KR101974979B1 - Automatic purifying apparatus for ice-binding protein - Google Patents

Abstract

The present invention relates to an ice-binding protein automatic purification device. The ice-binding protein automatic purification device (1) of the present invention includes: a chamber main body (100) which has an installation space (102) formed inside; a sample container (110) which is installed in the installation space (102) and stores cell lysate therein including ice-binding protein and interest protein combined with the ice-binding protein; a reaction container (300) which extracts the ice-binding protein and the interest protein combined with the ice-binding protein; a washing water container (310) which accommodates washing liquid to wash the reaction container (300); a cooling rod (400) which is arranged in the reaction container (300) and thermoelectrically cools cell lysate; a temperature control unit (410) which controls the temperature of the cooling rod (400); an unreacted sample container (510) which receives the cell lysate to store the same wherein the ice-binding protein and the interest protein combined with the ice-binding protein are removed from the cell lysate; and a treatment water container (520) to which washing liquid of washing the inside of the reaction container (300) is supplied. The present invention improves workability and productivity since automatic purification is possible without a necessity of a direct work of a worker to purify ice-binding protein and interest protein combined with the ice-binding protein.

Description

[0001] The present invention relates to an automatic purifying apparatus for ice-

The present invention relates to an automatic ice-binding protein purification apparatus, and more particularly, to an automatic ice-binding protein purification apparatus configured to automatically purify a protein of interest expressed in a state fused with an ice-binding protein and an ice- will be.

The production of ice crystals in vivo causes the dehydration of tissues as well as the physical damage of cell membranes and cell organelles caused by the growth of ice crystals, which seriously damages the organism. Polar living organisms express antifreeze proteins (AFP) to survive freezing below freezing temperatures.

Proteins that have the ability to bind directly to ice are called ice-binding proteins (IBPs). The ice-binding protein is a protein that binds to ice and inhibits the growth of ice crystals. It is known that living organisms such as polar, alpine, and hornbillies are biosynthesized in the body as a mechanism to overcome cold ailments. It is also called floating protein, anti-freezing protein etc. depending on the source of protein. So far, about 17 species in fish, other diatoms, carrots and plants in more than 27 species, bacteria from dozens of anti-icing protein gene sequence has been reported. This protein is highly valuable in the development of cold-resistant animals and plants, cryopreservation of cells, preservation of frozen foods, and fermentation materials.

Such an ice-binding protein can be obtained through manual refinement by a person skilled in the relevant field by connecting refrigerant circulating cold finger to the refrigerant circulating device, so that the price is as high as about 10 million won per gram There is a problem that it is difficult to obtain a large amount at a time.

Accordingly, there is a demand for an automatic ice-refining apparatus capable of automatically purifying ice proteins and producing them in large quantities.

Korean Registered Patent No. 10-1492434 (Feb.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above problems, and it is an object of the present invention to provide a method for automatically purifying a protein of interest expressed in a state fused with an ice- And an apparatus for automatically purifying an ice-binding protein capable of mass-producing proteins and proteins of interest.

The technical problem of the present invention is not limited to those mentioned above, and another technical problem which is not mentioned can be clearly understood by those skilled in the art from the following description.

According to an aspect of the present invention, there is provided an apparatus for automatically purifying ice-binding proteins, comprising: a chamber body having an installation space therein; A sample container installed in the installation space and storing therein a cell disruption solution containing interest proteins fused with ice-binding proteins and ice-binding proteins; A reaction container installed in the installation space for extracting a protein of interest contained in the cell lysate supplied from the sample container and fused with the ice-binding protein and the ice-binding protein; A washing water container installed in the installation space and containing therein a washing solution for washing the reaction container; A cooling rod disposed in the reaction vessel in a vertical direction to thermoelectrically cool the cell lysate; A temperature controller connected to one end of the cooling rod to control the temperature of the cooling rod; An unrefined sample container which is located at a lower portion of the reaction container and is supplied with and stored a cell disruption solution from which interest proteins fused with the ice-binding protein and the ice-binding protein are removed; A treatment water container positioned at a lower portion of the reaction container and supplied with a washing solution that has been washed in the reaction container; An eluent vessel disposed in the lower portion of the reaction vessel and supplied with the protein of interest fused with the purified ice-binding protein and the ice-binding protein from the reaction vessel; And a controller connected to the temperature control unit and controlling the temperature and the cooling time of the cooling rod by controlling the temperature control unit.

And the reaction vessel and the reaction vessel are connected to each other by a supply line, and the supply line is operated under the control of the controller, and the supply line is shielded Or a supply solenoid valve is provided for opening.

In addition, a connection pipe communicating with the inside of the reaction vessel is connected to the lower part of the reaction vessel, and the connection pipe is connected to the untreated sample vessel, And a connection solenoid valve for shielding or opening the connection pipe at predetermined time intervals according to the container and the eluent container.

The turntable further includes a turntable rotatably installed at a lower portion of the connection tube and having an upper portion of the untreated sample vessel, a treated water container, and an eluent container radially disposed around the connection tube, And is rotated under the control of the controller.

In addition, when the cell lysate is received in the reaction vessel, the temperature of the cooling rod is controlled by the controller to grow ice crystals in the cooling rod, so that the protein of interest fused with the ice-binding protein and the ice- Crystals. ≪ / RTI >

In addition, when the ice crystal growth is completed in the cooling rod, the ice-binding protein remaining in the reaction vessel and the unreacted sample from which the protein of interest fused with the ice-binding protein are removed are stored in the unrefined sample vessel.

When the ice crystal growth is completed in the cooling rod, the controller rotates the turntable so that the non-reacted sample container is positioned below the reaction container, so that the non-reacted sample is supplied to the unreacted sample container.

In addition, the washing liquid is supplied into the reaction vessel when the removal of the unreacted sample from the reaction vessel is completed, and the ice crystal of the cooling rod is maintained under the control of the controller while the washing liquid is supplied into the reaction vessel.

The controller is further configured to rotate the turntable so that the treatment water container is positioned below the reaction container when the washing solution is supplied into the reaction container, so that the washing solution is supplied to the treatment water container.

When the washing of the reaction vessel is completed, the temperature of the cooling rod is controlled by the controller so that the ice crystals of the cooling rod are melted and become an effluent solution containing interest proteins fused with the ice-binding protein and the ice- .

The controller rotates the turntable so that the eluent container is positioned below the reaction container when the ice crystals of the cooling rod are melted so that the eluted solution is supplied to the eluting solution container.

The temperature control unit may further include a thermoelectric element that cools or warms the cooling rod according to a voltage polarity.

The details of other embodiments are included in the detailed description and drawings.

According to the automatic refinement apparatus for ice-binding protein according to an embodiment of the present invention, it is possible to purify the interest protein fused with the ice-binding protein and the ice-binding protein automatically without requiring the operator to work directly, Can be improved, and the contamination and damage of the sample can be prevented.

In addition, even a non-expert can purify only the protein of interest from the protein of interest fused with the ice-binding protein and the ice-binding protein using an automatic ice-binding protein purification apparatus, thereby further improving workability and productivity.

Particularly, even if the kind of protein of interest is various, it is possible to easily purify a protein of interest only by the automatic refining apparatus of an ice-binding protein if the protein is fused with an ice-binding protein.

The effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned can be clearly understood by those skilled in the art from the description of the claims.

1 is a perspective view illustrating a configuration of an automatic ice-binding protein purification apparatus according to an embodiment of the present invention.
2 to 7 show a process of automatically purifying a protein of interest fused with an ice-binding protein from a cell lysate containing interest protein fused with an ice-binding protein through an automatic ice-binding protein purification apparatus according to an embodiment of the present invention Fig.
8 to 10 are operational diagrams illustrating a process of purifying only the protein of interest from the protein of interest fused with the ice-binding protein through the automatic device of the ice-binding protein according to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings, so that those skilled in the art can easily carry out the present invention.

In the following description of the embodiments of the present invention, descriptions of techniques which are well known in the technical field of the present invention and are not directly related to the present invention will be omitted. This is for the sake of clarity of the present invention without omitting the unnecessary explanation.

For the same reason, some of the components in the drawings are exaggerated, omitted, or schematically illustrated. Also, the size of each component does not entirely reflect the actual size. In the drawings, the same or corresponding components are denoted by the same reference numerals.

FIG. 1 is a perspective view of a configuration of an automatic ice-binding protein purification apparatus according to an embodiment of the present invention.

As shown in FIG. 1, the automatic ice-binding protein purification apparatus 10 includes an ice-binding protein (ice-binding protein) In order to purify the protein, the appearance and the skeleton are formed by the chamber main body 100.

In this embodiment, the chamber body 100 may be provided with a temperature control device (not shown). The temperature control device serves to maintain the temperature of the chamber body 100 at about 3 to 5 ° C. This is to minimize external influences in the process of refining ice-binding proteins. The configuration of the temperature control device may be the same as the configuration for maintaining the temperature of a general refrigerator compartment.

An installation space 102 is formed in the chamber body 100. The front surface of the installation space 102 may be opened to allow the equipment and the like to be housed. In the installation space 102, a sample container 110, a reaction container 300, a washing water container 310, a cooling rod 400, an untreated sample container 510, a treated water container 520, an effluent container 530, Etc. may be installed. As shown in FIG. 1, a plurality of cradles 104 and 105 may be installed inside the upper portion of the installation space 102, respectively. The cradles 104 and 105 are portions where the sample container 110, the compression container 122, the washing water container 310, and the like are mounted.

A door 101 may be installed on the open front of the installation space 102. The door 101 is rotatably coupled to the chamber body 100 through a hinge device (not shown). In the present embodiment, the door 101 can be formed of transparent plastic or a transparent glass material so that the installation space 102, which will be described below, can be seen with the naked eye.

As shown in FIG. 1, the sample container 110 is located in the installation space 102. The sample container 110 may be mounted on the cradle 104. The sample container 110 is a portion in which a cell disruption solution containing interest proteins fused with an ice-binding protein and an ice-binding protein is stored therein.

A cap 112 may be coupled to the upper portion of the sample container 110. The cap 112 selectively opens and closes the upper portion of the sample container 110. The cap 112 can be selectively opened and closed by a high pressure gas supplied from a gas supply assembly 120, which will be described below.

1, a gas supply assembly 120 may be installed at a position adjacent to the sample container 110. The gas supply assembly 120 serves to selectively supply a high-pressure gas from the sample container 110 so that the sample is moved to the reaction container 300 to be described below.

In this embodiment, the gas supply assembly 120 may be provided with a compression vessel 122. A high-pressure gas may be stored in the compression vessel 122.

In this embodiment, a high pressure gas is an inert gas suitable for use in the process, including nitrogen, argon, helium, and neon. Or other reactive gases such as oxygen, ozone, nitrous oxide, hydrogen, hydrogen sulfide, carbon tetrafluoride, methane, and ammonia may be used.

The compression vessel 122 may communicate with the sample vessel 110 by a connection pipe (not shown). At this time, a check valve (not shown) is built in the connection pipe, so that the space between the compression vessel 122 and the sample vessel 110 can be opened or shielded under the control of the controller C.

Meanwhile, a reaction vessel 300 may be installed in the installation space 102. The reaction vessel 300 may be fixed to the upper portion of the installation space 102 by a fixing member such as a hook (not shown). The reaction vessel 300 is a portion in which the cell lysate supplied from the sample vessel 110 is accommodated. In the reaction vessel 300, the protein of interest fused with the ice-binding protein and the ice-binding protein is extracted.

In the present embodiment, a connection pipe 302 communicating with the inside of the reaction vessel 300 may be connected to the lower part of the reaction vessel 300. The connection pipe 302 may extend vertically from a lower portion of the reaction vessel 300.

The connection pipe 302 is connected to the connection pipe 302 at a predetermined time according to the unreversed sample container 510, the process water container 520 and the eluent container 530, A connection solenoid valve 304 that shields or opens at intervals may be provided. The connection solenoid valve 304 is operated under the control of the controller C. [

Meanwhile, a washing water container 310 may be installed in the installation space 120. The washing water container 310 may be mounted on a holder 120 at a position opposite to the sample container 110. A washing liquid for washing the reaction container 300 is received in the washing water container 310. At this time, the washing solution may be purified water.

1, the space between the sample container 110 and the reaction container 300 and the space between the washing container 310 and the reaction container 300 are connected to each other by feed lines 320 and 330 Can be connected. The supply lines 320 and 330 serve to communicate between the sample container 110 and the reaction container 300 and between the wash water container 310 and the reaction container 300, respectively.

 The supply lines 320 and 330 may be provided with supply solenoid valves 340 and 350, respectively. The supply solenoid valves 340 and 350 are operated under the control of the controller C and serve to shield or open at predetermined time intervals.

Meanwhile, as shown in FIG. 1, a cooling rod 400 is disposed in a vertical direction in the reaction vessel 300. The cooling rod 400 thermally cools the cell lysate.

A temperature controller 410 is connected to one end of the cooling rod 400. The temperature controller 410 may be installed in the chamber body 100. The temperature control unit 410 controls the temperature of the cooling rod 400.

In this embodiment, the temperature control unit 410 may include a thermoelectric device (not shown). The thermoelectric element serves to cool or warm the cooling rod 400 according to voltage polarity. In the present embodiment, the thermoelectric element can cool or warm the cooling rod 400 while changing the current direction under the control of the controller C. [

On the other hand, an unredeemed sample vessel 510 is positioned below the reaction vessel 300. The unrefined sample container 510 is a portion in which a cell disruption solution from which interest proteins fused with the ice-binding protein and the ice-binding protein are supplied and stored.

A treatment water container 520 is positioned below the reaction vessel 300. The treatment water container 520 is a portion to which the washing solution that has been washed in the reaction container 300 is supplied.

In addition, an effluent container 530 is disposed below the reaction vessel 300. The eluent container 530 is a portion to which a protein of interest fused with the purified ice-binding protein and the ice-binding protein is supplied from the reaction container 300.

In this embodiment, the non-return sample vessel 510, the treatment water vessel 520, and the eluent vessel 530 are rotatably supported by the turntable 600. The turntable 600 is rotatably installed at a lower portion of the connection pipe 302 and the untreated sample vessel 510, the treatment water vessel 520 and the effluent vessel 530 are connected to the connection tube 302 in the radial direction. The turntable 600 may be rotated under the control of the controller C. [

The turntable 600 includes a rotating body 610 having an upper portion of the untreated sample vessel 510, a treated water container 520 and an effluent vessel 530 disposed radially with respect to the connecting tube 302, And a fixing body 620 for rotatably supporting the rotating body 610. [

A position sensing sensor (not shown) may be provided on the upper portion of the rotating body 610 to sense the presence or absence and position of the non-replenishment sample container 510, the process water container 520, and the eluent container 530 . The position sensing sensor senses the position of the non-return sample vessel 510, the process water container 520, and the eluent container 530, and transmits the result to the controller C.

A motor (not shown) for transmitting a rotational force to the rotating body 610 is incorporated in the fixed body 620, and the motor is rotated under the control of the controller C.

The controller C may be connected to the temperature controller 410. The controller C controls the temperature control unit 410 to adjust the cooling temperature and time of the cooling rod 400. The controller C also controls the supply solenoid valves 340 and 350 to control the amount of the cell disruption liquid and the amount of wash water delivered to the sample container 110. The connection solenoid valve 304 and the turntable 600 are also controlled so that the untreated sample vessel 510, the treated water vessel 520, and the eluent vessel 530 ) To be positioned.

Meanwhile, as shown in FIG. 1, a battery B may be installed in the installation space 102. The battery B serves to supply power to the temperature controller 410, the controller C, the connection solenoid valve 304, the supply solenoid valves 340 and 350, and the turntable 600.

In this embodiment, as shown in FIG. 1, the power supply control apparatus 700 may be installed in the installation space 102. The power supply control device 700 is a PLC (Programmable Logic Controller) and RELAY that controls the power supply to the controller B from the battery B or disconnects the power from the controller B, .

Next, the operation of the ice-binding protein automatic refining apparatus according to the present embodiment will be described with reference to the drawings.

In this embodiment, a process of purifying a protein of interest fused with an ice-binding protein from a cell lysate containing a protein of interest fused with an ice-binding protein will be described.

First, the operator stores a cell disruption liquid (L) containing interest protein (IP) fused with the ice-binding protein in the sample container (110). In this state, the controller C is operated.

2, the cell lysate L is supplied from the sample container 110 to the reaction vessel 300. As shown in FIG. At this time, it is preferable that the cooling rod 400 is locked to the reaction vessel 300 to a predetermined depth.

3, when the cell disruption liquid L is supplied to the reaction vessel 300, the cooling rod 400 is thermally cooled under the control of the controller C. As a result, 400) on the surface of ice crystals. At the same time, the protein of interest (IP) fused with the ice-binding protein is bound to the ice crystal.

When ice crystals are formed on the surface of the cooling rod 400 to a predetermined size, other proteins in which the protein of interest (IP) fused with the ice-binding protein are removed may remain in the reaction vessel 300 have.

At this time, the turn table 600 is rotated under the control of the controller C, and the unrequited sample vessel 510 is positioned below the reaction vessel 300. At the same time, by opening the connection solenoid valve 304, an unreversed sample is stored in the unfolded sample container 510 through the connection tube 302 as shown in FIG.

Next, as shown in FIG. 5, a washing liquid is supplied into the reaction vessel 300 from the washing water vessel 310 in the reaction vessel 300. This is to clean the remaining ice crystals by washing the surface of the ice crystals. At the same time, the turn table 600 is rotated by the control of the controller C, and the treatment water container 520 is positioned below the reaction container 300.

At this time, the ice crystal of the cooling rod 400 is maintained under the control of the controller C while the washing liquid is supplied into the reaction vessel 300. This is to prevent the ice crystals of the cooling rod 400 from melting.

The washing liquid that has been cleaned from the ice crystal surface of the cooling rod 400 and the reaction vessel 300 is discharged to the unrecorded sample vessel 510 through the connection pipe 302 by opening the connection solenoid valve 304 .

Next, the temperature of the cooling rod 400 is raised by the control of the controller C, and the ice crystals of the cooling rod 400 start to melt. In this case, as shown in FIG. 6, only the effluent containing the purified protein (IP) fused with the purified sample, that is, the ice-binding protein, remains in the reaction vessel 300.

At the same time, the controller (C) rotates the turntable (600) so that the eluant container (530) is positioned below the reaction container (300) so that the eluent is supplied to the eluant container (530).

The effluent in the reaction vessel 300 is stored in the effluent vessel 530 through the connection pipe 302 by opening the connection solenoid valve 304 as shown in FIG. do.

In the present invention, the protein of interest fused with the purified ice-binding protein is obtained but is not necessarily limited thereto. For example, only the protein of interest (P) can be isolated from the eluate containing the protein of interest (IP) fused with the ice-binding protein obtained using the ice-binding protein purification apparatus (10).

As shown in FIG. 8, an eluate containing interest protein (IP) fused with an ice-binding protein and proteolytic enzyme (PE) for decomposing an ice-binding protein are placed in the sample container 110, When the binding protein purification apparatus 10 is operated, as shown in FIG. 9, the protease fused with the ice-binding protein is formed of ice crystals on the surface of the cooling rod 40.

In this case, as shown in FIG. 10, only the protein of interest P remains in the reaction vessel 300. Therefore, only the protein of interest (P) can be obtained through the same process as described above.

On the other hand, only the ice-binding protein can be obtained from the cell disruption solution containing the ice-binding protein and other proteins through the same process as described above through the automatic ice-binding protein purification device 10.

When the ice-binding protein auto-purifying device 10 configured as described above is utilized, it is possible to purify the interest protein fused with the ice-binding protein and the ice-binding protein automatically without the need of a worker, Can be improved, and contamination and damage of the sample can be prevented.

In particular, even a non-expert can purify only the protein of interest from the protein of interest fused with the ice-binding protein and the ice-binding protein by using the automatic refining apparatus for ice-binding protein, so that workability and productivity can be further improved.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, And is not intended to limit the scope of the invention. It is to be understood by those skilled in the art that other modifications based on the technical idea of the present invention are possible in addition to the embodiments disclosed herein.

10: automatic ice-refining apparatus 100: chamber body
102: installation space 110: sample container
300: Reaction vessel 302: Connector
304: connection solenoid valve 310: wash water container
320, 330: supply line 400: cooling rod
410: temperature control unit 510: non-test sample container
520: Process water container 530: Eluent container
600: Turn table 610: Rotor body
620: Fixture 700: Power control device
C: Controller

Claims (12)

A chamber body having an installation space therein;
A sample container installed in the installation space and storing therein a cell disruption solution containing interest proteins fused with ice-binding proteins and ice-binding proteins;
A reaction container installed in the installation space for extracting a protein of interest contained in the cell lysate supplied from the sample container and fused with the ice-binding protein and the ice-binding protein;
A washing water container installed in the installation space and containing therein a washing solution for washing the reaction container;
A cooling rod disposed in the reaction vessel in a vertical direction to thermoelectrically cool the cell lysate;
A temperature controller connected to one end of the cooling rod to control the temperature of the cooling rod;
An unrefined sample container which is located at a lower portion of the reaction container and is supplied with and stored a cell disruption solution from which interest proteins fused with the ice-binding protein and the ice-binding protein are removed;
A treatment water container positioned at a lower portion of the reaction container and supplied with a washing solution that has been washed in the reaction container;
An eluent vessel disposed in the lower portion of the reaction vessel and supplied with the protein of interest fused with the purified ice-binding protein and the ice-binding protein from the reaction vessel;
And a controller connected to the temperature control unit and controlling the cooling temperature and time of the cooling rod by controlling the temperature control unit,
Wherein a space between the sample container and the reaction container and between the washing water container and the reaction container are connected by a supply line,
Wherein the supply line is provided with a supply solenoid valve which is operated under the control of the controller and which shields or opens the supply line at a set time interval,
A connection pipe communicating with the inside of the reaction vessel is connected to the lower portion of the reaction vessel,
Wherein the connection pipe is provided with a connection solenoid which is operated under the control of the controller and which shields or opens the connection pipe at a predetermined time interval in accordance with the non-return sample container, the process water container, A valve is installed,
A turntable in which the unrequited sample vessel, the treated water vessel, and the effluent vessel are arranged radially with respect to the connecting tube is rotatably installed on a lower portion of the connecting tube,
Wherein the turntable is rotated under the control of the controller. delete delete delete The method according to claim 1,
When the cell lysate is received in the reaction vessel, the temperature of the cooling rod is controlled by the controller to grow ice crystals in the cooling rod, and the protein of interest fused with the ice-binding protein and the ice- Wherein the at least one ice-binding protein is purified. 6. The method of claim 5,
Wherein the ice-binding protein remaining in the reaction vessel and the unreacted sample from which the protein of interest fused with the ice-binding protein are removed are stored in the unrefined sample container when the ice crystal growth is completed in the cooling rod. Device. The method according to claim 6,
Wherein the controller is configured to rotate the turntable so that the unrequited sample container is positioned below the reaction container when the ice crystal growth is completed in the cooling rod to supply the unrecorded sample to the unreacted sample container. Automatic refiner. 8. The method of claim 7,
Wherein the cleaning liquid is supplied into the reaction vessel when the removal of the unreacted sample from the reaction vessel is completed and the ice crystal of the cooling rod is maintained under the control of the controller while the washing liquid is supplied into the reaction vessel. Automatic refiner. 9. The method of claim 8,
Wherein the controller rotates the turntable so that the treatment water container is positioned below the reaction container when the washing solution is supplied into the reaction container so that the washing solution is supplied to the treatment water container.
10. The method of claim 9,
When the washing of the reaction vessel is completed, the temperature of the cooling rod is controlled by the controller so that the ice crystals of the cooling rod are melted and become an effluent solution containing interest proteins fused with the ice-binding protein and the ice- And an ice-binding protein auto-purification device. 11. The method of claim 10,
Wherein the controller rotates the turntable so that the eluent container is positioned below the reaction container when the ice crystals of the cooling rod are melted so that the eluted solution is supplied to the eluting solution container. The method according to claim 1,
Wherein the temperature control unit is provided with a thermoelectric element for cooling or warming the cooling rod according to a voltage polarity.

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