CN112552392A - Purification method of recombinant Exendin-4 polypeptide - Google Patents
Purification method of recombinant Exendin-4 polypeptide Download PDFInfo
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Abstract
The invention provides a purification method of a recombinant Exendin-4 polypeptide, which comprises the following steps: 1) carrying out affinity chromatography I on the crude recombinant Exendin-4 polypeptide to obtain an affinity chromatography solution I; 2) carrying out ultrafiltration on the affinity chromatography solution I to obtain an ultrafiltrate; 3) carrying out enzymolysis on the ultrafiltrate to obtain an enzymolysis liquid; 4) performing affinity chromatography II and anion exchange chromatography I on the enzymatic hydrolysate to obtain anion exchange chromatography solution I; 5) carrying out reverse phase chromatography on the anion exchange chromatography liquid I to obtain reverse phase chromatography liquid; 6) and (4) carrying out anion exchange chromatography II on the reversed-phase chromatography liquid to obtain anion exchange chromatography II liquid. An ultrafiltration process is added before the enzymolysis of the fusion protein, so that impure proteins such as uncrushed thallus residues in the bacteria-breaking clarified liquid are removed; the method improves the purity and recovery rate of target protein.
Description
Technical Field
The invention relates to the field of product purification, in particular to a purification method of a recombinant Exendin-4 polypeptide.
Background
Chinese patent document CN1635117A discloses a new process for preparing recombinant Exendin-4 polypeptide, wherein a purification method of recombinant Exendin-4 polypeptide is disclosed, comprising: affinity chromatography-enzymolysis protein-affinity chromatography-reversed phase chromatography-ion exchange chromatography, however, the scale, purity and yield of the recombinant Exendin-4 polypeptide protein obtained by the purification method are all required to be further improved, so that the commercial production is carried out.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a method for purifying the recombinant Exendin-4 polypeptide, and the purity and recovery rate of the recombinant Exendin-4 polypeptide produced by the method are high.
A purification method of a recombinant Exendin-4 polypeptide comprises the following steps:
1) carrying out affinity chromatography I on the crude recombinant Exendin-4 polypeptide to obtain an affinity chromatography solution I;
2) carrying out ultrafiltration on the affinity chromatography solution I to obtain an ultrafiltrate;
3) carrying out enzymolysis on the ultrafiltrate to obtain an enzymolysis liquid;
4) performing affinity chromatography II and anion exchange chromatography I on the enzymatic hydrolysate to obtain anion exchange chromatography solution I;
5) carrying out reverse phase chromatography on the anion exchange chromatography liquid I to obtain reverse phase chromatography liquid;
6) and (4) carrying out anion exchange chromatography II on the reversed-phase chromatography liquid to obtain anion exchange chromatography II liquid.
Optionally, gradient elution is adopted in the elution in the step 5), impurities are eluted by mixing the mobile phase A with the volume percentage of 67% and the mobile phase B with the volume percentage of 33%, and 1 column volume is eluted; then mixing the mobile phase A with the volume percentage of 45% and the mobile phase B with the volume percentage of 55% to elute the target protein; collecting reversed phase chromatographic liquid when the A280 absorption value is 0.4AU, and stopping collecting the reversed phase chromatographic liquid when the A280 absorption value is reduced to 0.4AU after the maximum peak value is reached; the mobile phase A is a solution containing 10mmol/L PB and 10% ethanol by volume, and has a pH of 7.5-8.5 (optionally, a pH of 8.0); the mobile phase B is 80 percent ethanol water solution by volume percentage.
Optionally, the ultrafiltration membrane has a pore size of 5-10 KD.
Optionally, a chromatographic column used in the affinity chromatography I is a nickel ion chelating affinity layer column, the sample loading amount is 2.60g-3.40g of crude pure/mL filler, and Buffer B is adopted for elution to obtain an affinity chromatography solution I; the Buffer B is a solution containing PB and imidazole, and the pH is 7.5-8.5 (the optional pH is 8.0); the concentration of PB is 8mmol/L-12mmol/L (optional 10 mmol/L); the concentration of imidazole is 45mmol/L-55mmol/L (optional 50mmol/L), and the absorption value of an elution peak collection range A280 is 0.3-0.1 AU.
Optionally, a step of gel filtration chromatography is also included;
sephadex G-25Fine is a gel filtration medium;
optionally, in the gel filtration chromatography, Buffer M is used as an eluent, the collection is started when the absorption value of A280 reaches 0.01AU, the maximum peak value is reached, and the collection is stopped when the absorption value of A280 is reduced to 0.01 AU; buffer M is 30mmol/L acetic acid Buffer solution, pH5.5; the elution flow rate was 30 mL/min.
Optionally, the chromatographic column of the reverse phase chromatography is Source 15 RPC; the loading amount is 5 mg/mL-8 mg/mL filler; the elution speed is 22mL/min-26 mL/min; and (3) starting to collect the reverse-phase chromatographic liquid when the A280 is 0.5AU, and stopping collecting the reverse-phase chromatographic liquid when the A280 absorption value is reduced to 0.05AU after the maximum peak value is reached.
Optionally, a chromatographic column used for affinity chromatography II and a chromatographic column used for anion exchange chromatography I are connected in series for sample loading; eluting the target protein by the eluent; the eluent is a solution containing NaCl and Tris-HCl, and the pH value is 7.5-8.5 (the optional pH value is 8.0); the concentration of NaCl is 170mmol/L-180mmol/L (alternatively 175mmol/L), and the concentration of Tris-HCl is 8mmol/L-12mmol/L (alternatively 10 mmol/L); collecting anion exchange chromatography liquid I with A280 absorption value in the range of 0.04AU-0.01 AU; the elution flow rate is 30 mL/min;
or, after loading the sample by the anion exchange chromatography II, fully eluting by using a buffer solution H, eluting by using a buffer solution I, eluting by using a buffer solution J, wherein the elution and elution flow rate is 24mL/min, and collecting the target protein; collecting when the A280 absorption value of the eluent reaches 0.01AU, reaching the peak value, and stopping collecting when the A280 absorption value of the eluent falls to 0.01 AU; the buffer solution H is 10mmol/L PB (phosphate buffer solution), and the pH value is 7.5-8.5; the buffer solution I is 8mmol/L-12mmol/L (optional 10mmol/L) PB (phosphate buffer solution), and the pH value is 6.5; buffer J is used as a solution containing 8mmol/L-12mmol/L (optionally 10mmol/L) PB and 180mmol/L-210mmol/L (optionally 200mmol/L) NaCl, pH 6.5.
The optional enzymolysis system is 1.5mmol/L-1.8mmol/L CaCl21.5mg/mL fusion protein and enterokinase; the addition amount of the enterokinase is 1U enterokinase added into every 2mg-3mg of fusion protein, and the system is more beneficial to enzymolysis of the fusion protein and improves the enzymolysis efficiency.
The optional preparation method of the crude recombinant Exendin-4 polypeptide comprises the following steps:
1) preparing the fermented thalli into a thalli heavy suspension, and homogenizing to obtain a bacteria breaking liquid;
2) centrifuging the bacteria-breaking liquid at 9000-12000 rpm at 4-6 deg.C for more than 45min, and retaining the supernatant; filtering with 0.4-0.5 μm filter to obtain bacteria-breaking clear liquid, i.e. crude pure.
The technical scheme of the invention has the following advantages:
1. a purification method of a recombinant Exendin-4 polypeptide comprises the following steps: 1) carrying out affinity chromatography I on the crude recombinant Exendin-4 polypeptide to obtain an affinity chromatography solution I; 2) carrying out ultrafiltration on the affinity chromatography solution I to obtain an ultrafiltrate; 3) carrying out enzymolysis on the ultrafiltrate to obtain an enzymolysis liquid; 4) performing affinity chromatography II and anion exchange chromatography I on the enzymatic hydrolysate to obtain anion exchange chromatography solution I; 5) carrying out reverse phase chromatography on the anion exchange chromatography liquid I to obtain reverse phase chromatography liquid; 6) and (4) carrying out anion exchange chromatography II on the reversed-phase chromatography liquid to obtain anion exchange chromatography II liquid. An ultrafiltration process is added before the enzymolysis of the fusion protein, so that impure proteins such as uncrushed thallus residues in the bacteria-breaking clarified liquid are removed, the enzymolysis of the fusion protein is facilitated, and the enzymolysis efficiency is improved; the method improves the purity and recovery rate of target protein.
2. The reverse phase chromatography continuous gradient elution section in the existing purification process has wider range, consumes time and has low purity and recovery rate. After optimization, the impurities are eluted by using a mixed gradient of 67% of mobile phase A and 33% of mobile phase B, and 1 column volume is eluted. The target protein is eluted by using the 45% mobile phase A and the 55% mobile phase B in a mixed gradient manner, so that the results show that the stage gradient elution saves time and improves the purity and the recovery rate.
3. Technical parameters in the prior art are not suitable for industrialized production, parameter design and optimization are carried out while the process amplification is completed, the purity and yield of the prepared recombinant Exendin-4 polypeptide are high, the protein purity is higher than 99.4%, and the recovery rate of the protein is more than 90%.
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 description of the embodiments or 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 other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 comparison of the results induced by different lactose concentrations;
FIG. 2 shows 4-hour electrophoretograms induced at different temperatures; in the figure, S1 represents an experiment number, which has no practical significance, and 20080417 and 20080422 are experiment times without other meanings;
FIG. 3 shows an electropherogram at different temperatures for 6 hours; in the figure, S1 represents an experiment number, which has no practical meaning, and 20080417 and 20080422 are experiment times without other meanings;
FIG. 430 ℃ is an electrophoretogram showing the expression level at different times; in the figure, S1 represents an experiment number and has no practical significance;
FIG. 5 electrophoresis chart of different media at 30 degree induction for 6 hours; in the figure, S1 represents an experiment number and has no practical significance
FIG. 6 electrophoretogram of lactose in different modes of addition;
FIG. 7 is a flow diagram of a fermentation process.
Detailed Description
The invention uses the following authorization notice numbers: CN100535111C recombinant Exendin-4 polypeptide was prepared from the engineered bacterium disclosed in example 1 (pET32a (+) -Exendin-4/BL21(DE 3)).
EXAMPLE 1 preparation of recombinant Exendin-4 polypeptide
The general process flow is shown in fig. 7, and the induction concentration, induction temperature and induction time of lactose are determined by experiment exploration in the example. The expression levels of TB, LB and LM9 in the three media were compared with each other using lactose as an inducer. After the selection of TB medium was determined, the effect of lactose addition on fermentation and expression was compared. According to the experimental data, the amplification and the research are carried out in the fermentation tank.
1. Determination of lactose Induction concentration
Inoculating the strain into 250mL shake flask (containing 50mL LB culture medium), and culturing at 37 deg.C to OD600Values 2, lactose addition at 5, 10, 20, 30, 40g/L (final concentration), induction culture for 4 hours sampling test, comparison of the effect of different lactose concentrations on expression, results show: when lactose is added above 30g/L, the expression level is reduced (FIG. 1 a); lactose is added according to 5, 10, 15 and 20g/L respectively for induction, sampling detection is carried out after 4 hours of induction culture, and the result shows that the expression amount of the added lactose is approximate to that of 5-20g/L (figure 1b), and the result of 5g/L lactose is slightly higher, so that the induction concentration of lactose is determined to be 5 g/L.
2. Determination of Induction temperature and Induction time
Inoculating the strain into 250mL shake flask (containing 50mL LB culture medium), and culturing at 37 deg.C to OD600Value 2, lactose was added at a final concentration of 5g/L, the cells were incubated at 25 ℃, 28 ℃, 30 ℃, 32 ℃, 37 ℃ and 40 ℃ respectively, and samples were taken 4 and 6 hours after induction for SDS-PAGE analysis (see FIGS. 2-4), and the semi-quantitative results of SDS-PAGE were statistically analyzed to obtain tables 1 and 2. The results showed that the expression level was highest after 6 hours of induction at 30 ℃ (tables 1 and 2), and therefore, induction culture at 30 ℃ was confirmed. By comparison at 30 ℃ for 4 to 8 hours of induction, expression was instead lower after 6 hoursThe temperature was decreased (Table 3, FIG. 4), and the induction temperature and time were determined to be 30 ℃ for 6 hours.
TABLE 1 lactose Induction of 4 hours expression at different temperatures
The expression amount is expressed as the mass of the fusion protein/the mass of the total bacterial protein × 100%.
TABLE 2-1 lactose induction for 6 hours of expression at different temperatures
TABLE 2 Induction of expression at 230 ℃ for various periods of time
The expression amount is expressed as the mass of the fusion protein/the mass of the total bacterial protein × 100%.
3. Determination of the culture Medium
Inoculating the strain into 250mL shake flasks containing 50mL TB, LB, LM9 culture medium, respectively, and culturing at 37 deg.C to OD600The value 2, adding lactose according to 5g/L, sampling after induction culture for 6 hours at 30 ℃ for SDS-PAGE analysis and biomass determination, repeating the experiment for 4 times, and the result shows that the expression level and biomass of the TB culture medium are higher than those of the other two culture media (table 3 and figure 5), and the TB culture medium is determined to be the TB culture medium because the TB culture medium has a more complete nutritional structure and is suitable for scale-up production.
TABLE 3-1 LM9 Medium
TABLE 3-2 TB Medium
TABLE 3-3 LB Medium
TABLE 3-4 comparison of expression levels in different media
The expression amount is expressed as the mass of the fusion protein/the mass of the total bacterial protein × 100%.
4. Effect of lactose addition on results
Lactose is used as an inducer and also as a carbon source, and when the concentration is excessively high, the growth of thalli and the expression of products are influenced. Therefore, under the condition that the total amount of lactose is not changed, the lactose is added in a plurality of times for expression comparison.
Transferring the strain to TB culture medium, and culturing at 37 deg.C to OD600The culture temperature is 2, the culture is transferred to 30 ℃ for induction culture, lactose is added in 1, 2, 4, 5 and 6 times on average (1 time is all added at the beginning of induction, 2 times are half added after the beginning of induction and three hours after induction, 4 times are 1/4 added every other hour from the beginning of induction and are supplemented in the first 4 hours, 5 times are 1/5 added every other hour from the beginning of induction and are supplemented in the first 5 hours, 6 times are 1/6 added every other hour from the beginning of induction), and the total addition amount of lactose is 5g of lactose added in each liter of TB culture medium. A final concentration of 0.5mM IPTG was added as a parallel control. The above experiment was repeated 2 times, i.e. experiment 1 and experiment 2, with 4 trials of the lactose addition regime, and 4 replicates were performed (see fig. 6a and b). The test results were averaged. The results of the experiment showed that the expression level and biomass were slightly higher in 5-fold average additions (Table 4, FIG. 6). It was therefore established that the induction was carried out with 1/5 volumes of lactose solution added per hour, with the total lactose added being constant, i.e. the induction was completed at hour 4 from the start of the induction.
TABLE 4 comparison of expression levels for different lactose addition modes
Note: the expression amount is the mass of the fusion protein/the mass of the total bacterial protein multiplied by 100 percent;
of these, 4 experiments with lactose addition were performed in 4 replicates (see fig. 6a and b), with a mean of 13.3; 2 of these are listed in the table.
5. Determination of lactose Induction timing
The induction time and the regulation and control of the feeding are further optimized to improve the density and the expression quantity of the bacteria and increase the yield of unit fermentation volume. The main optimization conditions are as follows:
1) according to the growth curve of the strain cultured in 5L fermenter, OD was taken600Inducing the engineering bacteria when the value is 3-5 and 8-12;
2) the content of glycerol in the TB culture medium is increased from 4mL/L to 8 mL/L;
3) feed medium (11.4g/L MgSO4·7H2O、100g/LTrytone100g/L Yeast extract (Yeast extract) and 20% (v/v) glycerol) the effects of 10% (v/v) and 20% (v/v) glycerol on expression and biomass were compared using glycerol as a carbon source.
The results show that: after the content of glycerol is increased in the initial TB culture medium, the early growth speed is accelerated; when inducing the timing OD600The value is 8-12, 20% of glycerol is added into a feed culture medium, and under the condition, the biomass and the expression quantity are relatively high (Table 5).
TABLE 5 comparison of the results of the fermentations in 5L fermentors under different conditions
Note: the expression amount is expressed as the mass of the fusion protein/the mass of the total bacterial protein × 100%.
6. Optimized result of fermentation process
And (3) combining the experimental results to preliminarily determine the fermentation process conditions: the fermentation medium is TB medium (Glycine)Oil volume of 8mL/L), culture temperature of 37 deg.C, pH 7.0, Dissolved Oxygen (DO) value of 30%, and induction time OD600The value is 8-12, the induction temperature is 30 ℃, the induction time is 6 hours, the lactose induction concentration is 5g/L (1/5 is added in each of 0, 1, 2, 3 and 4 hours of induction), and the culture medium is fed (11.4g/L MgSO)4·7H2O, 100g/L tryptone, 100g/L yeast extract, 20% glycerol (v/v)) were supplemented according to the dissolved oxygen and pH changes.
7. Fermentation process
The developed recombinant insulinotropic hormone secretagogue (rExendin-4) fermentation process was subjected to commercial scale studies.
1) First order seed liquid preparation
Taking one working seed, inoculating to 0.75L of first-class seed culture medium (TB culture medium) according to 1 ‰ (v/v) inoculum size, placing in a shaking table, culturing at 37 deg.C and 250rpm for 15-17 h, and measuring OD600Values (see table below).
2) Second stage seed liquid preparation
Transferring the first-class seed culture to a 50L fermentation tank, culturing with a culture medium (TB culture medium) volume of 30L at 37 deg.C under dissolved oxygen of 30% and rotation speed of 250rpm for 2-3 h, and measuring OD600And obtaining a secondary seed culture after the value reaches 3-5.
3) Fermentation of
30L of the secondary seed culture was transferred into a 500L fermenter (medium volume 200L) via a transfer line. The temperature is 37 ℃ plus or minus 2 ℃, the dissolved oxygen is 30 percent (the ventilation and the stirring are coupled), and the pH fluctuation range is 7.00 plus or minus 0.20. OD600And starting induction when the induction time is 8-12, namely supplementing 460mL of supplemented medium and 400mL of lactose solution, simultaneously reducing the culture temperature to 30 +/-2 ℃, supplementing 5 times of 400mL of lactose, supplementing 80mL of lactose each time, completing lactose supplementation within 4-5min each time, and completing the complete supplementation for the first 5 hours from the induction time of 0 hour to ensure that the final concentration of the lactose in the tank is 5 g/L. When the pH value is higher than 7.0 and the rising is continued, the feed liquid is supplemented until the fermentation is finished. Inducing for 5-6 h, OD600When the trend of the value changes to be flat and slow, the fermentation is stopped.
TABLE 6 results of fermentation Process study
The above experimental results show that: the protein expression amount is 14.1-14.6%, and the stable level can be ensured after the process is amplified.
Example 2 purification method of recombinant Exendin-4 polypeptide
Abbreviations: PB refers to phosphate buffer.
1. Coarse purity
10.0. + -. 1.0kg of fermented wet cells having a protein expression level of 10% or more (14.1% in this example, expression level. the amount is expressed as fusion protein mass/total cell protein mass. times.100%) were taken and resuspended in Buffer A (10mmol/L PB, 500mmol/L NaCl, 50mmol/L imidazole, pH8.0) in an amount of 5L per 1kg of cells to prepare a cell resuspension solution. Homogenizing for 3 times (can homogenize for 2-5 times) under 550-600 bar pressure by using a high-pressure homogenizer, wherein the temperature of the homogenized liquid is 30 ℃ (optionally 20-30 ℃) in the homogenizing process to obtain the bacteria breaking liquid.
Centrifuging the lysate solution with high-speed refrigerated centrifuge at 9000rpm and 4 deg.C for 45min, and collecting supernatant. Then, the mixture was filtered through a 0.45 μm filter to obtain a bacteria-removing supernatant.
2. Affinity chromatography I
And (3) performing affinity chromatography I on the bacteria-breaking clarified liquid in the last step, wherein instrument and equipment are shown in the following table. The filler loading capacity of the affinity chromatography I chromatographic column is 80mg/mL (can be 70-90 mg/mL), the chromatographic column is balanced to be stable at a base line by using Buffer A (10mmol/L PB, 500mmol/L NaCl, 50mmol/L imidazole, pH8.0), the sample loading amount is 3.00g (can be 2.60g-3.40g) of bacteria-breaking clarified liquid/mL filler, the polypeptide is eluted by using mobile phase Buffer B (10mmol/L PB, 50mmol/L imidazole, pH8.0), the flow rate of balance and elution are both 30mL/min, the elution peak collection range A280 is 0.3-0.1 AU, and the affinity chromatography liquid I is obtained.
TABLE 7 affinity chromatography instrumentation
The step is the first step of chromatographic purification in the purification process, is specific affinity chromatography, mainly enriches fusion protein in the bacteria-breaking clarified liquid, and the loaded bacteria-breaking clarified liquid has complex components. The results show that under the condition, the purity of the fusion protein in the affinity chromatography liquid I is more than 82 percent.
TABLE 8 summary of affinity chromatography data
3. Ultrafiltration
The affinity chromatography liquid I was diluted with Buffer C (pH8.0, 10mmol/L Tris-HCl) in an equal volume, and then ultrafiltered 7 times with a 5KD ultrafiltration membrane pack to remove imidazole and other substances from the affinity chromatography liquid, and the concentration and purity of the ultrafiltrate were measured by A280, and the results are shown in the following table.
TABLE 9 summary of ultrafiltrate results
4. Enzymolysis of fusion protein
Filtering ultrafiltrate with 0.2 μm filter for sterilization, diluting with Buffer C to final concentration of 1.5mg/mL, and adding CaCl2Is prepared by reacting CaCl2The final concentration is 1.5mmol/L, enzyme digestion liquid is obtained, the pH value of the enzyme digestion liquid is adjusted to 8.0, enterokinase is added according to the proportion of 2.5mg (can be 2.0-3.0 mg) of fusion protein which is cut by 1U enterokinase (Chengdu original valley biotechnology limited Y070), the stirring speed is 50rpm, the enzyme digestion is carried out for 16h at the temperature of 30 ℃, the enzyme cleavage rate is detected to be more than 90% after the enzyme digestion is finished, and the specific results are shown in the following table.
Note: the enzyme cutting ratio is enterokinase/fusion protein, and the ratio relation is U/mg.
5. Affinity chromatography II and anion exchange chromatography I
Removing thioredoxin by utilizing affinity chromatography II and anion exchange chromatography I, and enriching rExendin-4 protein, wherein the purification step is that the affinity chromatography II is connected with the anion exchange chromatography I in series. The apparatus used in affinity chromatography II and anion exchange chromatography I is shown in the following table. After the enzyme digestion, the fusion protein is digested into thioredoxin containing His label and rExendin-4 protein. When the enzyme digestion solution passes through the affinity chromatographic column II, thioredoxin containing His labels is combined on the affinity chromatographic column II, and negatively charged rExendin-4 protein flows through the affinity chromatographic column II, enters the anion exchange chromatography I and is combined with the anion column through the charge effect. The anion exchange chromatography uses an ion exchanger as a stationary phase, and the binding force between the rExendin-4 protein and the hybrid protein is different according to the difference of charges carried by the rExendin-4 protein and the hybrid protein in a mobile phase. When the method is used for elution by using counter ions, the hybrid protein with less negative charges than rExendin-4 protein is eluted first and is separated from the target protein; the hybrid protein with more negative charges than the rExendin-4 protein is eluted after the rExendin-4 protein is separated from the target protein, thereby achieving the effect of improving the purity of the target protein.
The affinity chromatography II and the anion exchange chromatography I are connected in series, the filler loading capacity of the affinity chromatography I is 80mg/mL (can be 80 mg/mL-90 mg/mL), and the filler loading capacity of the anion exchange chromatography I is 20mg/mL (can be 20 mg/mL-30 mg/mL). The enzyme digestion solution firstly flows through an affinity chromatographic column, the hetero-protein is adsorbed by the affinity chromatographic column, the penetrating solution is a solution containing the target protein, the penetrating solution passes through an anion exchange chromatographic column, Tris-HCl with the pH value of 8.010mmol/L is used as a balance solution of a chromatographic column before sample loading, the balance is carried out until the absorption value is stable, the target protein is adsorbed on the anion exchange chromatographic column, and 175mmol/L NaCl, 10mmol/L Tris-HCl and pH value of 8.0 are used as eluent of rExendin-4 protein. The elution flow rate was 30mL/min, and the eluate A280 was collected at 0.04AU-0.01AU to give anion exchange chromatography solution I, and the results are shown in the following Table.
TABLE 11 Instrument set in affinity chromatography II and anion exchange chromatography I
TABLE 12 summary of affinity chromatography II and anion exchange chromatography I results
Remarking: anion exchange chromatography recovery ratio (mg) of anion exchange chromatography solution I target protein/enzyme digestion solution target protein
6. Reverse phase chromatography
The reversed phase chromatography is a chromatography technique which uses a nonpolar reversed phase medium as a stationary phase and an aqueous solution of a polar organic solvent as a mobile phase to separate and purify proteins according to the difference of polarity (hydrophobicity). The rExendin-4 protein and the hybrid protein have different hydrophobicity due to the difference of amino acid composition, space conformation and the like, so that the rExendin-4 protein and the hybrid protein are effectively separated by using reverse phase chromatography.
Buffer F (10mmol/L PB, 10% (v/v) ethanol, pH8.0) was used as mobile phase A, and 80% (v/v) ethanol was used as mobile phase B. The Source 15RPC column was equilibrated with mobile phase A and loaded with 5mg/mL of packing. Gradient elution is adopted after loading: firstly, mixing 67% (v/v) of mobile phase A and 33% (v/v) of mobile phase B to elute impurities, and eluting 1 column volume; then mixing 45% (v/v) mobile phase A and 55% (v/v) mobile phase B to elute the target protein, wherein the equilibrium and elution speed is 24mL/min, and collecting the eluent with A280 of 0.4AU-0.04 AU. The process parameters are shown in the following table, and the results of the inversion studies of 3 consecutive batches are shown in the following table.
TABLE 13 reverse phase chromatography equipment parameters
TABLE 14 summary of the results of the reverse phase chromatography study
Remarking: the recovery rate of reverse phase chromatography (mg) of target protein/total amount of target protein in anion exchange chromatography solution I)
7. Anion exchange chromatography liquid II
The target protein diluent obtained by reverse phase chromatography is applied to a Q Sepharose High Performance column equilibrated with buffer H (10mmol/L phosphate buffer, pH7.5-8.5), and after application, the column is sufficiently eluted with buffer H, then with buffer I (10mmol/L PB, pH6.5), and eluted with buffer J (10mmol/L PB, 200mmol/L NaCl, pH6.5), at a flow rate of 24 mL/min. Collecting target protein, collecting A280 when reaching 0.01AU, reaching maximum peak value, and stopping collecting when reducing to 0.01 AU. The anion exchange chromatography fluid II equipment parameters and the results of the study are shown in the following table.
TABLE 15 anion exchange chromatography fluid II equipment parameters
TABLE 16 summary of anion exchange chromatography liquid II data results
Remarking: the recovery ratio of anion exchange chromatography liquid II was total amount of target protein (mg) of anion exchange chromatography liquid II/total amount of target protein (mg) of reverse phase chromatography.
8. Gel filtration chromatography
The gel filtration chromatography displacement buffer solution utilizes the principle of size exclusion, the rExendin-4 with the volume larger than that of a filler hole can not enter the hole, and inorganic salt micromolecules enter the hole and flow through a chromatographic column in a delayed manner, so that the displacement of a buffer system is realized. Sephadex G-25Fine from GE company is selected as gel filtration medium. The device parameter conditions of gel filtration chromatography are shown in the following table, and the specific process steps are as follows: and balancing 3CV by Buffer M until the base line is stable, wherein the sample loading solution is anion exchange chromatography solution II, the sample loading amount is 40-50 mg/mL filler, Buffer M (30mmol/L acetic acid Buffer solution, pH5.5) is used for eluting the target protein, the elution flow rate is 30mL/min, A280 reaches 0.01AU and starts to be collected, the maximum peak value is reached, and the collection is stopped when the value is reduced to 0.01 AU. The equipment parameters and the results of the study are shown in the following table.
TABLE 17 gel filtration chromatography equipment conditions
TABLE 18 summary of gel filtration chromatography findings
Note: gel filtration chromatography recovery ═ total protein (mg) of gel filtration chromatography order/total protein (mg) of anion exchange chromatography order II
The result shows that the purity of the rExendin-4 protein in the solution is more than 99.4% after the anion exchange chromatography liquid II is subjected to gel filtration chromatography, the recovery rate of the protein is more than 90%, the process is stable, the rExendin-4 protein can be effectively separated, and the product quality meets the requirements related to biological products for treatment in Chinese pharmacopoeia through detection and analysis of three batches of gel filtration chromatography liquid.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. A method for purifying a recombinant Exendin-4 polypeptide is characterized by comprising the following steps:
1) carrying out affinity chromatography I on the crude recombinant Exendin-4 polypeptide to obtain an affinity chromatography solution I;
2) carrying out ultrafiltration on the affinity chromatography solution I to obtain an ultrafiltrate;
3) carrying out enzymolysis on the ultrafiltrate to obtain an enzymolysis liquid;
4) performing affinity chromatography II and anion exchange chromatography I on the enzymatic hydrolysate to obtain anion exchange chromatography solution I;
5) carrying out reverse phase chromatography on the anion exchange chromatography liquid I to obtain reverse phase chromatography liquid;
6) and (4) carrying out anion exchange chromatography II on the reversed-phase chromatography liquid to obtain anion exchange chromatography II liquid.
2. The method for purifying recombinant Exendin-4 polypeptide of claim 1, wherein the elution in step 5) is performed by gradient elution, wherein 1 column volume is eluted by mixed elution of impurities with 67% by volume mobile phase A and 33% by volume mobile phase B; then mixing the mobile phase A with the volume percentage of 45% and the mobile phase B with the volume percentage of 55% to elute the target protein; collecting reversed phase chromatographic liquid when the A280 absorption value is 0.4AU, and stopping collecting the reversed phase chromatographic liquid when the A280 absorption value is reduced to 0.4AU after the maximum peak value is reached; the mobile phase A is a solution containing 10mmol/L PB and 10% ethanol by volume percentage, and the pH value is 7.5-8.5; the mobile phase B is 80 percent ethanol water solution by volume percentage.
3. The method for purifying recombinant Exendin-4 polypeptide of claim 1 or 2, wherein the ultrafiltration membrane used in ultrafiltration has a pore size of 5KD to 10 KD.
4. The method for purifying recombinant Exendin-4 polypeptide according to any of claims 1-3, wherein the chromatography column used in the affinity chromatography I is a nickel ion chelating affinity layer column, the sample loading amount is 2.60g-3.40g crude purity/mL filler, and the elution is carried out by using Buffer B to obtain an affinity chromatography solution I; the Buffer B is a solution containing 8mmol/L-12mmol/L PB and 45mmol/L-55mmol/L imidazole, and the pH value is 7.5-8.5; the absorption value of the elution peak collection range A280 is 0.3-0.1 AU.
5. The method for purifying recombinant Exendin-4 polypeptide of any of claims 1-4, further comprising the step of subjecting the anion exchange chromatography II solution to gel filtration chromatography.
6. The method for purifying recombinant Exendin-4 polypeptide of claim 5, wherein the gel filtration chromatography uses Buffer M as eluent, the A280 absorption value reaches 0.01AU, the collection is started, the maximum peak value is reached, and the collection is stopped when the A280 absorption value is reduced to 0.01 AU; buffer M is 30mmol/L acetic acid Buffer solution, pH5.5; the elution flow rate was 30 mL/min.
7. The method for purifying recombinant Exendin-4 polypeptide of claim 2, wherein the column of reverse phase chromatography is Source 15 RPC; the loading amount is 5 mg/mL-8 mg/mL filler; the elution speed is 22mL/min-26 mL/min; and (3) starting to collect the reverse-phase chromatographic liquid when the A280 is 0.5AU, and stopping collecting the reverse-phase chromatographic liquid when the A280 absorption value is reduced to 0.05AU after the maximum peak value is reached.
8. The method for purifying recombinant Exendin-4 polypeptide according to any of claims 1-7, wherein the chromatography column used in affinity chromatography II and the anion exchange chromatography I chromatography column are connected in series for loading; eluting the target protein by the eluent; the eluent is a solution containing NaCl and Tris-HCl, and the pH value is 7.5-8.5; the concentration of NaCl is 170mmol/L-180mmol/L, and the concentration of Tris-HCl is 8mmol/L-12 mmol/L; collecting anion exchange chromatography liquid I with A280 in the range of 0.04AU-0.01 AU; the elution flow rate is 30 mL/min;
or, after loading the sample by the anion exchange chromatography II, fully eluting by using a buffer solution H, eluting by using a buffer solution I, eluting by using a buffer solution J, and collecting the target protein; collecting when the A280 absorption value of the eluent reaches 0.01AU, reaching the peak value, and stopping collecting when the A280 absorption value of the eluent falls to 0.01 AU; the buffer solution H is 10mmol/L PB, and the pH value is 7.5-8.5; the buffer solution I is 8mmol/L-12mmol/L PB, and the pH value is 6.5; buffer J is a solution containing 8mmol/L-12mmol/L PB and 180mmol/L-210mmol/L NaCl, pH 6.5.
9. The method for purifying recombinant Exendin-4 polypeptide of any claim 1-7, wherein in the enzymolysis step, the enzymolysis system is 1.5mmol/L-1.8mmol/L CaCl21.5mg/mL fusion protein and enterokinase; the amount of enterokinase added is 1U enterokinase per 2mg-3mg of fusion protein.
10. The method for purifying the recombinant Exendin-4 polypeptide of any one of claims 1-9, wherein the crude purity of the recombinant Exendin-4 polypeptide is prepared by:
(I) preparing the fermented thalli into a thalli heavy suspension, homogenizing and crushing to obtain a bacteria breaking liquid;
(II) centrifuging the bacteria-breaking liquid at 9000-12000 rpm and 4-6 ℃ for more than 45min, and keeping the supernatant; filtering with pore size of 0.4-0.5 μm to obtain bacteria-breaking clear liquid, i.e. coarse purity.
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| CN116236819A (en) * | 2023-05-09 | 2023-06-09 | 成都佩德生物医药有限公司 | Method for purifying polypeptide toxins in batches and composite double-layer chromatographic column |
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| CN104232666A (en) * | 2014-09-01 | 2014-12-24 | 江苏海王生物制药有限公司 | Gene expressing recombinant exenatide and carrier thereof |
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