KR20150137440A - Antifreeze protein and the methods of inhibiting freezing by using thereof - Google Patents

Abstract

The present invention relates to an antifreeze protein derived from an antarctic toothfish. According to the present invention, the antifreeze protein is classified as a new type of antifreeze protein due to a big difference in a sequence in comparison with an existing antifreeze protein, thereby being useful in the development of organ cryopreservation, cold-weather damage prevention for plant, and frozen food. The present invention further provides a method for producing the protein, and a recombinant vector and a transformed cell for the method.

Description

[0001] The present invention relates to freezing proteins and methods for inhibiting freezing using the same,

The present invention relates to an anti-freeze protein and to prevention of frost prevention using the same.

More particularly, the present invention relates to an anti-freeze protein derived from Antarctic cod ( Notothenia coriiceps ), a gene encoding the same, a recombinant vector containing the same, a transformant thereof, and an anti-icing protein expressed from the gene, .

Antifreeze protein (AFP) is a protein that blocks the growth of ice crystals by binding to ice. It is also called ice-binding protein. It is also called "ice-binding protein" (DeVries, AL et al . (1969) Science 163: 1074-1075; DeVries, AL et al . (1970) Journal of Biological Chemistry 245: 2901-2913).

AFP, secreted out of the cell to adapt to cold temperatures, binds to ice crystals through hydrogen bonds. At the point where AFP does not adhere, ice crystals grow at an irregular level, creating a deep pit on the ice surface . By this action, clear boundaries can be observed between the areas where AFPs are attached and those where they are not.

Since the existence of anti-icing proteins from marine fishes in 1971 has been known, steady studies have revealed the presence and structure of one type of AFGPs (Antifreeze Glycoproteins) and four types of AFPs (DeVries, AL et al . :. 1074-1075; DeVries, aL et al (1970) Journal of Biological Chemistry 245: 2901-2913; Fletcher, GL (1981) Canadian Journal of Zoology 59:. 193-201; Chao, H. et al (1994) Davies, PL and Hew, CL (1990) FASEB Journal 4 (8): 2460-2468; Fletcher GL et al ., (1982) Canadian Journal of Zoology 60: 348-355; Protein Science 3 (10): 1760-1769; Xue, YQ et al . (1994) J. Mol. Biol. 237 (3): 351-353)

It is known that anti-freezing proteins are produced in 17 kinds of fish including cod and flounder, and it is known that anti-icing proteins are produced in more than 27 kinds of higher plants including polar diatoms, carrots and dandelions (DeVries, AL et . al (1970) Journal of Biological Chemistry 245:.. 2901-2913; Hincha, DK et al (1992) J. Plant Physiol 140:.. 236-240; Sun, XY et al (1995) Can J. Microbiol. 41 (9): 776-784; Griffith , M. et al (1997) Physiologia Plantarum 100 is 2:..... 327-332; Smallwood, M. et al (1999) Biochemical Journal 340: 385-391; Janech MG. Et al ., (2006) J. Phycol., 42, 410-416).

In general, ice is subjected to a re-crystallization process in which ice crystals are formed again in the process of melting, and large ice crystals formed at this time often cause irreversible damage to the cells. Thus, the primary role of AFP is to inhibit crystal formation and recrystallization of ice, thereby reducing the damage caused by ice. AFP inhibits the formation of ice crystals, which leads to the difference between the freezing point and the melting point of water. This phenomenon is called thermal hysteresis activity and is an indicator of quantitatively the activity of AFP. Thermal hysteresis can be easily measured with a device called a nanolitre osmometer. Anti-icing proteins bound to the ice surface inhibit the growth of thermodynamically favorable ice.

AFP and its genes can be usefully used and its application range is also broad. Plants expressing the fish AFP gene have been studied to have cold resistance and can also be used for cryopreservation of E. coli, yeast, platelets, erythrocytes, oocytes, sperm, rabbit liver, etc. (Davies, (1989) J. Plant Physiol. 135, 351-354; Parody-Morreale et al . (1988) Nature 333, 782-783; Rubinsky et al . (1992) Cryobiology 29 , 69-79; Carpenter & Hansen, (1992) Proc Natl Acad Sci USA 89, 8953-8957; Lee et al . (1992) Cryo-Letters 13, 59-66).

Recently, AFP has been reported to use AFP to selectively destroy cancer cells and tumors during cryosurgery using the property of forming hexagonal double pyramid (Koushafer et al . (1997) J Surg Oncol 66, 114-121). In addition, AFP can be used in a variety of applications such as ice cream, where the microstructure of the ice influences the texture, and frozen foods that require excellent preservation conditions during thawing (Margesin et al . (2007) Naturwissenschaften 94, 77-99). In particular, WO 92/22581 discloses a method of using AFP isolated from a plant to regulate the shape of ice crystals in an ice cream, and International Publication No. WO 99/37782 discloses a method for producing ice cream Or frozen yogurt.

AFP with the above characteristics can be used as a new functional biomaterial with high added value.

It is an object of the present invention to provide a sequence of an anti-freeze protein gene which is expressed by Antarctic cod to be adapted to a low temperature and cultivate a transformant containing this anti-icing protein gene to produce an anti- And a method using the same.

In order to achieve the above object, the present invention provides an AFP protein having an amino acid sequence of SEQ ID NO: 2 which encodes AFP and encodes a mature protein (mature AFP).

The present invention also provides a gene encoding the AFP protein of SEQ ID NO: 2. More specifically, it is a gene having a nucleic acid sequence of SEQ ID NO: 1.

The present invention also provides a recombinant vector comprising the AFP gene and a transformant transformed with the recombinant vector.

The present invention provides a composition for an anti-icing agent containing an anti-freeze protein according to the present invention.

The present invention provides a method of preventing icing using an anti-icing protein according to the present invention.

The present invention secures the AFP gene of Antarctic cod ( Notothenia coriiceps ) and expresses it by using E. coli to enable mass production. Antarctic cod is an organism that survives in the cryogenic temperature environment caused by the freezing phenomenon of the Antarctic Ocean. It is expected that the anti - icing protein synthesized from this fish will exhibit high freezing point descending activity. The anti-icing protein synthesized from the present invention exhibits the anti-freezing activity which can be used even though there is no post-translational modification step in the eukaryote, so that it can be usefully applied to the mass expression and production of the pilot scale have.

Figure 1 shows the result of protein expression and electrophoresis and staining. M is a protein ladder; 1 is an extract not expressed in E. coli ; 2 is an induced immature AFP protein; 3 is a derived mature AFP protein.
2 shows the anti-icing effect of the anti-icing protein according to the present invention. (A) shows the anti-icing effect by the anti-freezing protein isolated from E. coli expressing anti-icing protein according to the present invention.

The present invention provides an anti-icing protein of SEQ ID NO: 2 derived from Antarctic cod ( Notothenia coriiceps ). In addition, the present invention provides a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 2.

A second aspect of the present invention provides a recombinant vector comprising the anti-icing protein nucleic acid sequence and a host organism transformed with the recombinant vector. The recombinant vector of the present invention may be a recombinant vector such as various types of recombinant vectors known in the art, for example, plasmid, cosmid, phagemid, phage, virus and the like. A preferred vector is a vector capable of self-replication and expression of the nucleic acid to which it is coupled. As used herein, the term " transformation " means that the foreign DNA or RNA is absorbed into the cell and the genotype of the cell is changed. Such host cells include, but are not limited to, prokaryotic cells, yeast cells, and insect cells. Preferably, Escherichia coli is the most preferred among the prokaryotic cells.

A third aspect of the present invention provides a method for producing freezing protein using the transformed host organism. More particularly, the present invention provides a method for isolating a mutant polymerase protein by culturing a microorganism transformed with an expression vector containing a nucleic acid sequence encoding the anti-freeze protein and inducing expression of the recombinant protein.

The anti-icing protein of the present invention may also include its "functional equivalents ". Functional equivalents include amino acid sequence variants in which some or all of the amino acid sequences of the anti-freeze protein are replaced or portions of the amino acids are deleted or added. At this time, the amino acid substitution is preferably conservative substitution. Examples of conservative substitutions of amino acids present in nature include: (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur-containing amino acids (Cys, Met). In this case, it is preferable that the amino acid deletion is located in a portion not directly involved in the activity of the anti-freezing protein.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples illustrate the present invention, and it is obvious that the scope of the present invention is not limited thereto.

<Example 1> Construction and sequencing of cDNA library of Antarctic cod

Total RNA was isolated from Antarctic cod and cDNA library was prepared. About 2000 clones were obtained and sequenced. cDNA was sequenced to obtain the nucleotide sequence of the ORF (open reading frame) portion of the anti-freeze protein, and the length of the sequence was measured as 465 bp. This was translated into an amino acid sequence to obtain the amino acid sequence of SEQ ID NO: 2. As a result of comparison with other species of AFP using the amino acid, it was classified as belonging to type II. In addition, AFP type II Oreochromis niloticus ice-structured protein (Accession No. XP 005460680.1), which has the highest similarity when analyzed by NCBI Blastp, has only 46% identity, and AFP in Antarctic cod is a novel protein .

Example 2 Cloning of the gene obtained from Example 1

The ORF gene of the anti-freezing protein was inserted into the pET-28a (+) vector having the T7 lac promoter and the expression vector was transformed using BL21 (DE3) E. coli.

&Lt; Example 3 > Protein expression from transformed E. coli

The transformed E. coli (BL21 (DE3)) was added to 10 ml of LB medium containing ampicillin at a concentration of 50 μg / ml and cultured at 37 ° C at 150 rpm. After incubation for about 16 hours, the cells were transferred to 100 ml of LB medium containing 50 μg / ml of ampicillin and cultured until the O. D600 value reached 0.4-0.5. Then, IPTG was added at 1 mM and cultured for about 4 hours. Then, the cells were harvested by centrifugation and the supernatant was discarded. The collected cells were lysed with 10 ml of lysis buffer (20 mM Tris-HCl, pH 9.0) and vortexed. After sonicator operation was performed every 5 seconds, the supernatant The supernatant was then removed using a centrifuge and the supernatant thus obtained was loaded on an SDS-PAGE gel to confirm the overexpression of the protein (FIG. 1).

<Example 4>Prevention of freezing proteinMeasurements of activity and observation of ice crystals

The degree of change of the shape of the single ice crystal was measured using the supernatant containing the anti-freezing protein expressed by E. coli . Braun and Griffith (eds.), Nanoliter osmometer (Otago osmometers, New Zealand) were used (Kobashigawa et al ., 2005, A part of ice nucleation protein exhibits ice-binding ability. FEBS. 579, 1493-1497; ... 2005, Characterization of antifreeze activity in Antarctic plants J. Experiment Bot 56 (414), 1189-1196;... Gwak et al .2010 Antifreeze protein in Antarctic marine diatom, Chaetoceros neogracile Mar. Biotechnol 12, 630-639 ).

After injecting about 0.5 ul of sample into six sample chambers in a sample disc, the sample disc was placed on a thermal stage of a nanoliter osmometer and the temperature stage was adjusted to -20 deg. The sample was frozen. After the sample was frozen by an optical microscope (Zeiss Axiolab, Zeiss), the temperature of the temperature stage was slowly increased to confirm the formation of ice crystals. The temperature was elevated until all the ice crystals except the single ice crystal were melted, and the temperature was adjusted so that the single ice crystal was perfectly spherical. We measured the temperature at which the single ice crystal showed a specific shape of ice crystal form in spherical shape while lowering the temperature stage temperature at 0.01 ℃ / sec in the spherical single ice crystal state with minimum area. The ice crystal form was photographed (Canon A620) at intervals of 10 seconds while lowering the temperature, as shown in Fig. 2 (A). The spherical ice crystals appearing in the control group (Fig. 2 (B)) exhibit a shape that is larger in size while retaining the same spherical shape even at a freezing point, whereas when freezing inhibiting proteins are present, (Fig. 1). In addition, the ice cream was found to bind to the preferential ice surface of the anti-proliferative protein and exhibit a unique type of single ice crystal form. In the case of the freezing protein according to the present invention, ice crystals grow in a star shape or a snow flake shape to show the ice-binding property of the anti-freezing protein.

<110> Korea Institute of Ocean Science & Technology <120> Antifreeze protein and the methods of inhibiting freezing          using <130> PN140012 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 465 <212> DNA <213> Notothenia coriiceps <400> 1 atggctgccg tttcagagga agcagctgag aaagacctag cagttaagag tcatgtggtg 60 aagcgctctt tctactgccc cggtggatgg acctggatca acggacgctg tttctacttc 120 aaccccacac ccatgagttg ggctagagcc gagaaaatct gctcctccat ggggggacac 180 ctggcgtccg tgcacaactc gatggagtta tttgagatcg agaagatcac cataatcacc 240 acttatcaga tgccgctagc gtggatcgga ggctctgaat tacagaagct gaacgtctgg 300 acctggtctg atgggactcc tttccactac gcagactggt gtggtggaga gcccaatcag 360 gcaggcgtgc agaaatgcat acagataaat tactcagcgg cgaagtgctg ggataacatg 420 cattgtagct tgaagttgcc gtccgtctgt gtcaagggat cctga 465 <210> 2 <211> 154 <212> PRT <213> Notothenia coriiceps <400> 2 Met Ala Ala Val Ser Glu Glu Ala Ala Glu Lys Asp Leu Ala Val Lys   1 5 10 15 Ser His Val Val Lys Arg Ser Phe Tyr Cys Pro Gly Gly Trp Thr Trp              20 25 30 Ile Asn Gly Arg Cys Phe Tyr Phe Asn Pro Thr Pro Met Ser Trp Ala          35 40 45 Arg Ala Glu Lys Ile Cys Ser Ser Met Gly Gly His Leu Ala Ser Val      50 55 60 His Asn Ser Met Glu Leu Phe Glu Ile Glu Lys Ile Thr Ile Ile Thr  65 70 75 80 Thr Tyr Gln Met Pro Leu Ala Trp Ile Gly Gly Ser Glu Leu Gln Lys                  85 90 95 Leu Asn Val Trp Thr Trp Ser Asp Gly Thr Pro Phe His Tyr Ala Asp             100 105 110 Trp Cys Gly Gly Glu Pro Asn Gln Ala Gly Val Gln Lys Cys Ile Gln         115 120 125 Ile Asn Tyr Ser Ala Ala Lys Cys Trp Asp Asn Met His Cys Ser Leu     130 135 140 Lys Leu Pro Ser Val Cys Val Lys Gly Ser 145 150

Claims (10)

The anti-icing protein of SEQ ID NO: 2. A gene encoding the anti-icing protein of SEQ ID NO: 2. 3. The gene according to claim 2, wherein the gene has the nucleotide sequence of SEQ ID NO: 1. A recombinant vector comprising the gene of claim 2. A host cell transformed with the recombinant vector of claim 4. 6. The host cell according to claim 5, wherein the host cell is prokaryotic. 7. The host cell of claim 6, wherein the host cell is E. coli . A composition for preventing freezing, which comprises the icing protein of claim 1. The method for producing an iced protein according to claim 1, comprising culturing the host cell according to claim 4. A method for preventing icing, which comprises treating the icing protein of claim 1.

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