313), which increase the viability of organisms at low temperatures (Rubinsky et al. 1991, Hypothermic protection-a fundamental property of "antifreeze" proteins.Biochem Biophys Res Commun 180, 566-571).

Antifreeze proteins bind to ice crystals and inhibit the growth of ice crystals, thereby lowering the freezing point of water to prevent freezing injury (Duman et al. 1993, Duman, JA, and Olsen, TM Thermal hysteresis protein activity in bacteria, fungi and phylogenetically diverse plants.Cryobiology, 30 : 322-328.). Furthermore, thermal hysteresis, which is a phenomenon in which a difference in freezing point and melting point occurs due to the action of an anti-freezing protein, can be easily measured by a nanoliter osmometer. The antifreeze protein bound to the ice surface prevents thermodynamically favorable ice growth. To date, no studies have shown a clear indication of the anti-freeze function of ice-binding protein (IBP) known in Antarctic diatoms, and in particular have not been reported to measure the function of thermal phenomena.

As described above, genes related to proteins that prevent ice formation may be very useful for humans. For example, studies have shown long ago that plants are resistant to cold and cold damage when the plants are transformed with fish-free proteins extracted from fish (Davies, 1987, Antifreeze proteins: prospects for transferring freeze resistance.New biotechnol. 1, 1057-1063; Cutler, 1989, Winter flounder antifreeze proteins improves the cold hardiness of plant tissue.J. Plant Physiol. 135, 351-354). In addition, glycoproteins of the anti-freezing protein can inhibit the formation of proteins that induce the formation of ice crystals, thereby preventing the damage of plants caused by cold damage. nucleators by fish antifreeze glycoproteins.Nature 333, 782-783). It can also be used for cryopreservation of eggs, red blood cells, rabbit liver (Rubinsky et al. 1991; Carpenter & Hansen, 1992, Antifreeze protein modulates cell survival during cryopreservation: mediation through influence on ice crystal growth.Proc Natl Acad Sci USA 89 , 8953-8957; Lee et al. 1992, Hypothermic preservation of whole mammalian organs with "antifreeze" proteins. Cryo-Letters 13, 59-66), and can be used as a chemical adjuvant that facilitates selective cell destruction during cryosurgery (Koushafer et al. 1997, Chemical adjuvant cryosurgery with antifreeze proteins.J Surg Oncol 66, 114-121). In addition, it can be applied and applied in various fields such as food preservation and more effective low temperature fermentation (Margesin et al. 2007, Cold-loving microbes, plant, and animals-fundamental and applied aspects.Naturwissenschaften 94, 77-99). The development and application possibilities are endless.

Antarctic diatom, Chaetoceros neogracile , is one of the major biomass producers of polar microalgae. C. neogracile has an ice-binding protein (IBP) gene to adapt to low temperature and is known to use it for low temperature adaptation by secreting extracellular ice-binding protein. The polar diatom ice-binding protein gene is a unique gene that is not found in the genome of temperate diatoms. It is very inexpensive to obtain a large amount of ice-binding protein by culturing the diatoms in order to utilize these gene products. It is thought. In addition, the method of culturing the diatoms under refrigerated conditions and recovering the secreted ice-binding protein also requires a high cost.

In addition, in the case of Republic of Korea Patent No. 0543063, the gene is transformed into a plant to express and isolate and use a useful protein from the plant. Many other studies have carried out an activity experiment on proteins secreted from actual organisms. The ice-binding protein expressed in is a disadvantage that requires a glycosylation process. Therefore, there is a need for a method of expressing such a protein in large amounts in E. coli and applying the protein as it is.

Accordingly, one aspect of the present invention is to provide a gene encoding an ice-binding protein and derived from Chitoceros neogracile .

Another aspect of the present invention is to provide a recombinant vector comprising the gene.

Another aspect of the invention is to provide a protein encoded by the gene.

According to an aspect of the present invention, SEQ ID NO: 1 encoding an ice-binding protein and encoding a mature IBP. Or a gene represented by the nucleotide sequence of SEQ ID NO: 2 encoding a pre-mature IBP.

According to another aspect of the invention, there is provided a recombinant vector comprising a gene represented by the nucleotide sequence of SEQ ID NO: 1 or 2.

According to another aspect of the invention, the pre-mature protein (mature IBP) or the amino acid sequence of SEQ ID NO: 4 (pre-mature IBP) encoded by the gene and having the amino acid sequence of SEQ ID NO: 3 (pre- mature IBP).

The present invention isolates the ice-binding protein gene of the polar diatom chitoceros neogracile ( Chaetoceros neogracile ) and is then expressed using E. coli to enable mass production. In particular, unlike ice-binding proteins expressed in eukaryotes require a glycosylation process, when the ice-binding protein according to the present invention is expressed through Escherichia coli or the like, it is not subjected to post-translational modification. By showing sufficient activity even if it can be useful for mass expression and mass production.

The present invention reveals the sequence of the ice-binding protein (IBP) gene expressed by the polar diatoms, Chaetoceros neogracile , to adapt to low temperatures. The gene is expressed as a recombinant protein in Escherichia coli in large quantities, thereby enabling the production of an economically enhanced protein.

According to the present invention, a gene encoding an ice-binding protein, which is represented by the nucleotide sequence of SEQ ID NO: 1 or 2, is provided, and the polar diatoms used in the present invention are chitocerose neogramyl. ( Chaetoceros neogracile ) was purchased from Korea Ocean Polar Research Institute (KOPRI). A cDNA library was prepared by extracting mRNA from chitoceros neogracile and analyzing the nucleotide sequence of the cDNA to obtain the completed EST in NCBI (National Center for Biotechnology Information) EST database (EL620615-EL622495). Registered as.

To obtain a gene encoding an ice-binding protein of chitocerose neogracile ( C. neogracile ), a cDNA library was first prepared and the cDNA prepared above was maintained and stored in a pBluescript vector. The sequencing of the obtained cDNA of the Chaetoceros neogracile ( chaetoceros neogracile ) to secure the base sequence of the ORF (open reading frame) portion of the ice-binding protein, the length of this sequence measured by 849 bp And has the nucleotide sequence of SEQ ID NO: 2. A base sequence of 90 bp from the 5 'end of the sequence was identified as a sequence encoding a signal peptide, and the base sequence of the mature protein portion except this portion has a nucleotide sequence of SEQ ID NO: 1.

Southern blot analysis showed that ice-binding proteins exist in the multi gene family in C. neogracile , which can be seen in FIG. 5.

The present invention also provides a recombinant vector comprising a gene encoding the ice-binding protein. Three primers shown in Table 2 were prepared to insert the ORF gene of the ice-binding protein into the protein expression vector. For forward primers of pre-mature ice-defective protein (IBP) and mature ice-binding protein (IBP), the preceding partial sequence contains the initiation codon and is restricted for cloning. To use EcoRI as an enzyme, an EcoRI restriction enzyme site was added immediately before the start codon, and three bases in front of the EcoRI site of the pProEX vector were added to the restriction enzyme so that recognition of the restriction enzyme was stable. Reverse primers of pre-mature and mature ice-binding proteins are identical and contain stop codons. At the 5` end of this portion, the restriction enzyme XhoI site was also added for cloning, and three bases after the XhoI site of the pProEX vector were added to stably recognize the restriction enzyme. In the preparation of the primer, preferably, EcoRI, an XhoI restriction enzyme site are added to the front part to allow ligation to the protein expression vector, and further, NcoI, AvaI, StuI, SalI, SpeI, NotI, XbaI. Any restriction enzyme commonly used in the art, such as SphI, KpnI, and HindIII, may be used depending on the design of the primer.

In the case of pre-mature ice-binding protein (IBP), a signal peptide portion is present. The signal peptide has information for protein secretion after protein translation and is removed after secretion. The removed protein becomes active. In other words, when C. neogracile is exposed to ice, pre-maturation proteins are synthesized and then released when they are secreted out of the cell. In the present specification, both forms were prepared to compare the activity difference between the presence of a signal peptide and a mature ice-binding protein lacking this portion, and as a result of the actual pitting, It can be seen that the activity of mature ice-binding protein is excellent in both change and thermal hysteresis (TH) values.

That is, the cDNA of C. neogracile encodes a pre-mature protein, which is located immediately after the signal peptide portion as described above to obtain a mature protein. Primers were constructed from the gene sequence portion to prepare mature ice-binding proteins with high activity.

Any vector generally used in the art may be used to introduce a gene provided according to the present invention into various host cells, and the plasmid vector which may be used in the present invention is an essential component ori for replication of the vector. Moieties, antibiotic resistant moieties, multiple cloning sites, and promoter moieties for gene operation, and furthermore, for protein expression vectors, moieties that interact with the column during protein purification, such as Histaq or T7-tag for post-expression purification. And lac operon positions for expressing the gene of interest. For example, a pProEX HTa vector as shown in FIG. 2 can be used. pProEX HTa is a protein expression vector and includes an ori site, ampicillin resistance site, and multiple cloning site, and includes a 6XHis-Tag portion used for purification in front of the multiple cloning site. There is also a Trc promoter to initiate transcription of the gene of interest. FIG. 3 shows the insertion position at multiple cloning sites of the pProEX HTa vector, and FIG. 4 shows the structure of the vector with EcoR1 and Xhol cloning positions inserted with ice-binding proteins of polar diatoms.

More specifically, PCR was performed using the primers of Table 2 to amplify the ORF portion of the ice-binding protein gene, and then the amplified DNA and the vector were cleaved using restriction enzymes such as EcoRI and XhoI as restriction enzymes. Ligation using ligase. However, the restriction enzyme is not limited thereto, and any restriction enzyme used in the art may be used. That is, as shown in FIG. 4, the cloning site was cleaved using restriction enzymes such as EcoRI and XhoI, and then combined with a pre-mature form or mature ice-binding protein gene to produce a recombinant vector. Can be.

The recombinant vector can transform the microorganism, and the microorganism is preferably Escherichia coli. More preferably, the E. coli BL21 strain may be transformed.

In addition, according to the present invention, a recombinant ice-binding protein (Ice-binding) in which a gene identified in the cDNA library of Chitoceros neogracile is expressed in E. coli through a gene recombination process. protein), and the obtained ice-binding protein has a length of 282 amino acids and a molecular weight of 31 kDa, and can be represented by the amino acid sequence of SEQ ID NO: 4. Scanning with the Signal P3.0 Program It can be seen that 30 amino acid sequences from the N terminus of the amino acid sequence correspond to the signal peptide portion (FIGS. 10A and 10B), and the mature ice-binding protein except for the signal peptide portion is represented by the amino acid sequence of SEQ ID NO. 3. Can be. Mature proteins, excluding signal peptides, are 252 amino acids long and have a molecular weight of about 27 kDa.

Figure 1 shows the evolutionary phylogenetic tree of the amino acid sequence of the ORF gene of the ice-binding protein obtained from the chietoceros neogracile ( Chatoceros neogracile ), which is the reported Antarctic diatom The ice-binding protein (IBP ) of Navicula glaciei showed systematic similarity. The ice-binding protein of Navicula glaciei has the amino acid sequence of SEQ ID NO: 5.

Figure 11 shows the amino acid sequence alignment of proteins similar to the ice-binding protein. Multiple alignments were shown using an alignment program of the SDSC biology workbench (http://workbench.sdsc.edu.) To compare the similarity with amino acid sequences of other organisms known as ice-binding proteins. In the study, 61% of the same diatomic ice-binding protein, 47% of the antifungal protein of Typhula ishikariensis , and two other types of polar bacteria ( Colwellia) sp., and Psychromonas ingrahamii ) shows only 45-47% identity, and significant similarity when aligning the amino acid sequences of known anti-freeze, polar insects, and freeze stress proteins of plants. ) Was not seen.

Activity intensity of ice-binding protein expressed and purified in E. coli through recombination of ice-binding protein secreted into culture solution of Chaetoceros neogracile and genes identified in cDNA library 8A, 8B, 8C, and 8D show the results of morphological observations of growing ice surfaces, and the control (FIG. 8B) shows that the surface of the ice cubes is smooth, whereas the ice-binding protein It can be seen that the present buffer exhibits a pit-like shape on the ice surface, and the peripheral portion of the ice also changes sharply (FIGS. 8C and 8D). Furthermore, it can be seen that the ice-binding activity of the mature ice-binding protein (FIG. 8D) is superior to the pre-mature ice-binding protein (FIG. 8C). This can be confirmed by the degree of pit-like formation on the surface of the ice. On the other hand, the ice-binding protein secreted into the culture solution of chitoceros neogracile (Fig. 8a) has a clear pit shape compared to the control group, but the active strength was observed to be weak compared to the recombinant protein.

Also, The ability of ice-binding proteins secreted from chitoceros neogracile to change the crystal form of ice was measured and the ice crystal form was photographed (Canon A620), which is shown in FIGS. 9A and 9B. . The activity of changing the shape of the ice crystals on the recombinant ice-binding protein expressed in Escherichia coli was compared with the activity of the ice-binding protein secreted from the control group and chitoceros neogracile . In FIG. 9B, in the case of the control (upper left), the ice crystals are not angled and grow round, whereas in the presence of ice-binding protein (lower left and lower right), the ice-binding protein binds to the ice crystal surface to form an angle. You can see that. In the case of protein extracted from E. coli, which did not express the protein (top right), an irregular circle was formed than the control group, but the angle formed was slower than that of the ice-binding protein.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited to the following examples.

< Example  1> Quitoceros Neogramsil C. neogracile )of cDNA  library( library Production

One. Quitoceros Neogramsil Chaetoceros neogracile )of  culture

The polar diatoms used in this study were Chaetoceros neogracile and were sold by the Korea Ocean Polar Research Institute (KOPRI). Chitoceros neogracile ( C. neogracile ) is incubated in a 1: 1 mixture of F / 2 medium and Kalle's medium under a temperature condition of 4 ℃ and luminous conditions of 20μmol photons m ^-2 s ^-1 .

2. cDNA  library( library Production

To prepare the cDNA library of chietocerros neogracile ( Cetocoros neogracile ), total RNA of chitoceros neogracile ( C. neogracile ) was extracted. Total RNA was prepared using RNeasy midi kit from QIAGEN. All subsequent RNA extraction was carried out under the condition that the temperature of 4 ℃ is maintained. The cultured chitocerose neogramyl is aliquoted into a 50 ml tube and only cells are collected by centrifugation. And 3 ~ 4 times of RNAlater (QIAGEN) is added to the cell and reacted at 4 ° C. for 16-18 hours. Then, 2 ml of RLC buffer (buffer) was mixed well and centrifuged at a rate of 3,000 to 5,000 g for 5 minutes (RLC buffer is a mixture of 1 ml of RLT buffer and 10 μl of β-mercaptoethanol in the kit). The supernatant is then transferred to a 15 ml tube and 1.4 ml of 100% ethanol is added. The mixed solution is added to a MIDI column and centrifuged for 5 minutes. Then discard the supernatant and add 4 ml of RW1 buffer to the column and centrifuge again for 5 minutes. To wash the column, add 2.5 ml of RPE buffer, centrifuge for 5 minutes, and discard the supernatant. Once again add 2.5 ml of RPE buffer to the column and centrifuge for 5 minutes. Combine the column in a new 15 ml tube, add 150 μl of RNase free water in the center of the column, and let stand for 10 minutes to allow sufficient RNA to dissolve. The total RNA is then collected by centrifugation. Thereafter, electrophoresis was performed on 1.2% formaldehyde agarose gel to confirm total RNA.

Poly (A) + using 1mg / mL of RNA using Poly (A) Track mRNA isolation system (Promega, Madison, WI, USA) mRNA was generated and a cDNA library was prepared using a ZAP-cDNA synthesis kit and ZAP-cDNA Gigapack III Gold packaging extract (Stratagene, La Jolla, CA, USA). The total initial titer of each library is 6.5 × 10 ⁶ per plaque-forming unit. After amplification, a sample of the cDNA library was cut and used as a subclone insert to convert from lambda to a subclone phagemid vector. Phagemid libraries are plated in LB medium containing ampicillin (50 mg / L) in small amounts and colonies are grown in LB medium to extract plasmids.

3. Nucleotide Sequencing sequencing )

Plasmid DNA is extracted from 2,500 colonies randomly selected. Plasmid DNA was purified using GeneClean (Millipore, MA, USA), and then sequencing was performed by a specialized company.

4. cDNA  Analysis and functional classification

The analyzed sequences are removed using a computer program (Lasergene software, DNASTAR, Inc., Madison, Wis., USA). Edited EST sequences are divided into groups by comparing nucleotides with an alignment program (Lasergene software, DNASTAR, Inc., Madison, Wis., USA). The BlastX search was used as the basis for identifying the nucleotide sequence by selecting the one with the highest correlation and no duplicate accession number. E-Value is a parameter that shows the similarity between two sequences. In this study, the minimum value of E-Value was set to 10 ^-4 to find EST. The completed sequence is registered as an accession number (EL620615-EL622495) in the National Center for Biotechnology Information (NCBI) EST database.

5. cDNA library Using EST  analysis

The cDNA library of chitoceros neogracile ( C. neogracile ) was prepared by inserting it into Uni-ZAP vector using XhoI and EcoRI positions of pBluescript. 2,500 clones were randomly selected from the cDNA library, of which 1,881 clones were identified by enzyme fragments of XhoI and EcoRI.

The alignment program DNA STAR was used to obtain 1,694 non-overlapping Express Sequence Sequence Taq (EST) groups consisting of 154 contigs and 1,540 singletones (Table 1). The sequence of each EST was chosen to show the highest correlation in contrast to the results of the BLAST X search of NCBI. As a result of comparing the similarity with the open database by setting the minimum value of the E-Value as 10 ^-4 , it was found that 939 out of 1,694 ESTs are similar to genes registered in the database. 540 ESTs were found to be unknown, and the remaining 215 ESTs appeared to be new sequences that showed no similarity with known genes.

Table 1.EST of chitoceros neogracile ( C. neogracile )

Number of Contigs Number of clones Single 1,540 1,540 Group 154 342 Total 1,694 1,881

The cDNA prepared above was maintained and stored in a pBluescript vector.

Example 2 Cloning Cloning )

The sequencing of the obtained cDNA of the chitoceros neogracile ( chaetoceros neogracile ) to secure the base sequence of the ORF (open reading frame) portion of the ice-binding protein, which is represented by the nucleotide sequence of SEQ ID NO: 2 The length of the sequence was measured at 849 bp.

One. Of primer  making

First, primers were prepared to insert the ORF gene of the ice-binding protein into the protein expression vector, and the primer sequences used in this experiment are shown in Table 2 below. The primer was manufactured by requesting from Bionics based on the results obtained by analyzing the cDNA sequence, wherein the primers obtained in powder state were pre-mature pre-matured with DNAase free water. Add 191μl for form forward primer, 189μl for mature-form forward primer and 206μl for reverse primer. This made a primer material of 100 pmole, 1μl of the primer diluted to 10pmole was used.

Table 2. Primer sequences designed to express ice-binding proteins of polar diatoms in E. coli

designation order Pre-form Forward primer 5`- CCG GAATTC ATGAGTCTTATTACA-3` Mature-form Forward primer 5`-CCG GAATTC CTCCGTCAGGAGAAA- 3` Reverse primer 5`- TGC CCTGAG TTAGTTTGGTGC- 3`

In the preparation of the primer, EcoRI was added to the front portion and XhoI restriction enzyme site was added to the rear to allow ligation to the protein expression vector pProEX as follows. The sequence of the part indicated by the underlined bold font of the primer sequence indicates the restriction enzyme site, that is, GAATTC of the forward primer is EcoRI site and CCTGAG is XhoI site. The three nucleotide sequences before the portion indicated by the underlined bold font are nucleotide sequences present in the vector sequence in order to stably recognize restriction sites. To obtain a mature protein from a cDNA encoding a pre-mature protein having the amino acid sequence of SEQ ID NO: 4, primers were prepared from the gene sequence portion immediately following the above except for the signal peptide portion as described above. (Mature Forward Primer) A highly active mature ice-binding protein having the amino acid sequence of SEQ ID NO: 3 can be prepared.

2. Construction of Vector

PCR was performed using the primers prepared in Table 2 to amplify the ORF portion of the ice-binding protein gene. The conditions for performing the PCR were 1 μl of the primer, 1 μl of cDNA, 1 μl of reverse primer, 4 μl of dNTP (2.5 mM), 10 × i-Tag polymerase buffer for pre-mature form forward primer. 2 μl, i-Tag (intron) 0.5 μl, and 10.5 μl of DNAase free water (DNase free water) were mixed, 1 minute of pre-denature (95 ° C.), denature (95 ° C.) 30 seconds, annealing (59 ° C.) 45 seconds, extension (72 ° C.) 1 minute, and total extension (72 ° C.) 10 minutes. The extension step is repeated 30 times, and in the case of a mature form forward primer, the mature forward primer is used instead of the pre-mature forward primer, and the annealing step is performed at 63 ° C. for 45 seconds. Except that the same conditions as in the case of pre-mature form forward primer .

Subsequently, the amplified DNA and the vector were cleaved using the restriction enzymes EcoRI and XhoI, ligated using a ligase, and transformed into E. coli BL21 (DE3) strain. It was. That is, the cloning site of the pProEX HTa (Invitrogen) vector shown in FIG. 4 was cleaved using EcoRI and XhoI, and then recombined by binding to a pre-mature form ice-binding protein gene or a mature ice-binding protein gene. Vectors were produced.

<Example 3> Southern blot ( Southern Blot )

Southern blot analysis was performed to determine the number of copies of the gene encoding the ice-binding protein (IBP) in the nucleotide sequence of chitoceros neogracile ( C. neogracile ). 15 μg of each genomic DNA extracted from chitocerose neogracile was treated, and each of the three genomic DNA tubes was treated with EcoRV, NcoI, and SacI restriction enzymes. After reacting the tube for 2 hours or more, run on a 0.7% agarose big gel (agarose big gel) was transferred to the Hybond ^TM -N ⁺ membrane. The membrane was hybridized for 18 hours at 61 ° C using the ORF gene of the ice-binding protein and the p-32 radioisotope. Then, the number of copies of the ice-binding protein gene was confirmed by exposure to x-ray film for one day at -70 ℃.

The Southern blot analysis results are shown in FIG. 5, and it can be seen that the ice-binding proteins exist in a multi gene family in C. neogracile . The copy was confirmed.

<Example 4> Northern blot ( Northern Bot )

Northern blot analysis was performed to determine the ice-binding protein expression pattern at the mRNA level in actual freezing conditions. 200 ml of C. neogracile cells were subjected to freeze stress at -20 ° C in three states. The first condition is about 20 minutes at the time when the surface of the flask starts to freeze (Freeze start, FA), and the second condition is about 40 at the time when ice is floating in the culture (1/2 Freeze, 1 / 2F.). Minutes, the last condition stressed the cultures for about 50 minutes in the sherbet-like state (All Freeze, AF). RNA was extracted from the cells thus obtained using Plant RNeasy mini kit (QIAGEN). The total RNA extracted from the three conditions of stress was then quantified, run on 1.2% formaldehyde agarose gel, and then transferred to a Hybond ^TM -N ⁺ membrane. Since the process was the same as the Southern blot process of Example 3.

The three RNAs were extracted and run on a 1.2% formaldehyde agarose gel. The upper part of FIG. 6 shows a photograph of the quantified RNA, and the lower part of FIG. 6 shows the result of exposure to the film. As a result, it can be seen that IBP gene expression is the highest in lane 3, that is, ice floating in the culture (1/2 freezing).

Example 5 Expression of Ice-binding Protein

Transformed E. coli (BL 21 (DE3)) is placed in 10ml LB medium containing ampicillin at a concentration of 50μg / ml and incubated at 37 ° C 150rpm. After incubation for about 16 hours, transfer to 100 ml of LB medium containing 50 μg / ml of ampicillin and incubate until OD ₆₀₀ is 0.5 ~ 0.6. Thereafter, IPTG is added to 1 mM and incubated for about 4 hours. The cells are then harvested by centrifugation and the supernatant is discarded. Collected cells were stored at −20 ° C. for about 16 hours, and 10 ml of lysis buffer (50 mM NaH ₂ PO ₄ , 200 mM NaCl, 10 mM imidazole, pH 8.0) was added to the frozen cells. Loosen frozen cells. The cells are then disrupted with a sonicator until the supernatant becomes clear. Then, remove the supernatant using a centrifuge. The supernatant thus obtained is loaded onto an SDS-PAGE gel to confirm overexpression of the protein.

The protein expression results are shown in FIG. 7, in which A is a protein mixture of E. coli overexpressed with an ice-binding protein, B is a protein mixture of E. coli without overexpressing an ice-binding protein, and a protein of E. coli overexpressed with a C protein The result of refine | purifying a mixture is shown.

Example 6 Purification of Proteins

After confirming the overexpression of the protein, add 1 ml of 50% Ni-NTA (Qiagen, Germany) per 4 ml of the supernatant and mix well at 4 ° C. for 1 hour. After the mixture is added to the column, the target protein bound to Ni-NTA remains on the column and the rest falls down. The remaining agarose was washed twice with ₄ ml of wash buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 20 mM imidazole, pH 8.0), followed by elution buffer (50 mM NaH ₂ PO ₄ , 200 mM NaCl). , 250mM imidazole, pH 8.0) 0.5ml each time more than 4 times to obtain protein. The obtained pre-mature ice-binding protein has a length of 282 amino acids and a molecular weight of 31 kDa, and the mature ice-binding protein has a length of 252 amino acids and a molecular weight of 27 kDa.

The evolutionary phylogeny of the amino acid sequence of the ORF gene of the ice-binding protein obtained from Chaetoceros neogracile is shown in FIG. 1, which is an Antarctic with an amino acid sequence of SEQ ID NO: 5 previously reported. Very similar to the ice-binding protein (IBP) of the diatom, Navicula glaciei .

Example 7 Presence and Activity of Ice Binding Proteins

The presence of ice-binding protein purified by expressing the ice-binding protein secreted into the culture solution of chitoceros neogracile and the gene identified in the cDNA library in E. coli through genetic recombination. And its active strength. The morphology of growing ice surfaces, one of the methods of measuring the activity of ice-binding proteins, was measured (Raymond et al. 1989, Inhibition of growth of nonbasal planes in ice by fish antifreezes. PNAS. 86, 881-885; Raymond and Fristsen 2001, Semipurification and ice recrystallization inhibition activity of ice-active substances associated with Antarctic photosynthetic organisms. Cryobiology. 43, 63-70).

First, in the case of chitoceros neogracile (FIG. 8a) in a general culture state, a pit shape such as a weak hexagonal shape was observed on the ice surface. In addition, the shape of the ice growing at the tip of the ice cube shows a rounded shape similar to the leaves of hardwoods, indicating that the activity of the ice-binding protein is weak. In addition, in the elution buffer of the E. coli without elution buffer and the ice-binding protein (Fig. 8b) as a control, a small number of pits (no pits or rounded shapes) were observed. It can be determined that it does not exist. In the case of the general culture state and the control group, observation was performed in the elution buffer salt concentration of 702 mmol / kg, temperature of -1.5 ° C, the salt concentration of the protein solution of E. coli protein without expression of 508 mmol / kg, the temperature of -1.5 ° C, and the salt concentration of the culture solution of 1056 mmol / kg. The kg temperature is made at -2 ° C.

The salt concentration of the sample was measured to determine the freezing temperature of the ice-binding protein that was expressed and purified in E. coli through genetic recombination (VAPRO5520, Wescor Ltd., USA). As a result, the salt concentration was measured as 520 mmol / kg (pre-mature IBP) and 524 mmol / kg (mature IBP), respectively. As a result, the temperature of the water bath was set to -1.5 ° C based on the measured salt concentration. It was. The sample tube was placed in a chamber adjusted to the set temperature. The sample was placed in the sample tube after the temperature of the sample tube was matched to the temperature in the chamber. Ice cubes prepared in advance at -20 ° C were placed in a sample tube and the surface of the ice was observed with an optical microscope (Olympus SZ61, Japan), and the shape was photographed (Olympus Camedia C-5060, Olympus). Are shown in FIGS. 8C and 8D. In the buffer containing the ice-binding protein, a large number of deep and steep pits were observed on the ice surface unlike the above two cases, and it was confirmed that sharp ice similar to coniferous leaves was growing at the tip of the ice surface. In particular, in the case of mature protein (FIG. 8D), a deeper pit was observed than the pre-mature protein (FIG. 8C), and the shape also showed a hexagonal shape. As shown in the figure on the right of FIG. 8D, very sharp ice crystals were formed at the tip of the ice surface, indicating that the activity of the mature protein was higher than that of the mature protein.

8a, 8b, 8c and 8d show whether ice-binding proteins directly bind to ice to inhibit ice growth. If there is no ice-binding protein or its activity is weak, a round shape without deep pits appears on the surface of the ice, and rounded ice similar to the shape of the leaves of hardwoods grows at the end of the ice surface. On the other hand, the strongly active ice-binding protein is strongly bound to the ice surface, and the ice-binding protein inhibits the growth of ice. Due to this action, deep and steep pits are formed on the ice surface, and at the tip of the ice surface, a pointed ice similar to the shape of the leaves of conifers grows.

Example 8 Measurement of Form Change of Ice Crystal

The nanoliter osmoometers (Otago osmometers, New Zealand) were used to measure the extent to which the ice-binding protein expressed in E. coli and purified by genetic recombination changes the shape of the ice crystals. (kobashigawa et al. 2005, A part of ice nucleation protein exhibits the ice-binding ability.FEBS. 579, 1493-1497; Bravo and Griffith 2005, Characterization of antifreeze activity in Antarctic plants.J. Experiment.Bot. 56 (414 ), 1189-1196).

After filling the immersion oil (Olympus) in the sample chamber, the sample to be measured was placed on the immersion oil. The sample chamber was placed on a stage of the nanoliter osmometer and the sample inside the sample chamber was frozen at -20 ° C. After confirming whether the sample was frozen by an optical microscope (Zeiss Axiolab, Zeiss) and slowly raising the temperature of the stage was confirmed the formation of ice crystals. The temperature was raised until all the ice crystals melted and the temperature was adjusted so that the ice crystals were completely spherical. The shape of the ice crystals was photographed to investigate the change in shape when a single ice crystal grows when the temperature of the sample decreases while the stage temperature is lowered to 0.01 ° C / sec in a spherical ice crystal state (Canon A620). This is shown in Figures 9a and 9b.

9A and 9B show a phenomenon in which ice-binding protein (IBP) changes the shape of ice crystals known as one of anti-freezing activity. In FIG. 9B, the elution buffer without ice-binding protein was used as a control (top left in FIG. 9B), in which case the ice crystals were angled and grow round, whereas in the presence of ice-binding protein (bottom left of FIG. 9B). And bottom right) It can be seen that the ice-binding protein is bonded to the ice crystal surface to form an angle. In addition, the conformation of ice crystals with supernatant crushed E. coli cells that did not express ice-binding proteins for more robust proof . The phenomenon was tested (upper right of FIG. B), resulting in an irregular circular shape than the control but no angular ice crystals were observed as in the presence of ice-binding proteins. It is known that the crystal shape of the mature ice-binding protein and the pre-mature ice-binding protein is due to the structure of the protein, and the activity of the mature ice-binding protein is superior to the pre-mature ice-binding protein. .

FIG. 9a illustrates a phenomenon in which ice-binding protein secreted into a culture solution of Chaetoceros neogracile in a normal culture state changes the shape of ice crystals, and is only an irregular angle on the surface of ice crystals. In comparison with the recombinant protein of the present invention, which forms an angular form, it can be seen that the activity is significantly reduced.

<Example 9> Ice-binding protein ( IBP ) Freeze protection anti - freeze ) Measurement of activity

To measure the antifreeze activity of the recombinant protein, a nanoliter-osmometer instrument coupled to an optical microscope was used (Otago nanolitre-osmometer). It is a device that can control the temperature of sample immediately. It can measure melting point, freezing point and thermal hysteresis (TH) of sample. Place 0.1-0.5 μl of the sample into the sample chamber and freeze the sample by lowering the temperature of the sample chamber to -20 ° C. Keep the sample frozen for 5 minutes and raise the temperature to about -5 ° C. The temperature of the sample chamber is then raised until a single ice crystal is produced. The phase change of the single ice crystal is observed under an optical microscope while the temperature of the single ice crystal stops at a temperature of 0.01 ° C./sec at the stopped temperature (melting point, Tm). Measure the temperature (freezing temperature, freezing temperature) at which a single ice crystal begins to grow when the temperature drops. Thermal hysteresis is a phenomenon in which the temperature difference between the melting point and the freezing point occurs, which is expressed as the temperature difference between the melting point and the freezing point.

Table 2. The value of thermal stress (TH) per sample and the degree of change in the shape of ice crystals per sample

TH (℃) (thermal stress phenomenon) Degree of change of ice crystal ^b C. neogracile IBP ^a secreted into culture 0.05 Round disc type with angle Unexpressed crude protein 0.01 Rounded Pr-emature 0.03 Indeterminate Mature 0.18 hexagon

^{a The} culture solution incubated at 2 ° C. for 5 days is concentrated by filtration.

^b shape of ice crystals

Table 2 shows the results of comparing the TH value and the ability to change the shape of ice crystals by sample by quantifying thermal hysteresis (TH), which may be the basis for estimating anti-freeze activity. . In the absence of IBP protein activity or the absence of IBP, thermal hysteresis is negligible, and the shape of a single ice crystal is in the form of a round disc. On the other hand, when ice-binding protein is active, single ice crystals grow in the form of a hexagonal or double pyramid. When the TH activity was observed using a sample of mature IBP, it has the highest value, and it can be seen that the ice crystals show a hexagonal shape. These results confirmed that diatom-derived ice-binding protein also exhibited anti-freeze activity.

Example 10 Signal Of peptides  Presence of presence and cleavage site

Signal P (3.0) program was used to predict the presence and cleavage site of the signal peptide, and the results confirmed that the cleavage site in most amino acid sequence 30-31 (AEG-LR) site. The NN and HMM results differ in how they interpret the database, that is, by how they organize the data. For the same reason, the Y-score, C-score, and S-score also differ depending on how the analyzed data are interpreted. Initially, the score is mainly used and the version is upgraded to analyze the exact result. In the latest version, the S-score is used. For other values, the S-score can be used to confirm the presence of signal peptides.

1. Signal Peptide Presence  Measure( Signalp - NN result )

Signal P (3.0) program was used to predict the presence of a signal peptide, the results are shown in Figure 10a and Table 3 below.

Table 3. Measurement result of presence of signal peptide

Measure Position Value Cutoff Presence of Signal Peptides max. C 31 0.534 0.32 YES max. Y 31 0.651 0.33 YES max. S 13 0.988 0.87 YES mean S 1-30 0.852 0.48 YES D 1-30 0.751 0.43 YES

2. Prediction of cleavage site of signal peptide ( SignalP - HMM result )

The signal P (3.0) program was also used to predict the cleavage site of the signal peptide, the results of which can be seen in Figure 10b. As a result, the signal peptide probability was 0.917, the signal anchor potential was 0.082, and the maximum cleavage site probability was 0.559, which was identified between amino acid sequences 30-31.

Figure 1 shows that the ice-binding protein of C. neogracile is similar in amino acid sequence to Ice-binding protein (IBP) of Navicula glaciei . A phylogenetic analysis showing the division into different clades.

Figure 2 shows a map of pProEX HTa, the expression vector used for the expression of the ice-binding protein of the present invention.

3 shows the multiple cloning site and insertion site of pProEX HTa, the expression vector used for the expression of the ice-binding protein protein of the present invention.

Figure 4 shows the structure of the pProEX HTa vector labeled with the EcoRI and XhoI cloning sites in which ice-binding proteins of polar diatoms are inserted.

FIG. 5 shows Southern bot analysis of the ice-binding protein gene, lane M is a marker, lane 1 is chiDeros of chitocerose neogramyl digested with EcoRV, and lane 2 is chitocerose digested with NcoI Neograsyl gDNA, lane 3 shows chitocerose neograsyl gDNA cleaved with SacI, and shows how many copies exist by using the ice-binding protein gene as a probe to the cleaved gDNA. .

Figure 6 shows the mRNA expression of the ice-binding protein gene in the freezing conditions, lane 1 is the control RNA, lane 2 RNA at the time when the flask surface begins to freeze (Freeze start, FA), lane 3 is ice in the culture medium RNA at floating time point (1/2 Freeze, 1 / 2F.), Lane 4 shows RNA in a state where the culture medium is sherbet (All Freeze, AF).

Figure 7 shows the result of overexpressing the ice-binding protein, lane A is a protein mixture of E. coli overexpressing ice-binding protein, lane B is a protein mixture of E. coli not overexpressing ice-binding protein, lane C is pre-maturation Purification of the protein mixture of Escherichia coli overexpressed with ice-binding protein, and lane D shows the purification of mature IBP.

8A to 8D show the activity of the ice-binding protein, FIG. 8A is a general culture protein, FIG. 8B is a control, E. coli eluate with the left side of the elution buffer and the right side of the protein, and FIG. 8C the pre-maturation (Pre -mature) ice-binding protein, Figure 8d shows the activity of mature ice-binding protein, respectively.

Figures 9a and 9b measured the change in the shape of the ice crystals, Figure 9a shows the case of IBP secreted in the culture, in Figure 9b using elution buffer as a control (upper left), the upper right is the ice-binding protein The unexpressed E. coli protein (Un-Induced) mixture is shown, and the lower left and lower right shows the degree to which pre-mature IBP and mature IBP change the shape of the ice crystals, respectively.

10a and 10b respectively predict the presence and cleavage site of the signal peptide using the Signal P (3.0) program.

Figure 11 shows amino acid sequence alignment of proteins similar to ice-binding proteins.

<110> Industry-University Cooperation Foundation Hanyang University          Korea ocean research and development institute <120> Ice-binding protein gene derived from Chaetoceros neogracile,          recombinant vector comprising the gene, and protein encoded by          the gene <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 759 <212> DNA <213> Chaetoceros neogracile <400> 1 ctccgtcagg agaaacgtgg aggtcttagg cgtcagctcg atgacgaacc actatctgct 60 gtcaagctac tcacagcagg aaggtttgcc attttgacaa aaactggtgt gacgaccacc 120 ggtccaacag atctgaaagg tgatatgggc acgagtccta tcacaggcgc ggccatcacg 180 ggatttggtt tgattacgga tcccagcgat accactttct ccacatcctc ccttgtgaca 240 ggacaggtat tcgcatcaga ctacacatcc cccacaccca atatgctaac tgtagcagtc 300 ctcgacatgc aggccgcata tgttgatgct gcaggccgcc ctgaccccga ctacgttgag 360 cttggtgctg gaaacattga gggcctcact cttgaacctg gcctatacaa gtgggggact 420 gatgtcggct tcaccaacag tctcaccttc gatggttctg acactgatat ttggatcttg 480 cagatcgatg gggatgtcac agcaggaagc ggtgcaaaag tcaaactcat taacgatgcc 540 aaggctgaga acatcttttg gcagattgcg ggcaagactg atctaggcac cacgtcccat 600 gttgagggtg tgttcctctg tagcacagca atcactttca aaactggaag cagcatgaat 660 ggtgctgcac tagcacagac agcagtcacg ttggattccg ctacgatcgt gaaggagtcc 720 gtatgcgatg tggatgttgg gtgtgtggca ccaaactaa 759 <210> 2 <211> 849 <212> DNA <213> Chaetoceros neogracile <400> 2 atgagtctta ttacaattaa tcataccctc gttgtaaccg ccctactatt cgctgcagtg 60 gcacttctag gagtaccaat ggcagagggt ctccgtcagg agaaacgtgg aggtcttagg 120 cgtcagctcg atgacgaacc actatctgct gtcaagctac tcacagcagg aaggtttgcc 180 attttgacaa aaactggtgt gacgaccacc ggtccaacag atctgaaagg tgatatgggc 240 acgagtccta tcacaggcgc ggccatcacg ggatttggtt tgattacgga tcccagcgat 300 accactttct ccacatcctc ccttgtgaca ggacaggtat tcgcatcaga ctacacatcc 360 cccacaccca atatgctaac tgtagcagtc ctcgacatgc aggccgcata tgttgatgct 420 gcaggccgcc ctgaccccga ctacgttgag cttggtgctg gaaacattga gggcctcact 480 cttgaacctg gcctatacaa gtgggggact gatgtcggct tcaccaacag tctcaccttc 540 gatggttctg acactgatat ttggatcttg cagatcgatg gggatgtcac agcaggaagc 600 ggtgcaaaag tcaaactcat taacgatgcc aaggctgaga acatcttttg gcagattgcg 660 ggcaagactg atctaggcac cacgtcccat gttgagggtg tgttcctctg tagcacagca 720 atcactttca aaactggaag cagcatgaat ggtgctgcac tagcacagac agcagtcacg 780 ttggattccg ctacgatcgt gaaggagtcc gtatgcgatg tggatgttgg gtgtgtggca 840 ccaaactaa 849 <210> 3 <211> 252 <212> PRT <213> Chaetoceros neogracile <400> 3 Leu Arg Gln Glu Lys Arg Gly Gly Leu Arg Arg Gln Leu Asp Asp Glu   1 5 10 15 Pro Leu Ser Ala Val Lys Leu Leu Thr Ala Gly Arg Phe Ala Ile Leu              20 25 30 Thr Lys Thr Gly Val Thr Thr Thr Gly Pro Thr Asp Leu Lys Gly Asp          35 40 45 Met Gly Thr Ser Pro Ile Thr Gly Ala Ala Ile Thr Gly Phe Gly Leu      50 55 60 Ile Thr Asp Pro Ser Asp Thr Thr Phe Ser Thr Ser Ser Leu Val Thr  65 70 75 80 Gly Gln Val Phe Ala Ser Asp Tyr Thr Ser Pro Thr Pro Asn Met Leu                  85 90 95 Thr Val Ala Val Leu Asp Met Gln Ala Ala Tyr Val Asp Ala Ala Gly             100 105 110 Arg Pro Asp Pro Asp Tyr Val Glu Leu Gly Ala Gly Asn Ile Glu Gly         115 120 125 Leu Thr Leu Glu Pro Gly Leu Tyr Lys Trp Gly Thr Asp Val Gly Phe     130 135 140 Thr Asn Ser Leu Thr Phe Asp Gly Ser Asp Thr Asp Ile Trp Ile Leu 145 150 155 160 Gln Ile Asp Gly Asp Val Thr Ala Gly Ser Gly Ala Lys Val Lys Leu                 165 170 175 Ile Asn Asp Ala Lys Ala Glu Asn Ile Phe Trp Gln Ile Ala Gly Lys             180 185 190 Thr Asp Leu Gly Thr Thr Ser His Val Glu Gly Val Phe Leu Cys Ser         195 200 205 Thr Ala Ile Thr Phe Lys Thr Gly Ser Ser Met Asn Gly Ala Ala Leu     210 215 220 Ala Gln Thr Ala Val Thr Leu Asp Ser Ala Thr Ile Val Lys Glu Ser 225 230 235 240 Val Cys Asp Val Asp Val Gly Cys Val Ala Pro Asn                 245 250 <210> 4 <211> 282 <212> PRT <213> Chaetoceros neogracile <400> 4 Met Ser Leu Ile Thr Ile Asn His Thr Leu Val Val Thr Ala Leu Leu   1 5 10 15 Phe Ala Ala Val Ala Leu Leu Gly Val Pro Met Ala Glu Gly Leu Arg              20 25 30 Gln Glu Lys Arg Gly Gly Leu Arg Arg Gln Leu Asp Asp Glu Pro Leu          35 40 45 Ser Ala Val Lys Leu Leu Thr Ala Gly Arg Phe Ala Ile Leu Thr Lys      50 55 60 Thr Gly Val Thr Thr Thr Gly Pro Thr Asp Leu Lys Gly Asp Met Gly  65 70 75 80 Thr Ser Pro Ile Thr Gly Ala Ala Ile Thr Gly Phe Gly Leu Ile Thr                  85 90 95 Asp Pro Ser Asp Thr Thr Phe Ser Thr Ser Ser Leu Val Thr Gly Gln             100 105 110 Val Phe Ala Ser Asp Tyr Thr Ser Pro Thr Pro Asn Met Leu Thr Val         115 120 125 Ala Val Leu Asp Met Gln Ala Ala Tyr Val Asp Ala Ala Gly Arg Pro     130 135 140 Asp Pro Asp Tyr Val Glu Leu Gly Ala Gly Asn Ile Glu Gly Leu Thr 145 150 155 160 Leu Glu Pro Gly Leu Tyr Lys Trp Gly Thr Asp Val Gly Phe Thr Asn                 165 170 175 Ser Leu Thr Phe Asp Gly Ser Asp Thr Asp Ile Trp Ile Leu Gln Ile             180 185 190 Asp Gly Asp Val Thr Ala Gly Ser Gly Ala Lys Val Lys Leu Ile Asn         195 200 205 Asp Ala Lys Ala Glu Asn Ile Phe Trp Gln Ile Ala Gly Lys Thr Asp     210 215 220 Leu Gly Thr Thr Ser His Val Glu Gly Val Phe Leu Cys Ser Thr Ala 225 230 235 240 Ile Thr Phe Lys Thr Gly Ser Ser Met Asn Gly Ala Ala Leu Ala Gln                 245 250 255 Thr Ala Val Thr Leu Asp Ser Ala Thr Ile Val Lys Glu Ser Val Cys             260 265 270 Asp Val Asp Val Gly Cys Val Ala Pro Asn         275 280 <210> 5 <211> 242 <212> PRT <213> Navicula glaciei <400> 5 Met Met Phe Leu Ala Lys Thr Val Thr Leu Leu Val Ala Leu Val Ala   1 5 10 15 Ser Ser Val Ala Ala Glu Gln Ser Ala Val Asp Leu Gly Thr Ala Gly              20 25 30 Asp Phe Ala Val Leu Ser Lys Ala Gly Val Ser Thr Thr Gly Pro Thr          35 40 45 Glu Val Thr Gly Asp Ile Gly Thr Ser Pro Ile Ala Ser Thr Ala Leu      50 55 60 Thr Gly Phe Ala Leu Ile Lys Asp Ser Ser Asn Thr Phe Ser Thr Ser  65 70 75 80 Ser Leu Val Thr Gly Lys Ile Tyr Ala Ala Asp Tyr Thr Ala Pro Thr                  85 90 95 Pro Ser Lys Met Thr Thr Ala Ile Ser Asp Met Ser Thr Ala Phe Thr             100 105 110 Asp Ala Ala Gly Arg Ser Asp Pro Asp Phe Leu Glu Leu Gly Ala Gly         115 120 125 Ser Ile Glu Gly Glu Thr Leu Val Ala Gly Leu Tyr Lys Trp Gly Thr     130 135 140 Asp Val Ser Phe Thr Ser Ser Leu Val Phe Asp Gly Ser Ala Thr Asp 145 150 155 160 Val Trp Ile Leu Gln Val Ala Lys Asp Phe Ile Val Gly Asn Gly Ala                 165 170 175 Gln Met Tyr Leu Thr Gly Thr Ala Lys Ala Glu Asn Ile Phe Ile Gln             180 185 190 Val Ser Gly Ala Val Asn Ile Gly Thr Thr Ala His Val Glu Gly Asn         195 200 205 Ile Leu Ser Ala Thr Ala Ile Ala Leu Gln Thr Gly Ser Ser Leu Asn     210 215 220 Gly Lys Ala Leu Ser Gln Thr Ala Ile Thr Leu Asp Ser Val Thr Ile 225 230 235 240 Val Ser          

Claims (6)

A gene encoding an ice-binding protein, represented by the nucleotide sequence of SEQ ID NO: 1. A gene encoding an ice-binding protein, represented by the nucleotide sequence of SEQ ID NO: 2. Recombinant vector comprising a gene represented by the base sequence of claim 1 or claim 2. The recombinant vector of claim 3, wherein the recombinant vector comprises an ori site, ampicillin resistance site, multiple cloning site, 6XHis-Tag site, and a Trc promoter. Ice-binding protein consisting of the amino acid sequence of SEQ ID NO: 3. Ice-binding protein consisting of the amino acid sequence of SEQ ID NO: 4.

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