CN218058999U - Protein fermentation liquor pretreatment device for recombinant escherichia coli expression system - Google Patents
Protein fermentation liquor pretreatment device for recombinant escherichia coli expression system Download PDFInfo
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Abstract
The utility model relates to a recombinant protein purification technical field, concretely relates to a protein fermentation liquid preprocessing device for recombining escherichia coli expression system. The protein fermentation liquor pretreatment device for the recombinant escherichia coli expression system comprises a buffer solution storage container, a fermentation liquor storage container with adjustable temperature, a tangential flow filtration device and a functional storage container; the buffer solution storage container is communicated with the fermentation liquor storage container with adjustable temperature through a first pipeline; a first pump body is arranged on the first pipeline; a second pipeline and a return pipeline are communicated between the fermentation liquor storage container with adjustable temperature and the tangential flow filtration device; a second pump body is arranged on the second pipeline; the tangential flow filtering device is communicated with the functional storage container through a third pipeline; an ultrafiltration membrane package is arranged in the tangential flow filtration device. The device can sequentially and continuously complete the buffer solution replacement of the fermentation liquor, the high-temperature cell disruption and the collection of the supernatant/inclusion body containing the target protein, simplify the process and improve the efficiency.
Description
Technical Field
The utility model relates to a recombinant protein purification technical field particularly, relates to a protein fermentation liquid preprocessing device for recombining escherichia coli expression system.
Background
Escherichia coli is the first host cell for recombinant protein production, and has the advantages of clear genetic background, simple culture operation, fast production and propagation, low cost, capacity of fast large-scale production of target protein, etc. and makes it possible to use widely recombinant protein expression system.
The main expression modes of the target protein in the escherichia coli mainly comprise intracellular soluble expression, inclusion body expression, periplasmic space expression and the like. Most recombinant proteins are expressed in escherichia coli mainly by intracellular soluble or inclusion bodies, and antibody fragments such as Fab, scFv and the like are mostly expressed in the periplasmic space of cells through signal peptides. The target protein requires pretreatment of the fermentation broth before entering the downstream purification process. The efficiency of pretreatment has a great influence on the yield of downstream purification.
The main steps of the traditional fermentation liquor treatment process comprise fermentation liquor transfer collection, thallus cell centrifugal collection, thallus buffer solution heavy suspension, thallus high-pressure homogenizing and crushing, centrifugal collection of supernatant/inclusion bodies, and the middle relates to a plurality of discontinuous treatment processes such as sample transfer, centrifugation and the like, and the process is relatively complex.
In view of this, the utility model discloses it is special.
SUMMERY OF THE UTILITY MODEL
An object of the utility model is to provide a protein fermentation liquid preprocessing device for recombining escherichia coli expression system to it is complicated to solve traditional escherichia coli fermentation liquid preprocessing process flow, and the step is many, the problem of inefficiency.
In order to realize the above purpose of the utility model, the following technical scheme is adopted:
the protein fermentation liquor pretreatment device for the recombinant escherichia coli expression system comprises a buffer solution storage container, a fermentation liquor storage container with adjustable temperature, a tangential flow filtration device and a functional storage container;
the buffer solution storage container is communicated with the fermentation liquor storage container with the adjustable temperature through a first pipeline; a first pump body is arranged on the first pipeline;
a second pipeline and a return pipeline are communicated between the fermentation liquor storage container with the adjustable temperature and the tangential flow filtration device; a second pump body is arranged on the second pipeline;
the tangential flow filtration device is communicated with the functional storage container through a third pipeline;
and an ultrafiltration membrane package is arranged in the tangential flow filtration device.
In one embodiment, the buffer storage container comprises at least one of a beaker, a storage bottle, a storage bag, a stainless steel drum, and a liquid reservoir.
In one embodiment, the temperature-adjustable fermentation broth storage vessel comprises a storage vessel body structure and an adjustable temperature stirrer.
In one embodiment, the storage vessel body structure comprises at least one of a beaker, a storage bottle, and a fermentor.
In one embodiment, the adjustable temperature mixer includes a temperature control assembly and a mixing assembly.
In one embodiment, the storage vessel body structure is a storage bottle or a fermentor; the top end of the storage container body structure is provided with a first reserved interface, a second reserved interface and a sampling interface;
the tangential flow filtration device comprises a tangential flow filtration device inlet end, a tangential flow filtration device return end and a tangential flow filtration device permeate end;
the first reserved interface is communicated with the first pipeline;
the second reserved connector is communicated with the return end of the tangential flow filtering device through the return pipeline;
the sampling connector is communicated with the inlet end of the tangential flow filtration device through the second pipeline;
the tangential flow filtration device permeate end is in communication with the functional storage container.
In one embodiment, the liquid inlet of the second pipeline is close to the inner bottom surface of the storage container body structure.
In one embodiment, the ultrafiltration membrane package is one having a molecular weight cut-off greater than or equal to 1000KD.
In one embodiment, the functional storage container comprises a waste liquid storage container and/or a sample collection container.
In one embodiment, the waste fluid storage container comprises at least one of a beaker, a storage bottle, a storage bag, a stainless steel drum, and a liquid storage tank.
In one embodiment, the sample collection container comprises at least one of a beaker, a storage bottle, a storage bag, a stainless steel bucket, and a liquid reservoir.
In one embodiment, the number of the waste liquid storage containers and the number of the sample collection containers are one or more.
Compared with the prior art, the beneficial effects of the utility model are that:
the treatment device in the utility model is suitable for the pretreatment of high-heat stable protein fermentation liquor expressed in escherichia coli, simplifies the complex steps of fermentation liquor transfer collection-thallus cell centrifugal collection-thallus buffer solution heavy suspension-high-pressure homogeneous crushing-centrifugal collection of supernatant/inclusion body and the like in the pretreatment process of the escherichia coli fermentation liquor, and can greatly improve the working efficiency; meanwhile, the device has the advantages of easily obtained materials, convenient assembly and simple amplification, and is suitable for industrial amplification production of specific products.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the technical solutions in the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a schematic structural diagram of a front view of a protein fermentation broth pretreatment device for a recombinant Escherichia coli expression system;
FIG. 2 is a schematic diagram of a temperature-adjustable fermentation broth storage vessel;
FIG. 3 is a schematic diagram of a tangential flow filtration device.
Reference numerals:
1-buffer storage container, 2-first peristaltic pump, 3-fermentation liquor storage container, 4-second peristaltic pump, 5-tangential flow filtration device, 6-ultrafiltration membrane package, 7-waste liquid storage container, 8-sample collection container, 9-temperature control component, 10-stirring component, 11-first reserved interface, 12-second reserved interface, 13-sampling interface, 14-tangential flow filtration device inlet end, 15-tangential flow filtration device return end, 16-tangential flow filtration device permeation end, 17-first pipeline, 18-second pipeline, 19-return pipeline, 20-third pipeline, 21-fermentation tank, 22-functional storage container.
Detailed Description
In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", and the like indicate the position or positional relationship based on the position or positional relationship shown in the drawings, or the position or positional relationship which is usually placed when the product of the present invention is used, and are only for convenience of description and simplification of the description, but do not indicate or imply that the device or element referred to must have a specific position, be constructed and operated in a specific orientation, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first," "second," "third," and the like are used solely to distinguish one from another and are not to be construed as indicating or implying relative importance.
In the description of the present invention, it should also be noted that, unless otherwise explicitly specified or limited, the terms "disposed," "mounted," "connected," and "connected" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meaning of the above terms in the present invention can be understood in specific cases to those skilled in the art.
The protein fermentation liquor pretreatment device for the recombinant escherichia coli expression system comprises a buffer solution storage container, a fermentation liquor storage container capable of adjusting the temperature, a tangential flow filtration device and a functional storage container;
the buffer solution storage container is communicated with the fermentation liquor storage container with the adjustable temperature through a first pipeline; a first pump body is arranged on the first pipeline;
a second pipeline and a return pipeline are communicated between the fermentation liquor storage container with the adjustable temperature and the tangential flow filtration device; a second pump body is arranged on the second pipeline;
the tangential flow filtration device is communicated with the functional storage container through a third pipeline;
and an ultrafiltration membrane pack is arranged in the tangential flow filtration device.
The processing device combines the characteristics of high-heat-stability protein, assembles a plurality of common experimental devices together, and can sequentially and continuously complete the buffer solution replacement of fermentation liquor, the high-temperature cell disruption and the supernatant/inclusion body collection containing target protein (the inclusion body protein needs to be collected by one step of centrifugation), thereby simplifying the flow and improving the efficiency. The device has the advantages of easily available materials, convenient assembly and simple amplification, and is suitable for industrial amplification production of specific products.
In one embodiment, the first pipeline, the second pipeline, the third pipeline and the return pipeline are made of silicone tubes respectively. In one embodiment, the first and second pumps are each peristaltic pumps. The specification of the peristaltic pump is adapted to the test scale; the first pipeline and the second pipeline are matched with the peristaltic pump. The flow rate of the buffer solution is controlled by the first pump body. The sampling flow rate and the inlet end pressure of the tangential flow filtration device are controlled by the second pump body. In one embodiment, the peristaltic pump is a Lange BT300-2J peristaltic pump on a bench scale.
In one embodiment, the buffer storage container comprises at least one of a beaker, a storage bottle, a storage bag, a stainless steel barrel, and a liquid reservoir. In one embodiment, the buffer storage container may be a PETG bottle. The specification of the buffer storage container is suitable for the experimental scale. In one embodiment, in a bench scale, the buffer storage vessel is a 5L beaker.
In one embodiment, the temperature-adjustable fermentation broth storage vessel comprises a storage vessel body structure and an adjustable temperature stirrer. In one embodiment, the storage vessel body structure comprises at least one of a beaker, a storage bottle, and a fermentor. In one embodiment, the adjustable temperature mixer includes a temperature control assembly and a mixing assembly. The specification of the storage container body structure is adapted to the experimental scale; in one embodiment, the storage vessel body configuration is a 2L scale fermentor on a pilot scale. In one embodiment, when the storage container body is a beaker, the pipeline connected with the beaker is fixed.
In one embodiment, the tangential flow filtration device is a Pall corporation Centramate TM Provided is a system. In one embodiment, the ultrafiltration membrane is one having a molecular weight cut-off of greater than or equal to 1000KD, which can be 1050KD, 1100KD, 1200KD, 1250KD, etc., e.g., 1000-1300 KD. In one embodiment, the ultrafiltration membrane package has a membrane area of0.09m 2 The molecular weight cut-off is 1000KD.
In one embodiment, the functional storage container comprises a waste fluid storage container and/or a sample collection container. The waste reservoir and sample collection containers are replaced according to different test phases. In one embodiment, the waste fluid storage container comprises at least one of a beaker, a storage bottle, a storage bag, a stainless steel drum, and a liquid storage tank, and the waste fluid can be directly discarded if the waste fluid meets the discharge standard. In one embodiment, the sample collection container comprises at least one of a beaker, a storage bottle, a storage bag, a stainless steel bucket, and a liquid reservoir. The number and volume of the waste liquid storage containers and the sample collection containers can be adjusted according to the test silicon film. In one embodiment, the number of the waste liquid storage containers and the number of the sample collection containers are one or more. In one embodiment, the waste storage vessel is a 5L beaker on a bench scale. In one embodiment, the sample storage container is a 5L beaker on a bench scale.
In one embodiment, the liquid inlet port of the first pipeline extends into the buffer storage container and is close to the bottom surface; can adopt arbitrary fixed mode to fix the feed liquor port of first pipeline to guarantee to carry out good imbibition process. The top or the upper end of the container body structure of the fermentation liquid storage container is provided with a first reserved interface, a second reserved interface and a sampling interface. The tangential flow filtration device is provided with a tangential flow filtration device inlet end, a tangential flow filtration device return end, and a tangential flow filtration device permeate end. The first reserved interface is communicated with the buffer solution storage container through a first pipeline; the second reserved connector is communicated with the return end of the tangential flow filtering device through a return pipeline; the sampling interface is communicated with the inlet end of the tangential flow filtration device through a second pipeline, the liquid inlet port of the second pipeline is a fermentation tank sampling tube, and is close to the inner bottom surface of the fermentation liquid storage container with adjustable temperature, namely: the inlet end of the tangential flow filtration device is connected with the inside of the liquid level of the fermentation liquor storage container with adjustable temperature, and the return end is connected with the inside of the fermentation liquor storage container with adjustable temperature. In one embodiment, the tangential flow filtration device permeate end of the tangential flow filtration device is connected to the waste storage container and the sample collection container, respectively, at different stages of the experiment. In one embodiment, the number of the permeation ports of the tangential flow filtration device can be two, the permeation ports are respectively provided with a valve and are respectively connected with two silicone tubes, one of the silicone tubes is connected with the waste liquid storage container, the other silicone tube is connected with the sample collection container, and the waste liquid storage container or the sample collection container is selectively opened according to different experimental stages.
Furthermore, the escherichia coli expression protein suitable for the device has good thermal stability within the range of less than or equal to 80 ℃. The production scale can linearly amplify the device by equal volume according to the volume of a sample to be processed.
The following is further illustrated with reference to specific examples.
Example 1
The protein fermentation liquor pretreatment device for the recombinant escherichia coli expression system comprises a buffer solution storage container 1, a fermentation liquor storage container 3 with adjustable temperature, a tangential flow filtration device 5 and a functional storage container 22.
The fermentation liquid storage container 3 with the adjustable temperature comprises a fermentation tank 21 and an adjustable temperature stirrer, wherein the adjustable temperature stirrer comprises a temperature control component 9 and a stirring component 10; the top end of the fermentation tank 21 is provided with a first reserved interface 11, a second reserved interface 12 and a sampling interface 13;
the tangential flow filtration device 5 comprises a tangential flow filtration device inlet end 14, a tangential flow filtration device return end 15, and a tangential flow filtration device permeate end 16;
the buffer solution storage container 1 is communicated with a first reserved interface 11 of a fermentation tank 21 through a first pipeline 17; a first peristaltic pump 2 is arranged on the first pipeline 17; a sampling connector 13 of the fermentation tank 21 is communicated with an inlet end 14 of the tangential flow filtration device 5 through a second pipeline 18, a second reserved connector 12 of the fermentation tank 21 is communicated with a return end 15 of the tangential flow filtration device 5 through a return pipeline 19, and a liquid inlet port of the second pipeline 18 is close to the inner bottom surface of the fermentation liquid storage container 3 with adjustable temperature; a second peristaltic pump 4 is arranged on the second pipeline 18; the tangential flow filtration device permeate end 16 of the tangential flow filtration device 5 is in communication with the functional storage vessel 22 via a third conduit 20;
an ultrafiltration membrane pack 6 is arranged inside the tangential flow filtration device 5, and the molecular weight cut-off of the ultrafiltration membrane pack 6 is greater than or equal to 1000KD;
the buffer solution storage container 1 is a beaker;
the functional storage container comprises a waste liquid storage container 7 and a sample collection container 8;
the waste liquid storage container is a beaker; the sample collection container is a beaker.
Example 2
The pretreatment method of the protein fermentation liquor of the recombinant escherichia coli expression system comprises the following steps:
(a) Installation of equipment
After the fermentation culture is finished, the aeration operation is cancelled, and the electrodes and pipelines irrelevant to the subsequent test are removed, so that the air pressure inside and outside the fermentation tank 21 is ensured to be consistent. Only the temperature electrode, the temperature control assembly 9 and the cooling pipeline are reserved. The device was connected prior to the experiment according to the reference figures 1, 2 and 3. First, a 5L buffer storage vessel 1 (beaker) containing a formulated resuspension buffer was connected to a 2L fermentor 21 containing fermentation broth via a first line 17. One end of the first pipe 17 is extended to the bottom of the 5L buffer storage vessel 1 and fixed, and the other end is connected to the fermentation tank 21 through the first reserved port 11 at the top of the fermentation tank 21. The first line 17 controls the flow rate of the solution by means of a first peristaltic pump 2 of the BT300-2J type. The 1000KD ultrafiltration membrane package 6 is installed and fixed in the tangential flow filtration device 5 (the cleaning process is completed before the membrane package is used), the second pipeline 18 is connected to the bottom of the fermentation liquid through the top sampling interface 13 of the buffer storage container 1, the return pipeline 19 is connected into the fermentation tank 21 through the second reserved interface 12 at the top of the fermentation tank 21, the tangential flow filtration device 5 is connected into the waste liquid storage container 7 (5L beaker) or the sample collection container 8 (5L beaker) through the third pipeline 20 at the end, and the sample storage container and the waste liquid storage container are replaced according to different test stages.
(b) Buffer replacement of fermentation broth
The purpose of this step is to remove the culture medium in the fermentation broth, replace it with a heavy suspension buffer solution, and complete concentration, buffer solution exchange and thallus washing by the tangential flow filtration device 5, including two stages of concentration and solution exchange. Before the test, the temperature of the tank body is controlled to be less than or equal to 18 ℃ through the temperature control assembly 9, and the stirring speed of 100RPM is maintained through the stirring assembly 10. And in the concentration stage, the second peristaltic pump 4 is started, the tangential flow filtration device 5 is started, the TMP (trans-module pressure) range is controlled to be 0.3-0.5 bar, the fermentation liquor in the fermentation tank 21 is concentrated by 5 times, and the fermentation liquor is collected through the waste liquor storage container 7 at the permeation end. And in the liquid changing stage, starting the first peristaltic pump 2, adding the heavy suspension buffer solution in the buffer solution storage container 1 into the fermentation tank 21, controlling the flow rate to be consistent with the flow rate of the permeation end of the tangential flow filtration device 5 through the first peristaltic pump 2, and carrying out liquid changing with a constant volume of 7 times to clean the bacterial cells. After the step is finished, the first peristaltic pump 2 and the second peristaltic pump 4 are closed, and the permeation liquid is discarded.
(c) Thermal treatment
The purpose of this step is to heat-treat the E.coli cells concentrated and washed in the previous step to release the protein of interest and precipitate most of the host cell proteins. First, the agitation speed in the fermentation tank 21 was controlled to 350RPM, the first peristaltic pump 2 was operated to add the resuspended buffer in the buffer storage container 1 to the fermentation tank 21, and the cell after the liquid exchange was diluted 5 times by volume. The temperature of the bacterial cell suspension in the fermentation tank 21 is then raised to the target temperature (. Ltoreq.80 ℃) within 60 minutes and maintained for. Ltoreq.5 minutes, and then the temperature in the fermentation tank 21 is cooled to the target temperature (. Ltoreq.25 ℃) within about 60 minutes. The step is carried out while the ultrafiltration membrane forming module is cleaned.
(d) Collection of target protein
The purpose of the step is to remove most cell fragments and precipitated host proteins generated after heating the thalli, complete primary collection of target proteins and prepare for subsequent purification, and the steps comprise two stages of thalli cell suspension concentration and percolation. First, the temperature of the fermentation tank 21 is controlled to 18 ℃ or less by the temperature control unit 9 and the stirring speed of 100RPM is maintained by the stirring unit 10. The tangential flow filtration device inlet end 14 and the tangential flow filtration device return end 15 are reconnected to the fermenter 21 tube top first sampling port 13, second reserved port 12, respectively, and the permeate ends into a new sample collection vessel 8. And in the concentration stage, the second peristaltic pump 4 is started, the tangential flow filtration device 5 is started, the TMP (trans-module pressure) range is controlled to be 0.2-0.8 bar, the thallus cell suspension in the fermentation tank 21 is concentrated by 5 times, and the permeation end is collected through the sample collection container 8. And in the infiltration stage, the first peristaltic pump 2 is started, the heavy suspension buffer in the buffer storage container 1 is added into the fermentation tank 21, the flow rate is controlled by the first peristaltic pump 2 to be consistent with the flow rate of the permeation end of the tangential flow filtration device 5, and 5 times of constant volume infiltration is carried out. After the test is finished, the collected liquid can be directly purified, and can also be subpackaged into liquid storage bags at the temperature of between 15 ℃ below zero and 20 ℃ below zero for freezing storage for later use.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications or substitutions do not depart from the scope of the invention in its corresponding aspects.
Claims (10)
1. The protein fermentation liquor pretreatment device for the recombinant escherichia coli expression system is characterized by comprising a buffer solution storage container, a fermentation liquor storage container capable of adjusting temperature, a tangential flow filtration device and a functional storage container;
the buffer solution storage container is communicated with the fermentation liquor storage container with the adjustable temperature through a first pipeline; a first pump body is arranged on the first pipeline;
a second pipeline and a return pipeline are communicated between the fermentation liquor storage container with the adjustable temperature and the tangential flow filtration device; a second pump body is arranged on the second pipeline;
the tangential flow filtration device is communicated with the functional storage container through a third pipeline;
and an ultrafiltration membrane pack is arranged in the tangential flow filtration device.
2. The apparatus of claim 1, wherein the buffer storage container comprises at least one of a beaker, a storage bottle, a storage bag, a stainless steel barrel and a liquid storage tank.
3. The apparatus of claim 1, wherein the temperature-adjustable fermentation broth storage container comprises a storage container body structure and a temperature-adjustable stirrer.
4. The apparatus of claim 3, wherein the storage vessel comprises at least one of a beaker, a storage flask, and a fermentor;
the adjustable temperature stirrer comprises a temperature control assembly and a stirring assembly.
5. The pretreatment device of protein fermentation broth for recombinant Escherichia coli expression system of claim 3, wherein the storage vessel body structure is a storage bottle or a fermentation tank; the top end of the storage container body structure is provided with a first reserved interface, a second reserved interface and a sampling interface;
the tangential flow filtration device comprises a tangential flow filtration device inlet end, a tangential flow filtration device return end and a tangential flow filtration device permeate end;
the first reserved connector is communicated with the first pipeline;
the second reserved connector is communicated with the return end of the tangential flow filtering device through the return pipeline;
the sampling connector is communicated with the inlet end of the tangential flow filtration device through the second pipeline;
the tangential flow filtration device permeate end is in communication with the functional storage container.
6. The apparatus of claim 5, wherein the inlet of the second pipeline is close to the bottom of the storage container.
7. The pretreatment device of protein fermentation broth for recombinant Escherichia coli expression system of claim 1, wherein the ultrafiltration membrane is an ultrafiltration membrane with molecular weight cut-off greater than or equal to 1000KD.
8. The apparatus of claim 1, wherein the functional storage container comprises a waste liquid storage container and/or a sample collection container.
9. The apparatus of claim 8, wherein the waste liquid storage container comprises at least one of a beaker, a storage bottle, a storage bag, a stainless steel barrel and a liquid storage tank;
the sample collection container includes at least one of a beaker, a storage bottle, a storage bag, a stainless steel bucket, and a liquid storage tank.
10. The apparatus of claim 8, wherein the number of the waste liquid storage containers and the number of the sample collection containers are one or more.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117417814A (en) * | 2023-12-18 | 2024-01-19 | 中国海洋大学 | Full-automatic virus extraction system and extraction method |
| CN117417814B (en) * | 2023-12-18 | 2024-04-12 | 中国海洋大学 | Full-automatic virus extraction system and extraction method |
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