CN213012655U

CN213012655U - Inclusion body renaturation intensive device

Info

Publication number: CN213012655U
Application number: CN202022196233.7U
Authority: CN
Inventors: 朱希灿
Original assignee: Beijing Zhongyuan Heju Biotechnology Co ltd
Current assignee: Beijing Zhongyuan Heju Biotechnology Co ltd
Priority date: 2020-09-29
Filing date: 2020-09-29
Publication date: 2021-04-20
Anticipated expiration: 2030-09-29

Abstract

The utility model discloses an intensive device of inclusion body renaturation, renaturation liquid pump setting gets into after being connected with the denaturization liquid pump and strains in the filter core of a section of thick bamboo, in the time of the clarification particulate matter, the albumen after diluting is kept in and has been prolonged renaturation time in straining a section of thick bamboo, renaturation albumen after the clarification filters out the pump control through filtering out the pump control and gets into ultrafiltration circulating pump concentration and ultrafiltration stock solution and mix the back and get into the ultrafiltration storage bag through getting into behind the hollow fiber ultrafiltration column, the hollow fiber ultrafiltration column still sets up and is connected with the ultrafiltration effusion pump. The utility model discloses can enlarge the inclusion body renaturation intensive device that is fit for diluting 1000L pilot scale and production scale, the renaturation inclusion body is diluted in the controllable proportion, and clarification concentration renaturation does have active target protein, gets rid of renaturation failure inclusion body granule, host DNA and thallus piece granule simultaneously.

Description

Inclusion body renaturation intensive device

Technical Field

The utility model relates to an inclusion body renaturation intensive technical field especially relates to, an inclusion body renaturation intensive device.

Background

The recombinant protein is expressed by the escherichia coli, and the method has the advantages of high growth speed, high expression yield, large production capacity and low cost; however, most of the recombinant proteins expressed by the escherichia coli are in the form of inclusion bodies, and when the inclusion bodies are efficiently expressed by the escherichia coli, the newly-generated polypeptides in cells are too high in concentration and cannot be correctly folded into natural structures in time, so that high-density insoluble protein particles are formed. The inclusion body is inactive protein, so it is necessary to use denaturant such as guanidine hydrochloride/urea to dissolve the inclusion body, called denaturation, and then add renaturation liquid to fold correctly in the controlled renaturation environment to recover the protein activity, called renaturation. Renaturation of inclusion bodies: there are two forms of inclusion body renaturation: the renaturation is diluted, a large amount of liquid is required to be diluted by hundreds of times, the volume is increased greatly, and the renaturation protein concentration is low; the other uses dialysis or ultrafiltration to remove denaturants. Dialysis is suitable for small-volume renaturation in a laboratory and cannot be amplified; ultrafiltration renaturation is a method commonly used in the existing production process, but if precipitation of renaturation failure exists in the renaturation process, the ultrafiltration method is easy to generate aggregation denaturation on a membrane (aggregation is caused by irreversible denatured protein, cell debris and host DNA), and the membrane is blocked. And (3) dilution renaturation: directly adding water or buffer solution, and standing overnight, with the disadvantages of large volume increase and diluted protein concentration after renaturation; the large volume of mixing does not lead to the denaturant removal process being unstable, difficult to control, and easy to have high local concentration and generate aggregation and precipitation. The inclusion body renaturation use stage proportion dilution renaturation of industrial scale is limited by the volume limitation of low temperature and huge dilution tank, can only use small multiple, step-by-step dilution renaturation, can not use 1: the 100-time efficient dilution renaturation method influences the recovery of active protein.

Accordingly, the prior art is deficient and needs improvement.

SUMMERY OF THE UTILITY MODEL

The utility model provides an inclusion body renaturation intensive device has solved the problem that exists among the prior art.

The technical scheme of the utility model as follows: an inclusion body renaturation intensive device, the renaturation liquid pump setting of the storage renaturation liquid links with denaturant liquid pump of the storage inclusion body denaturalization liquid, after mixing denaturant inclusion body and renaturation liquid of the same volume, the denaturant inclusion body begins to fold and enters the filter core in the filter cartridge after entering the renaturation liquid, will filter the renaturation liquid, filter the impurity in the renaturation liquid, while clarifying the particulate matter, the protein after diluting is kept in the filter cartridge temporarily and lengthened the renaturation time; the clarified renaturation protein is controlled by a filtering pump to filter out speed, filtrate enters a hollow fiber ultrafiltration column through an ultrafiltration circulating pump to be concentrated and then enters an ultrafiltration storage bag to be subjected to closed-loop circulating ultrafiltration, and an ultrafiltration outlet pump is arranged at the outlet end of the hollow fiber ultrafiltration column to control the ultrafiltration outlet speed.

In the above, the filtering and filtering pump and the ultrafiltration discharge pump are double-pump head pumps, and the filtering and filtering pump and the ultrafiltration discharge pump are controlled to have the same speed and are driven by a motor.

In the above, the renaturation liquid pump and the denaturant liquid pump respectively use two peristaltic pumps to mix the denatured inclusion body fluid and the renaturation liquid.

In the filter cartridge, the filter element is arranged in the center of the filter cartridge, the bottom of the filter element is provided with a sealing ring, the filter element is inserted into the central socket of the filter cartridge, and the sealing ring at the bottom of the filter cartridge is provided with a clamping hoop for clamping connection.

In the above, the upper part of the device shell is also provided with a pressure gauge and a release valve for adjusting the internal pressure.

In the above, the hollow fiber ultrafiltration column has 10Kd and inner diameter of fiber of 0.5mm, and the hollow fiber column has a shell and a stainless steel jacket for membrane-packed ultrafilter is omitted.

In the above, the hollow fiber column had a fiber internal volume of 660 ml.

Adopt the technical scheme of the utility model, can enlarge the inclusion body renaturation intensive device that is fit for diluting 1000L pilot scale and production scale, the renaturation inclusion body is diluted in proportion controllable, and clarification concentration renaturation does have active target protein, gets rid of renaturation failure inclusion body granule, host DNA and thallus piece granule simultaneously. The intensive design energy renaturation ultrafiltration 1000L system can be placed into a laboratory double-door chromatography refrigerator.

Drawings

Fig. 1 is a schematic view of an intensive apparatus according to an embodiment of the present invention.

Detailed Description

In order to facilitate understanding of the present invention, the present invention will be described in more detail with reference to the accompanying drawings and specific embodiments. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Example one

The utility model provides an inclusion body renaturation intensive device, as shown in figure 1, renaturation liquid pump 1 setting of the renaturation liquid of storage in figure 1 is connected with the denaturization liquid pump 2 of storage inclusion body denaturalization liquid, after denaturization inclusion body and the renaturation liquid with the same volume mix, in the filter core 9 that the denaturization inclusion body began folding entering to strain in the section of thick bamboo 8 after getting into renaturation liquid, will filter renaturation liquid, the impurity in the filtration renaturation liquid, including thallus piece, host DNA and the not totally insoluble inclusion body granule nature impurity of renaturation, renaturation liquid is further correctly folded renaturation in straining a section of thick bamboo 8 simultaneously, when the clarification particulate matter, the renaturation time has been prolonged in straining a section of thick bamboo 8 to the protein after diluting; the filtering speed of the clarified renaturation protein is controlled by a filtering pump 3, the filtrate enters a hollow fiber ultrafiltration column 6 through an ultrafiltration circulating pump 4 and is concentrated, and then enters an ultrafiltration storage bag 7 for closed loop circulating ultrafiltration, and an ultrafiltration discharge pump 5 is arranged at the discharge end of the hollow fiber ultrafiltration column 6 to control the ultrafiltration discharge speed.

Furthermore, the speed pumped by the end ultrafiltration permeation pump 5 is used as a speed control marker post of the integrated continuous system, the rear end is used for ultrafiltration to remove a large amount of waste liquid, the front end of the device enters mixed inclusion body denatured liquid and renaturation liquid with the same volume, the denatured inclusion body enters the renaturation liquid and then begins to fold, the correctly folded inclusion body forms dissolved active protein, particles still precipitate without correct folding, turbid renaturation liquid enters the filter cylinder 8, the renaturation liquid after filtration and clarification enters the ultrafiltration system through the ultrafiltration circulating pump 4 for concentration, and the volume of the active protein is reduced (can be concentrated to 1/10 of the original modified volume). Because the device is small enough to put into 4 degrees refrigerator-freezers, guaranteed the good protein activity of renaturation, there is no restriction to the duration of production, the system can dilute renaturation inclusion body constantly in succession, and concentrate active protein, the system handles renaturation liquid volume and can far exceed 1000L.

The above-mentioned clear particles are formed by mixing denatured liquid and renaturation liquid, and then it takes a short time for denatured protein to change its structure, and the denatured protein is bent and folded into an active three-dimensional structure, including disulfide bond and hydrogen bond, from its linear structure. Some denatured proteins do not fold into the native active structure during folding of the protein structure, and misfolding into inactive insoluble contaminants requires filtration of insoluble contaminant particles by a filter.

In the above, because the ultrafiltration speed is far slower than the filter speed of the filter element, in order to keep the balance of the inlet and outlet liquid of the ultrafiltration system and improve the ultrafiltration effect, the control filtration pump 3 and the ultrafiltration pump 5 have the same speed, and the filtration pump 3 and the ultrafiltration pump 5 adopt double-pump-head pumps driven by a motor to ensure the same speed of the liquid entering and exiting the ultrafiltration system. The sum of the speed of the renaturation liquid pump 1 and the speed of the denaturation liquid pump 2 is equal to the speed of the filtering-out pump 3 and the speed of the ultrafiltration permeation pump 5 is equal, and the flux balance of the system is ensured.

The renaturation liquid pump 1 and the denaturation liquid pump 2 use double-pump constant-current controllable large-proportion mixed denaturation liquid/diluent, and the denaturation liquid/diluent is completely mixed in the filter cylinder 8, so that the problems of non-uniform pouring dilution mixing proportion and poor process repeatability of a direct dilution renaturation method are solved. Renaturation liquid pump 1 and denaturant liquid pump 2 use two peristaltic pumps respectively with inclusion denaturant liquid and renaturation liquid pump after-mixing, and denaturant liquid pump 2 speed is 1 renaturation liquid pump 1 speed 99, guarantees 1: and (3) mixing and renaturating at a ratio of 100.

The filter cartridge 8 and the filter element 9 in the filter cartridge 8, the filter element 9 is a folding filter element for filtering, clarifying and filtering impurities in renaturation liquid, preventing an ultrafiltration column from being blocked, and comprises thallus fragment host DNA and renaturation incomplete insoluble inclusion body granular impurities, meanwhile, the renaturation liquid is further correctly folded and renatured in the filter, and when the particles are clarified, the diluted protein is temporarily stored in the filter cartridge to prolong the renaturation time. An inlet at one side of the bottom of the filter cylinder 8 and an outlet at the center of the bottom, a filter element is arranged at the center of the filter cylinder, a sealing ring is arranged at the bottom of the filter element, the filter element is inserted into a socket at the center of the filter cylinder, the socket seals the filter element to separate before and after filtration, and a pressure gauge and a vent valve are arranged at the upper part of the shell and used for adjusting the internal pressure; the sealing ring at the bottom of the filter cylinder is provided with a clamping hoop for clamping connection.

The ultrafiltration concentration process is carried out on an ultrafiltration column, the liquid flows through a hollow fiber tube, the pressure in the tube is high, small molecules in the liquid permeate through a membrane, the volume of the liquid in the hollow fiber tube is reduced and is equal to concentration, the concentrated liquid flows back into a liquid storage container 7 from the upper part of the hollow fiber tube, the hollow fiber ultrafiltration column 6 has the Kd of 10Kd, the inner diameter of the fiber is 0.5mm, and the area of the hollow fiber membrane for treating 1000L renaturation liquid is 2.5m²The volume in the fiber is 660ml, redundant renaturation liquid can be removed through ultrafiltration concentration, the concentration of active protein is improved, the volume of target protein is 1/10 before renaturation, a hollow fiber column with a shell omits a stainless steel clamping sleeve of a membrane-covered ultrafilter, and the space and the cost are saved; the inside of the fiber tube of the hollow fiber ultrafiltration column 6 is through, even if fine particles exist, the blockage can not occur (the volume of the inner fiber of the hollow fiber column 6 is only 660 ml); the device with the processing capacity of 1000L can be placed in a low-temperature cabinet with the temperature of 4 ℃, so that the low-temperature environment of the inclusion body renaturation process is ensured, and the device is suitable for pilot-scale test and amplification production in a laboratory.

The inclusion body renaturation of the utility model is a very complicated process and is restricted by a plurality of factors. Factors such as the concentration of various reagents and proteins, the operation temperature and the renaturation time, the components of a buffer solution, the pH value and the like in the experiment play a crucial role in the experiment result. The utility model discloses intensive renaturation system in succession, each factor is mastered to the accuracy, can repeatedly obtain stable high yield's active protein.

The technical scheme of the utility model is further explained by the following experimental data:

inactive recombinant inclusion body protein generated by the induced expression of the escherichia coli producing strain is dissolved in 10 liters of denatured liquid urea for denaturation and then is diluted and renatured in a large proportion. The protein concentration, renaturation temperature, pH value, renaturation buffer components and reducing agent concentration all have an influence on the renaturation effect of target protein. Specifically, the method comprises the following steps: as shown in fig. 1, the renaturation liquid pump 1 and the denaturant liquid pump 2 are connected after being pumped, and the modified inclusion body 1: 100, diluting and renaturing the mixture, then putting the mixture into a filter cylinder 8, filtering impurities in renaturation liquid by a filter element 9, wherein the impurities comprise thallus fragment host DNA and renaturation incomplete insoluble inclusion body granular impurities, simultaneously, the renaturation liquid is further correctly folded and renatured in the filter cylinder 8, and the renaturation time is prolonged by temporarily storing the diluted renaturation protein in the filter cylinder 8 while clarifying the granular matters; the clarified renaturation protein enters an ultrafiltration system by controlling the filtering speed through a filtering pump 3, filtrate and an ultrafiltration stock solution are mixed and then enter a hollow fiber ultrafiltration column 6 for concentration, and then enter an ultrafiltration storage tank 7, and the hollow fiber ultrafiltration column 6 is also provided with an ultrafiltration permeation pump 5 for controlling the permeation speed of the ultrafiltration system.

Dissolving 1000g of inclusion body protein in 10 liters of 7mol/L urea with the pH value of the denaturant liquid at 4 ℃ of 9.5, and forming a denaturant inclusion body storage liquid containing 5mmol/L beta-mercaptoethanol to be connected with a denaturant liquid pump 2; preparing purified water renaturation liquid with the room temperature of 25 ℃ and the pH value of 8.0 outside the refrigerator, and connecting the purified water renaturation liquid with the interior renaturation liquid pump 1 of the refrigerator; the pump speed of the denaturant pump 2 is adjusted to be 12.5ml/min, and the pump speed of the renaturation pump 1 is adjusted to be 1237.5 ml/min;

the filter element 9 in the filter cylinder 8 is a 20-inch 0.22um folded filter element, and the filtering speed is controlled to 1250ml/min by the filtering pump 3;

the hollow fiber ultrafiltration column 6 has 10Kd 2.5M²The volume of the inner cylinder with the fiber inner diameter of 0.5mm is 370ml, the ultrafiltration circulation speed of the ultrafiltration circulation pump 4 is 10L/min, the volume of the ultrafiltration storage tank is 7L, the volume of the ultrafiltration discharge pump 5 is 30L, the discharge speed of the ultrafiltration discharge pump 5 is 1250ml/min, and the discharge speed of the hollow fiber ultrafiltration column 6 and the filtration speed of the filter element 9 (the feed supplement speed of the ultrafiltration discharge pump 5 and the filtration pump 3) are both controlled by the double pump heads of the ultrafiltration discharge pump 5 and the filtration pump 3 to be 1250 ml/min. Diluting and renaturing in a freezer after 14 hours, filtering and clarifying, ultrafiltering and concentrating to obtain the highest renaturation activity, wherein the activity yield is over 80 percent, and finally, the target recombinant protein concentrated to 5 liters is obtained. A large-scale dilution renaturation method suitable for pilot plant test and scale-up production is established.

Adopt the technical scheme of the utility model, can enlarge the inclusion body renaturation intensive system who is fit for diluting 1000L pilot scale and production scale, the renaturation inclusion body is diluted in proportion controllable, and clarification concentration renaturation does have active target protein, gets rid of renaturation failure inclusion body granule, host DNA and thallus piece granule simultaneously. The intensive design energy renaturation ultrafiltration 1000L system can be placed into a laboratory double-door chromatography refrigerator.

It should be noted that the above technical features are continuously combined with each other to form various embodiments which are not listed above, and all the embodiments are regarded as the scope of the present invention described in the specification; moreover, modifications and variations will occur to those skilled in the art in light of the foregoing description, and it is intended to cover all such modifications and variations as fall within the true spirit and scope of the invention as defined by the appended claims.

Claims

1. An inclusion body renaturation intensive device is characterized in that a renaturation liquid pump for storing renaturation liquid is connected with a denatured liquid pump for storing inclusion body denatured liquid, denatured inclusion bodies and renaturation liquid with the same volume are mixed and then enter a filter element in a filter cylinder, clarified renaturation protein enters a hollow fiber ultrafiltration column through a filtering pump filtrate through an ultrafiltration circulating pump and then is concentrated, and then enters an ultrafiltration storage bag for closed-loop circulating ultrafiltration, and an ultrafiltration outlet end of the hollow fiber ultrafiltration column is provided with an ultrafiltration outlet pump for controlling the ultrafiltration outlet speed; the hollow fiber ultrafiltration column is provided with a shell.

2. The inclusion body renaturation and enrichment facility as claimed in claim 1, wherein the filtration pump and the ultrafiltration permeate-out pump are double-pump head pumps, and the filtration pump and the ultrafiltration permeate-out pump are controlled to have the same speed and are driven by a motor.

3. The inclusion body renaturation and enrichment facility as claimed in claim 2, wherein the renaturation liquid pump and the denaturant liquid pump respectively use two peristaltic pumps to mix the denatured inclusion body fluid and the renaturation liquid after being pumped.

4. The inclusion body renaturation and accumulation means as claimed in claim 3, wherein said filter cartridge has an inlet on one side of the bottom and an outlet at the center of the bottom, a filter element in the center of the cartridge, a gasket on the bottom of the filter element, the filter element being inserted into the central socket of the cartridge, and a clamp on the gasket on the bottom of the cartridge for clamping the connection.

5. The inclusion body renaturation and intensification device of claim 4, wherein a pressure gauge and a release valve are further provided at the upper part of the cartridge housing for adjusting the internal pressure.

6. The inclusion body renaturation and accumulation means as claimed in claim 5, wherein said hollow fiber ultrafiltration column has 10Kd and inner diameter of fiber of 0.5mm, and the hollow fiber column has a housing and a stainless steel jacket for membrane-packed ultrafiltration is omitted.

7. The inclusion body renaturation and accumulation means of claim 6 wherein the hollow fiber column fiber internal volume is 660 ml.