KR101766382B1

KR101766382B1 - The composition comprising protein from Leucosporidium sp for cryopreservation and preventing cryoinjury method thereof

Info

Publication number: KR101766382B1
Application number: KR1020150065232A
Authority: KR
Inventors: 이성구; 고혜연; 이준혁; 한세종; 박현
Original assignee: 한국해양과학기술원
Priority date: 2015-05-11
Filing date: 2015-05-11
Publication date: 2017-08-08
Also published as: KR20160132563A

Abstract

The present invention relates to a composition for cryopreservation comprising a microorganism derived from a microorganism belonging to the genus Lucosporium, and a method for preventing freezing damage using the same. The microbial protein derived from Luciferidium of the present invention effectively protects protozoa such as diatoms from freezing damage which may occur during the freezing and thawing process, and thus can be effectively used for preserving and utilizing the objective sample.

Description

[0001] The present invention relates to a composition for cryopreservation comprising a microorganism derived from a microorganism belonging to the genus Lucosporidium and a method for preventing freezing damage using the same,

The present invention relates to a composition for cryopreservation comprising a microorganism derived from a microorganism belonging to the genus Lucosporium, and a method for preventing freezing damage using the same.

Diatoms play a very important role in the carbon and silicon circulation and photosynthesis of most marine ecosystems. Phaeodactylum tricornutum (Bacillariophyceae), one of the pennate diatoms, is one of the most common diatoms found in most coastal areas, especially in small estuaries and between rocks. They have been developed in different forms according to various environmental conditions, and recent studies show that morphological changes of P.tricornutum can be induced by changes in culture conditions. Because of these characteristics, P.tricornutum has become an important model for studying the reaction of diatoms to the environment.

Diatoms can usually be sustained by continuous subculture, but this process is time consuming and involves genetic or physiological changes and potential contamination risks. Cryopreservation of cells is a technique that preserves unique cell traits and allows the same material to be shared among different researchers despite the differences in time and space. Therefore, cryopreservation is known to be the most suitable method for long - term preservation of microalgae. However, such cryopreservation may cause freezing damage to microalgae. However, there is not much research on freezing damage. However, cryoprotective agents have been used to reduce such freezing damage. In particular, methanol, DMSO (dimethylsulfoxide) and glycerol are widely used for cryopreservation of most marine microalgae. They reduce intracellular water content and lower the cytoplasmic crystallization rate, thereby improving the osmotic pressure that causes freezing damage to the cells. Other freezing preservatives include polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), hydroxyethylenestarch (HES), 2-methyl-2,4-pentadiol, ethylene glycol (EG) and propylene glycol (PG) Are also rare, but can be used for cryopreservation of microalgae. These cryopreservatives dehydrate the cell substrate and thereby minimize the formation of ice crystals inside the cell. Despite the availability of these cryopreservants, cytotoxicity can be induced by treatment with cryoprotectants at high concentrations. Therefore, there is a need for a non-toxic cryoprotectant. In recent years, research has been conducted to develop a cryoprotectant that is free of toxicity by using biologically-derived substances rather than chemical components. However, it has been reported that cryoprotectants capable of effectively protecting diatoms, There is a need for this as it has not been widely reported.

It is an object of the present invention to provide a composition for cryopreservation using a nontoxic microorganism-derived component so as to protect them from freezing damage that may occur during freezing and thawing of a prototype such as diatoms, and a method for preventing freezing damage using the composition .

Accordingly, an object of the present invention is to provide a composition for cryopreservation using a microorganism derived from a microorganism belonging to the genus Luciferidium, and a method for preventing freezing damage.

In order to accomplish the above object, the present invention provides a composition for cryopreservation comprising the polypeptide of SEQ ID NO: 1.

The present invention also provides a cryoprotective agent comprising the polypeptide of SEQ ID NO: 1.

The present invention also relates to a method for producing a polypeptide comprising the steps of: adding a polypeptide represented by SEQ ID NO: 1 to a freezing medium; The present invention provides a method for preventing freezing damage.

The microbial protein derived from Luciferidium of the present invention effectively protects protozoa such as diatoms from freezing damage which may occur during the freezing and thawing process, and thus can be effectively used for preserving and utilizing the objective sample.

FIG. 1 shows the results of comparing cell concentrations using DMSO, glycerol, propylene glycol (PG), ethylene glycol (EG) alone treatment group and 0.1 mg / ml LeIBP addition group (p> 0.01).
FIG. 2 is a diagram showing the results of observation of cell morphology by optical microscope using DMSO, glycerol, propylene glycol (PG), ethylene glycol (EG) alone treatment group and 0.1 mg / ml LeIBP addition group.
FIG. 3 is a graph showing the degree of cell damage observed by SEM using DMSO, glycerol, propylene glycol (PG), ethylene glycol (EG) alone treatment group and 0.1 mg / ml LeIBP addition group.

The present invention provides a composition for cryopreservation comprising the polypeptide of SEQ ID NO: 1.

The present invention also provides a cryopreservation agent represented by SEQ ID NO: 1.

Hereinafter, the present invention will be described in detail.

The polypeptide of SEQ ID NO: 1 is an anti-freeze protein (hereinafter referred to as AFP) called Ice-binding protein which is a protein derived from microorganism belonging to the genus Lucosporidium. The prototype such as cryopreserved diatoms is frozen and thawed It can be effectively used to effectively preserve and utilize a target sample.

The AOP protein derived from Luciferidium sp. Microorganism of the present invention was collected from a lake ice of the Arctic Svalbard archipelago, and then purified from the plate medium and purified by ITS1 (ribosomal internal transcribed spacer 1), derived from the yeast named AY30 in Luocoidolium according to the results of ITS2 and 5.8S gene analysis (Connell et al. (2008) Microb Ecol 56, 448-459) It can be used interchangeably with AFP. It also preferably comprises a protein having the amino acid sequence of SEQ ID NO: 1 and a functional equivalent of said protein. Is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70%, more preferably at least 90%, more preferably at least 90% Refers to a protein having a homology of at least 95% and exhibiting a physiological activity substantially equivalent to that of the protein represented by SEQ ID NO: 1.

The AFP protein of the present invention includes not only a protein having a native amino acid sequence thereof but also an amino acid sequence variant thereof, within the scope of the present invention. A variant of an AFP protein refers to a protein having a sequence that differs by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, with an AFP native amino acid sequence and one or more amino acid residues. Amino acid exchanges in proteins and peptides that do not globally alter the activity of the molecule are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly. And may be modified by phosphorylation, sulfation, acetylation, glycosylation, methylation, farnesylation, and the like in some cases.

The polypeptide of SEQ ID NO: 1 of the present invention can prevent damage to protists from freezing damage, which may occur, in particular, in cryopreservation of protists, and thus can be a composition for cryopreservation of protist organisms.

The term " protist organism " is a unicellular organism having one nucleus, including diatom, Abbe, euglena, dinoflagellates, water fungi, etc. In the present invention, (Chromaveolata), such as the subspecies can refer to the protozoa.

The diatoms are one of the photosynthetic phytoplankton, and the cell walls are highly silicified, and they can be characterized by various minute and complex holes, that is, a vacuum. It has a lot of cell fluids in its body and it has a chlorophyll and a saccharide as a pigment. In the present invention, the term " diatom " means any plant belonging to the same category and may include, without limitation, protists having the above-mentioned characteristics.

The polypeptide represented by SEQ ID NO: 1 of the present invention can be effectively used for preventing cell deformation such as deformation of cell walls, separation of epitheca and hypotheca, cell shape damage such as cell wall breakage and chlorophyll damage, So that it is preferably useful for cryopreservation of protozoa, particularly preferably diatoms. Accordingly, the present invention provides a composition for cryoprotectant preservation comprising a polypeptide represented by SEQ ID NO: 1.

In addition, the present invention may further comprise a cryopreservation agent in addition to the polypeptide represented by SEQ ID NO: 1, and the cryopreservation agent may include all of chemical substances and biologically-derived substances without limitation, and preferably DMSO, glycerol , Propylene glycol and ethylene glycol, more preferably a cryoprotectant which is propylene glycol or ethylene glycol. When the above-mentioned cryopreservation agent is further contained, the synergistic effect of the present invention can more effectively protect the sample from freezing damage. In particular, the amount of the cryopreservation agent, which is a chemical substance, is relatively decreased, , It is possible to reduce the toxicity of the sample and effectively protect the sample from freezing damage.

The above-mentioned freezing medium may include, without limitation, those widely used in the art for cryopreservation of a sample. The method for preventing freeze damage can be carried out more specifically by some modifications of the method developed by Mitbavkar and Anil.

The freezing injury may include damage caused by a rapid or non-rapid freezing method. Rapid freezing may be performed by a one-step freezing method performed by using a liquefied nitrogen gas, and a non-rapid freezing Means a freezing method comprising two or more steps such as slowly freezing the sample by cooling at a predetermined temperature using a control freezer at a predetermined time unit, and then freezing the sample by using liquid nitrogen again.

For example, the freeze damage prevention method of the present invention

1) adding a mixture of recombinant LeIBP or LeIBP and a cryoprotectant (CPA), which is the polypeptide of SEQ ID NO: 1, to the sterilized freezing medium;

2) suspending a sample, preferably a protist, more preferably a diatom, in the medium;

3) transferring the suspended cells to a cold vial and cooling by rapid or non-rapid freezing method; Lt; / RTI >

The frozen samples can be thawed using a water bath, after which the cryopreservation agent can be removed, washed, and used for cell culture again.

The concentration of the recombinant LeIBP added to the freezing medium can be appropriately adjusted so long as it does not cause physical and physiological changes in the sample, preferably 0.01 to 10 mg / ml, more preferably 0.05 to 5 mg / ml, May be from 0.1 to 1 mg / ml, but is not limited thereto, so long as the object of the present invention of protecting the sample from freezing damage can be achieved.

In order to prevent the cryoprotectant of the present invention, the cryoprotectant may further include a cryoprotective agent in addition to the polypeptide represented by SEQ ID NO: 1, and the cryopreservation agent may include any chemical substance or biologically-derived substance without limitation , Preferably one or more cryopreservatives selected from the group consisting of DMSO, glycerol, propylene glycol and ethylene glycol, more preferably propylene glycol or ethylene glycol.

The term " freezing injury " may include without limitation various physical, chemical, and physiological changes that can be caused to the sample by rapid or non-rapid freezing and subsequent thawing processes, Such as changes in cell morphology, cell wall damage, loss of cell resilience or cell viability as identified by a decrease in cell proliferation potency, damage of chlorophyll, separation of epitheca and hypotheca, and the like.

Accordingly, the present invention provides a method for producing a polypeptide comprising the steps of: adding a polypeptide represented by SEQ ID NO: 1 to a freezing medium; , Cell wall damage, reduction of cell viability, separation of epitheca and hypotheca, and chlorophyll damage.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Statistical analysis of the embodiment of the present invention was performed using Excel, Student's t test was used for statistical significance determination, and p <0.01 was judged to be significant. All experiments were performed at least three times independently and repeatedly. The experimental results are shown as mean ± 1 standard deviation.

Example 1. Preparation of recombinant LeIBP for cryopreservation

Recombinant LeIBP was produced and purified from Pichia pastoris, a yeast strain that consumes methanol as a nutrient. Regarding the separation and purification of LeIBP, Korean Patent No. KR 13969125 can be referred to in its entirety, and the protein sequence of LeIBP is shown in SEQ ID NO: 1.

Recombinant Pichia pastoris major X33 cells were transformed with pPICZaA containing the mature LeIBP gene and allowed to grow for 2 days at 30 ° C in a 3-l flask containing yeast-peptone-dextrose medium. Expression of recombinant LeIBP was induced by adding 5 ml methanol daily to the medium, and the culture supernatant was placed on an ion exchange chromatography (QFF) column and eluted using 50 mM Tris-HCl buffer, pH 8.0, containing 400 mM NaCl. The eluted fractions were collected and further purified using a pH 8.0 Superdex 200 size exclusion column (Thermo Fisher, USA) containing 150 mM NaCl buffer and equilibrated with 50 mM Tris-HCl using a flow rate of 1 ml / min, LeIBPs were confirmed using SDS-PAGE and Western blotting. The protein concentration was determined by measuring the absorbance at 26930 M ^-1 · cm ^-1 at 280 nm absorbance. SDS-PAGE and Western blotting confirmed the production of recombinant LeIBP from Pichia pastoris, which was used in subsequent experiments.

Example 2. Confirmation of protection effect of freezing damage of marine diatoms

2.1 Freezing and Thawing Process

Marine diatom (diatom) P. tricornutum (CCAP 1055/1 ) gained from the CCAP (Collection of Algae and Protozoa, http://www.ccap.ac.uk), this freeze-thawing by freezing by each preservative P. freezing damage protection of tricornutum was compared. P. tricornutum cells were grown in batches and cultured in flasks at a rate of 60-80 μmol m ^-2 · s ^-1 photon fluence, at which time the period of 16: 8 light (L / D) was controlled with white fluorescence . Cells were continuously maintained in F / 2 + Si medium and subcultured every month.

The cryopreservation process was carried out with some modification of the method developed by Mitbavkar and Anil. DMSO, glycerol, propylene glycol (PG) and ethylene glycol (EG) were prepared as cryoprotective agents (CPA) in a 10% v / v seawater solution. F / 2 + Si cells without CPA were used as a negative control. These frozen media were sterilized with a 0.2 μm filter. P. tricornutum cells in logarithmic growth phase were obtained by centrifugation at 1200 x g for 5 minutes at 12 ° C. Approximately 1 × 10 ⁶ cells were resuspended in 1 ml of freezing medium, supplemented or not supplemented with LeIBP, respectively. The final concentration of LeIBP was 0.1 mg / ml, and the cells were then transferred to a 2 ml low temperature cryovial. P. tricornutum cells were frozen in two different freezing processes, fast freezing and two-stage freezing. The low temperature vial was placed in liquefied nitrogen (LN2) for rapid freezing and rapid freezing was induced with LN2 gas. For the two-step freezing process, the low-temperature vial containing the cells was cooled to 20-0 0 C at -5 [deg.] C / min and then cooled to -1 [deg.] C / (Kryo 560, Planner, UK) in a controlled-rate freezer. The cells were then rapidly cooled with liquefied nitrogen. Each experiment was repeated three times.

After 2 weeks of storage, the low-temperature vials were thawed for 2 minutes at 30 ° C in a water bath and centrifuged at 1200 xg for 5 minutes at 12 ° C to remove supernatant. Cells were washed 3 times with F / 2 + Si medium to remove residual CPA and finally washed cells were transferred to fresh F / 2 + Si medium in 24 well plates and grown under the normal culture conditions described previously Respectively.

2.2 Cryopreservation effect of LeIBP - Improvement of cell survival rate after freezing - thawing

Cells of P. tricornutum , a marine diatom, were frozen using various combinations of chemical CPA and LeIBP as described in 2.1 above. Cells were grown for 11 days and cell viability was analyzed daily. In order to observe cell viability, 1 μl of a neutral red solution (Sigma, Korea) was added to 9 μl of the cells, and the cells were stained for 10 minutes at 12 ° C. in a dark environment. Were counted using a hemocytometer under a microscope (BX-51, Olympus, Japan) to confirm the cell concentration. The results are shown in Fig.

As shown in Fig. 1, there was a clear difference in cell concentration between the CPA-alone test group and the CPA-supplemented LeIBP-treated cells in the cells frozen by the two-stage freezing method. CPA alone showed no significant cell recovery in post-freezing cultures up to day 11, and the cell concentration at 11 days after thawing was higher in the freezing medium containing DMSO, glycerol, PG and EG compared with non-frozen control cells , Respectively, and 5, 2, 75, and 34%, respectively. However, the cell concentration of the experimental group supplemented with 0.1 mg / ml LeIBP was 68, 48, 100, and 100% in the freezing medium containing DMSO, glycerol, PG and EG, respectively, compared with the non- 100%. The addition of LeIBP markedly improved the protection effect of freezing damage and the cytotoxicity was lower than that of conventional chemical CPA alone. In particular, the combination of PG, EG and LeIBP showed 100% cell survival rate and showed the best cell survival rate.

2.3 Cryopreservation effect of LeIBP - Chlorophyll concentration

To determine if LeIBP can effectively protect chlorophyll that could be damaged during freeze-thawing, changes in chlorophyll concentration after freezing-thawing were analyzed by fluorescence measurement. 1 ml of each sample was periodically obtained during the incubation period, and then in vivo fluorescence measurement was performed using a Trilogy laboratory fluorometer (Turner Designs, USA). The results of evaluation of the chlorophyll content of the frozen cells are shown in Table 1.

[Table 1]

As shown in Table 1, the concentration of chlorophyll increased to 14, 48, 1.6 and 8.8-fold in DMSO, glycerol, PG, and EG, respectively, in the LeIBP-containing experimental group compared to the control without LeIBP. The smallest increase was in the PG experimental group, but the total chlorophyll concentration was highest in these experimental groups. In the EG group in the presence of LeIBP, an increase of about 9-fold was observed. In particular, the concentration of chlorophyll in these cells was similar to that of the control cells before freezing, indicating that the chlorophyll- It was confirmed that the cells could be protected. In the DMSO and glycerol experimental groups, 14 and 48 fold increases were observed, respectively, but the total chlorophyll concentration was slightly lower compared to the non-frozen control cells. These results indicate that LeIBP can protect chlorophyll effectively from freezing damage during freezing process.

2.3 Cryopreservation effect of LeIBP - Protection of cell morphology

Diatom cells are composed of rigid silicon cell walls which, since they are inflexible, can break during shrinkage and expansion in the freezing medium during the freeze-thaw process. It is therefore important to protect them from morphological changes and cell wall breakage during the freezing and thawing process. Therefore, the morphology of cells was observed in the freeze-thawed group supplemented with CPA and LeIBP. First, on day 11 after freeze-thaw culture, the morphology of diatom cell was observed through an optical microscope, and the result is shown in FIG.

As shown in Fig. 2, in the cell group treated with CPA alone, abnormal cell deformation such as theca showing separation of the cell wall and particles surrounding the cell appeared, and after the freezing, .

To observe cell wall damage more specifically, SEM was used. The cells were placed on a Petri dish and covered with a 1 cm round glass cover slip. 1% glutaraldehyde was spotted several times in the culture medium and fixed for 30 minutes, washed three times with distilled water and air-dried at room temperature. After that, it was coated with a gold-palladium layer and observed with an SEM (JSM-6610LV, JEOL Ltd., Japan). The results are shown in Fig. 3, and cell images are representative of each experimental group.

As shown in Fig. 3, in most of the cells, cell wall breakage and morphological changes were observed. The cell walls were damaged by the freezing-thawing process, and large and small pores were identified in the central and pole portions of the siliceous frustules. Frozen cells in the presence of PC using the two - stage freezing method were observed to have protruding nodules on the surface of the cell wall. In addition, the internal appearance of these cells was observed. As a result, it was confirmed that a double row of cell surface showing the separation of epitheca and hypotheca due to freezing of cell wall appeared. However, in the cells supplemented with LeIBP and PG and EG, the cell wall was almost the same as that of the control without the freezing.

These results suggest that chemical CPA is effective in protecting diatomic cells from freezing damage, but diabetic cells can be protected from cell damage by the presence of LeIBP. Particularly, in experimental group treated with both EG and PG and LeIBP, And it showed the best protection effect from freezing damage.

<110> Korea Institute of Ocean Science & Technology <120> The composition comprising protein from Leucosporidium sp cryopreservation and preventing cryoinjury method thereof <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 241 <212> PRT <213> Leucosporidium sp <400> 1 Gln Arg Asp Leu Ser Val Glu Leu Gly Val Ala Ser Asn Phe Ala Ile 1 5 10 15 Leu Ala Lys Ala Gly Ile Ser Ser Val Pro Asp Ser Ala Ile Leu Gly 20 25 30 Asp Ile Gly Val Ser Pro Ala Ala Ala Thr Tyr Ile Thr Gly Phe Gly 35 40 45 Leu Thr Gln Asp Ser Ser Thr Thr Tyr Ala Thr Ser Pro Gln Val Thr 50 55 60 Gly Leu Ile Tyr Ala Ala Asp Tyr Ser Thr Pro Thr Pro Asn Tyr Leu 65 70 75 80 Ala Ala Ala Gla Ala Gla Ala Gla Thl 85 90 95 Phe Val Asp Pro Asp Phe Leu Glu Leu Gly Ala Gly Glu Leu Arg Asp 100 105 110 Gln Thr Leu Val Pro Gly Leu Tyr Lys Trp Thr Ser Ser Val Ser Ser 115 120 125 Pro Thr Asp Leu Thr Phe Glu Gly Asn Gly Asp Ala Thr Trp Val Phe 130 135 140 Gln Ile Ala Gly Gly Leu Ser Leu Ala Asp Gly Val Ala Phe Thr Leu 145 150 155 160 Ala Gly Gly Ala Asn Ser Thr Asn Ile Ala Phe Gln Val Gly Asp Asp 165 170 175 Val Thr Val Gly Lys Gly Ala His Phe Glu Gly Val Leu Leu Ala Lys 180 185 190 Arg Phe Val Thr Leu Gln Thr Gly Ser Ser Leu Asn Gly Arg Val Leu 195 200 205 Ser Gln Thr Glu Val Ala Leu Gln Lys Ala Thr Val Asn Ser Pro Phe 210 215 220 Val Pro Ala Pro Glu Val Val Gln Lys Arg Ser Asn Ala Arg Gln Trp 225 230 235 240 Leu

Claims

A polypeptide represented by SEQ ID NO: 1; A composition for cryoprotectant storage of diatom including a propylene glycol or ethylene glycol as a cryopreservation agent.

The composition according to claim 1, wherein the polypeptide is a polypeptide derived from a lucosporidium genus AY30.

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A polypeptide represented by SEQ ID NO: 1; A diatomaceous cryopreservation agent comprising propylene glycol or ethylene glycol as a cryopreservation agent.

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A polypeptide represented by SEQ ID NO: 1; Adding a diatomaceous cryopreservation composition comprising propylene glycol or ethylene glycol as a cryopreservation agent to a freezing medium; / RTI > A method of preventing diatomaceous freezing damage.

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The method of claim 7, wherein the freezing injury is at least one selected from the group consisting of cell morphology, cell wall damage, cell viability reduction, epitheca and hypotheca separation, and chlorophyll damage.