CN105483193B - The method and kit of purifying protein from the fusion protein containing restriction enzyme site - Google Patents
The method and kit of purifying protein from the fusion protein containing restriction enzyme site Download PDFInfo
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Abstract
The method of this application discloses a kind of from the fusion protein containing restriction enzyme site purifying protein, includes the following steps: that the enzyme of the restriction enzyme site specificity digestion will be fixed on the first chromatographic column；It will be fixed with the first chromatographic column and the second chromatography column in series of enzyme, second chromatographic column can adsorb the non-destination protein part after the fusion protein digestion, and will not adsorb destination protein；By fusion protein followed by the first chromatographic column and the second chromatographic column.Method of the invention uses an one-step chromatography, saves purification step, and improve purification efficiency.
Description
Technical field
This application involves a kind of methods from fusion protein purification albumen, and in particular to a kind of enzyme for saving program and cost
The method for cutting purifying protein.
Background technique
Enzyme immobilization technology has the precedent used, such as biodiesel technology in field of biotechnology, and food engineering gives up
Water process etc., but relevant technical report is had not found in pharmaceutical field.Fusion protein technology is to obtain a large amount of standards
Albumen and the purposive Gene Fusion carried out can be constructed and be expressed with the new of multiple functions using fusion protein technology
Type destination protein.For example, some fusion proteins are thioredoxin and purpose containing fibrin ferment or enterokinase cleavage site
Thioredoxin and destination protein are cut in subsequent purification using fibrin ferment or enterokinase, and adopted by protein fusion expression
It is chromatographed with multistep column and separates both albumen, reach expected purification effect.Therefore to obtain destination protein need to be divided into two
Step: digestion and purifying.There is the fusion protein technology of special marking highly developed by genetic engineering means expression at present,
In the prior art using static enzyme cutting method, destination protein is replaced into corresponding digestion solution, the static digestion regular hour,
Then achieve the purpose that isolate and purify with different chromatographic columns.
The static enzyme cutting method that current enzyme incision technology generallys use is after mixing well enzyme and substrate protein, suitable
Digestion under the conditions of enzyme by fusion protein be cut into destination protein and fusion head, at this point, enzyme only to the albumen that can be touched around it
Digestion effect is generated, digesting efficiency is low, and enzyme dosage is big, in addition, enzyme and albumen are dissolved in simultaneously in a kind of solvent, subsequent purpose egg
The problem of enzyme residual is had in white purifying.And the digestion time is long, and enzyme and albumen are dissolved in simultaneously in a kind of solvent, after not only giving
Continuous destination protein purifying affects, and remaining enzyme has further digestion to albumen, makes the bad control of digestion terminal, enzyme
The variability cut increases.It is usually necessary to use further chromatographies to be purified for albumen by digestion, if next purifying
It cannot be removed effectively enzyme, then remaining enzyme will continue to play a role, to introduce additional impurity, can not effectively purify purpose
Albumen.It is more complex by ingredient in the protein solution of digestion, if according to conventional chromatography method, destination protein is adsorbed on chromatography
On medium, then destination protein and foreign protein are separated further according to physical properties such as its different surface charge and hydrophobicitys, gesture
More chromatographic enrichment is moved through, each step chromatographic column requires at least five kinds of solution to complete chromatographic step, entirely chromatographs technique ratio
It is more complex.One step column of every increase chromatography, will increase many workloads, while the rate of recovery of albumen can also decline, therefore using up can
The few chromatographic step of energy reaches the best boundary that identical purification effect is purifying process.
Summary of the invention
In order to overcome the above-mentioned problems of the prior art, in protein purification technique, inventor dexterously sets the application
Having counted one kind can be in the method for step purifying: once purifying destination protein using concatenated two chromatographic columns, is greatly reduced into
Sheet and time.Specifically, then medium is attached in chromatographic column firstly, enzyme is fixed on medium, digestion chromatography is made
The chromatographic column for being fixed with enzyme and another are had the chromatography column in series of particular medium by column, when fusion protein is in specific buffer bar
The concatenated device is flowed through under part, destination protein is directly flowed through from columns in series, and foreign protein is adsorbed on chromatography media, such step
Chromatography can be obtained by the destination protein of high-purity.
In one embodiment, the present invention provides one steps of one kind, and egg is purified from the fusion protein containing restriction enzyme site
White method, includes the following steps:
The enzyme of the restriction enzyme site specificity digestion will be fixed on the medium in the first chromatographic column；
It will be fixed with the first chromatographic column and the second chromatography column in series of enzyme, second chromatographic column can adsorb the fusion
Non- destination protein (hereinafter also referred to as fusion head) part of albumen, and destination protein will not be adsorbed；
By the sample containing the fusion protein followed by the first chromatographic column and the second chromatographic column.It, can according to the method
The destination protein that high-purity is obtained with a step, is greatly saved cost, and avoid the generation of a variety of foreign proteins.
In one preferred embodiment, the enzyme is fibrin ferment, enterokinase, papain, pepsin or pancreas
Protease.In actual operation, the structure of fusion protein can be designed according to the restriction enzyme site of these enzymes, so as to utilize
Above-mentioned enzyme effectively cuts to obtain destination protein.
In a further preferred embodiment, the medium in first chromatographic column is selected from the agar of high crosslinking
One of sugar, glucan and cellulose.Using fixing means appropriate, enzyme can be fixed on these media.
In another preferred embodiment, second chromatographic column, which is selected from, contains the affine of label protein for purifying
One of chromatographic stuffing, ion exchange class filler and molecular sieve filler.Specifically, the choosing of the medium in the second chromatographic column
Select several situations:
Contain label when expressing fusion protein, affinity chromatography of the second chromatographic column selection for purification tag albumen is filled out
Material when purifying BMP label protein, selects Dextrin for example, just selecting Ni affine filler when purifying his label protein
The filler of Sepharose series when purifying Strep (II) label protein, selects StrepTactin Sepharose series to fill out
Material selects Glutathione Sepharose FF series filler when purifying GST label protein；
When expressing fusion protein does not have label, to the non-destination protein part after fusion protein, destination protein and digestion
It is analyzed, when destination protein isoelectric point meta-alkalescence, and the non-destination protein isoelectric point meta-acid after overall length fusion protein and digestion
Property, then it can choose medium of the anion exchange series medium as the second chromatographic column, (such as strong anion exchanges Q
Sepharose series, weak anionic DEAE Sepharose FF)；When destination protein isoelectric point slant acidity, and overall length merges egg
Non- destination protein isoelectric point meta-alkalescence after white and digestion then can choose cation and exchange serial medium as the second chromatographic column
Medium (such as strong cation chromatography media SP Sepharose series and weak cation CMSepharoseFF).
When destination protein and widely different fusion protein molecule amount after digestion, the second chromatographic column can choose molecular sieve
Medium, when mobile phase is passed through, collect different components (such as: Superdex series and Sephacryl series).First chromatographic column
Described in enzyme be fixed on 4 ± 2 DEG C, carry out 12-20 hours under the conditions of pH8-8.5, can be chromatographed according to specific enzyme and first
The condition of the specific optimization immobilized enzyme of the selection of column, such as preferable 4 DEG C, pH8.0 fixes 16 hours.
The present invention also provides a kind of kits of purifying protein, including the first chromatographic column and the second chromatographic column, wherein
Medium in one chromatographic column is fixed with the enzyme that destination protein can be obtained from fusion protein digestion, the medium energy in the second chromatographic column
The non-destination protein part after the fusion protein is digested enough is adsorbed, and the destination protein will not be adsorbed, the reagent
Box is sequentially connected in series the first chromatographic column and the second chromatographic column when in use.In enzyme and the optional type of chromatographic column such as the above method
It is described.
Above-mentioned purification process may be implemented using the kit, also, for specific fusion protein, there is set of layer
Analysis column is present in kit, and the step of preparing above-mentioned specific chromatographic column is omitted, further saves the time of purifying.
Purification process of the invention compared with the existing technology in purification process have the advantage that 1, improve digestion effect
Rate, reduces the enzyme reaction time, and enzyme can recycle；2, enzyme passes through immobilization, will not pollute to subsequent technique, without increasing
Add removal residual enzyme step；3；Digestion system is identical with concatenated chromatographic column equilibrium condition, reduces solution type and dosage；4,
Column chromatography after digestion is using mode is penetrated, and destination protein is directly flowed through from column, and entire purifying process is simple and compact.
Detailed description of the invention
Fig. 1 is schematic flow chart of the invention；
Fig. 2 is the chemical equation of exemplary enzyme and medium coupling；
Fig. 3 be in the embodiment of the present invention 1 digesting efficiency quantitative measurment as a result, determining in the method for embodiment 1 different
The digestion effect picture of flow velocity, wherein each swimming lane is described as follows: pre: before digestion；1, digestion on 0.5ml/min flow velocity column, when reservation
Between 10min；2, digestion on 1ml/min flow velocity column, retention time 5min；3, digestion on 2ml/min flow velocity column, retention time
2.5min；4, digestion on 5ml/min flow velocity column, retention time 1min；
Fig. 4 is that digestion effect picture is repeated in embodiment 1, wherein each swimming lane is described as follows: pre: before digestion；1, digestion first
It is secondary；2, digestion the 2nd time；3, digestion the 3rd time；
Fig. 5 is the enterokinase digestion effect picture of different digestion times in embodiment 2, and each swimming lane is described as follows: 1, merging egg
White, 2, the digestion time 1 minute；3, digestion time 12 minutes；4, digestion time 10 minutes；5, digestion time 2 minutes；6, when digestion
Between 4 minutes；7, digestion time 6 minutes；8, digestion time 8 minutes；
Fig. 6 is the electrophoretogram according to the blood coagulation digestion column of embodiment 3 and consummate Q column series connection sample after purification；
Fig. 7 is that column and Ni column series connection digestion purification effect figure are cut according to the enterokinase of embodiment 4, wherein each swimming lane is distinguished
Are as follows: 1, molecular weight marker；2, Ni elutes miscellaneous peak；3, the destination protein 1 that Ni column penetrates；4, the destination protein 2 that Ni column penetrates；Each item
Band ingredient: a, remaining fusion protein；B, GH destination protein；C, noggin is merged；
Fig. 8 is that PTH fusion protein conventional purification process and immobilization digestion are connected purifying process flow chart pair with anion
Than.
Specific embodiment
In order to which technical solution of the present invention is described in more detail, below by this hair of specific embodiment exemplary illustration
It is bright, it will be understood by a person skilled in the art that, the implementation of these embodiments only to illustrate the invention is not constituted to of the invention
The restriction of protection scope.
Protein engineered expression in the prior art, can directly give expression to destination protein, and miscellaneous without other
The case where matter, is seldom, is in many cases usually first to give expression to fusion protein, then passes through digestion for the non-mesh on fusion protein
Protein part digestion fall, then destination protein is obtained by purification step.It is not only time-consuming due to complex steps, the entire production line
Cost it is also relatively high, and purity often ideal requirement is not achieved.
The present invention in view of the deficiencies of the prior art, designs new purification strategy: enzyme is fixed on solid state medium, not with
Destination protein dissolves each other, therefore will not isolate and purify and affect to following protein.Also, enzyme is after being fixed on medium, digestion
Efficiency improves, while the stability of enzyme improves, and after over cleaning, can be used for multiple times.
According to embodiment of the present invention, it before working out the entire strategy of protein production, first has to selection and closes
Suitable expression and way of purification, expression vector and expression way determine the ingredient of fusion protein, to determine the type of enzyme.
It is preferred that label is added in the fusion protein, such as histidine tag, GST, Strep (II) and BMP label, and in label and purpose
Digestion recognition site is added in design between albumen, for example, the restriction enzyme site of fibrin ferment is Ile-Glu-Gly-Arg, enterokinase
Restriction enzyme site is DDDDK；The restriction enzyme site of papain is arg, lys, gly, L-Citrulline；The digestion of pepsin
Site is the hydrophobic amino acids such as phe, trp, tyr, and the restriction enzyme site of trypsase is the Lys and Arg after K-Lys and R-Arg；,
In this way after digestion, by specifically binding above-mentioned label for the second chromatographic column of label design, reach more easily pure
Change.
The selection of first chromatographic column can be carried out according to the enzyme of wanted immobilization.And root is then wanted in the selection of the second chromatographic column
According in fusion protein label, the difference of non-destination protein part and destination protein selects in fusion protein.For example, when expression
Fusion protein when having histidine tag, contain label when expressing fusion protein, the second chromatographic column is selected for purifying
The affinity chromatography filler of label protein.For example, just selecting the affine filler of Ni when purifying his label protein；Purify BMP label egg
Bai Shi selects the filler of Dextrin Sepharose series；When purifying Strep (II) label protein, StrepTactin is selected
Sepharose series filler；Glutathione Sepharose FF series filler is selected when purifying GST label protein.
When expressing fusion protein does not have label, to the non-destination protein part after fusion protein, destination protein and digestion
It is analyzed: when destination protein isoelectric point meta-alkalescence, and the non-destination protein isoelectric point meta-acid after overall length fusion protein and digestion
Property, then can choose anion exchange as the second chromatographic column, such as strong anion exchange Q sepharose series, weak yin from
Sub- DEAE Sepharose FF；When destination protein isoelectric point slant acidity, and the non-destination protein after overall length fusion protein and digestion
Isoelectric point meta-alkalescence then can choose cation exchange as the second chromatographic column, such as strong cation chromatography media SP
Sepharose series and weak cation CMSepharoseFF.
When destination protein and widely different fusion protein molecule amount after digestion, the second chromatographic column can choose molecular sieve
Medium when mobile phase is passed through, collects different components.Such as: Superdex series and Sephacryl series.
By selecting suitable chromatography series connection digestion, it can be changed without buffer, flow through destination protein, merge head and not
The fusion protein cut is adsorbed on column, and a step purifies the destination protein that can be obtained by high-purity.Flow chart is referring to Fig. 1.
Below in conjunction with specific embodiment exemplary illustration technical concept of the invention.
The preparation method (agent activating and enzyme coupling) of preparation example immobilised enzymes
The large-meshed net agarose rich in hydroxyl that relatively appropriate biological macromolecular can be selected in enzyme immobilizatio matrix is carrier,
Enzyme immobilization process is completed, as shown in Figure 2 with the amino coupled of zymoprotein again after hydrogen bromide activates.Medium rich in hydroxyl
After being sufficiently swollen in acidic aqueous solution, imines carbonic acid active group is generated, which can be with protein or primary amine NH2It turns over
It translates, generates isourea derivatives, externally show as protein and medium is integrally formed.Medium low ph value and high ph-values after coupling
Buffer, which replaces, to be washed, and the free and unstable enzyme of combination is removed.
Immobilised enzymes medium is loaded into suitable chromatographic column to get to the first chromatographic column.
Preparation example 1: the preparation of immobilization fibrin ferment column (the second chromatographic column)
1. the Sepharose 4B filler powder 300ml 1mM HCl for taking 2.1g CNBr to activate is swollen by several times, then use
The solution repeated flushing filler (final packing volume is 5ml)；
2. taking (1000IU/ bottles) of 1 bottle of fibrin ferment to use 3ml ddH2O dissolution, divides 2 times and changes liquid with HiTrap desalting column (5ml)
To 0.1M NaHCO3pH 8.3；500mM NaCl finally obtains the fibrin ferment 5ml after changing liquid；
3. by the filler 50ml 0.1M NaHCO after the 5ml activation in step 13PH 8.3,500mM NaCl are sufficiently flat
After weighing apparatus, 5ml is added and changes the fibrin ferment after liquid, 4 DEG C are slowly stirred overnight；
4. using 0.1M Tris-HCl, pH8.0 solution closes the site of unbonded fibrin ferment with the flow velocity of 2ml/min.
Preparation example 2: the preparation of immobilization enterokinase column
1. the Sepharose 4B filler powder 1mM HCl swelling for taking 1.5g CNBr to activate, with the 1mM HCl of 200ml
Repeated flushing filler in batches (final packing volume is 3.75ml)；
2. take enterokinase 3.0ml (0.9mg/ml lot number be " 0909043.6U/ul), points 2 times with HiTrap Desalting
Column (5ml) desalination is to 0.1M NaHCO3PH 8.3,500mM NaCl finally obtain the enterokinase solution 5ml after changing liquid；
Final concentration of A280 is 0.6OD/ml
3.3.75ml addition 3.75ml changes the intestines after liquid after 3.75ml Coupling Buffer is added in the filler after activating
Kinases (EK enzyme), 4 DEG C are slowly stirred overnight, and the concentration A280 of enterokinase is 0.15OD/ml in repetition measurement supernatant；
4. using 0.1M Tris-HCl, pH8.0 solution closes the site of unbonded EK enzyme with the flow velocity of 2ml/min.
The digestion of immobilised enzymes
It needs to quantify immobilised enzymes progress digesting efficiency before use, measuring method is as follows:
Substrate protein solution is flowed through to the chromatographic column of immobilised enzymes with certain flow velocity, when recording contact of the enzyme with albumen
Between, according to digesting efficiency, determine the suitable digestion time, it is subsequent when repeating digestion, control the identical digestion time.
According to the digesting efficiency of measurement, substrate protein solution and flow velocity (i.e. the time of contact of enzyme) are adjusted, collection flows through peak,
Up to the mixed liquor after digestion.
Embodiment 1: immobilization fibrin ferment digestion thioredoxin-PTH fusion protein (Trx-PTH)
Purpose: by Trx-PTH with the immobilization fibrin ferment column different in flow rate prepared by preparation example 1, suitable enzyme is determined
Flow velocity is cut, with the determination suitable digestion time.
Analysis: 1, thioredoxin-PTH fusion protein contains the restriction enzyme site of fibrin ferment, without label；Applicable first
Chromatographic column selects the medium (Sepharose 4B) of hydrogen bromide activation, by the method for the immobilised enzymes of above-mentioned introduction, by blood coagulation
Enzyme is fixed on medium, in first chromatographic column；2, it since the destination protein isoelectric point after digestion is greater than 9.0, and merges
The isoelectric point of albumen and non-destination protein is below 7.5, therefore the second chromatographic column selects anionic exchange medium (Q
SepharoseBigBeads)。
Chromatographic column: solidify the Sepharose 4B XK16/20 column of fibrin ferment, 5ml
Equilibrium liquid: 25mM Tris, pH8.0；50mM NaCl
Sample: PTH fusion protein Trx-PTH (1140401-2)
Buffer: 25mM Tris, pH8.0；50mM NaCl；1M urea), OD 5.0mg/ml
Loading 1:0.5ml/min, retention time 10min, loading temperature are 25 DEG C
Loading 2:1ml/min, retention time 5min, loading temperature are 25 DEG C
Loading 3:2ml/min, retention time 2.5min, loading temperature are 25 DEG C
Loading 4:5ml/min, retention time 1min, loading temperature are 25 DEG C
Collection flows through, electrophoresis detection as shown in figure 3, from electrophoresis result can be seen that digestion condition be 25mM Tris,
pH8.0；50mM NaCl digestion 1-5 minutes at room temperature, fusion protease can be cut away 90% or more.
It may be reused to verify immobilization digestion column.It is 2.5 minutes in digestion retention time, 2.0ml/min stream
Immobilized enzyme column is reused many times under speed.Digestion effect does not change.As a result as shown in Figure 4.
Immobilization fibrin ferment column carries out digestion to Trx-PTH under 5ml/min flow velocity below, and 80% fusion protein is by enzyme
It cuts, it is as shown in table 1 below to compare static digestion data:
Table 1: the digestion of fibrin ferment traditional static and the immobilization digestion fusion protein table of comparisons
As seen from the above table, for fibrin ferment, in the usage amount of time cost and enzyme, immobilization digestion of the invention
Method is all significantly better than static digestion.
Embodiment 2: immobilization enterokinase digestion thioredoxin-GH fusion protein (Trx-GH)
Analysis: enterokinase cleavage site DDDDK and the His purification tag for including in thioredoxin-GH fusion protein,
Therefore the medium (Sepharose 4B) of the first chromatographic column selection hydrogen bromide activation, passes through the side of the immobilised enzymes of above-mentioned introduction
Enterokinase is fixed on medium by method, and in first chromatographic column, the non-destination protein part after fusion protein and digestion is equal
With His label, therefore the second chromatographic column selects Ni affinity column (NI Sepharose FF chromatography)；
Chromatographic column: the Sepharose 4B XK16/20 column of immobilization enterokinase, 5ml
Equilibrium liquid: 25mM Tris, pH8.0；50mM NaCl
Sample: GH fusion protein PA precipitates 1.2g, (H401-2), with Buffer:50mM Tris, pH8.0；50mM NaCl
Sufficiently dissolution, centrifugation removal precipitating, OD 7.5OD/ml.
Whether complete in order to detect digestion, repetition loading is multiple, for increasing the digestion time, in order to judge different digestions
The digestion gradient effect of time, and electrophoresis detection its digestion result.
Loading 1: loading flow velocity 5ml/min, retention time 1min, loading temperature is 25 DEG C
Loading 2: loading flow velocity 2.5ml/min, retention time 2min, loading temperature is 25 DEG C
Loading 3: it collects loading 2 and penetrates peak, repeat loading, flow velocity 2.5ml/min, retention time 4min, loading temperature
It is 25 DEG C
Loading 4: collect loading 3 flow through liquid repeat loading it is primary, retention time 6min, loading temperature be 25 DEG C
Loading 5: collect loading 4 flow through liquid repeat loading it is primary, retention time 8min, loading temperature be 25 DEG C
Loading 6: collect loading 5 flow through liquid repeat loading it is primary, retention time 10min, loading temperature be 25 DEG C
Loading 7: collect loading 6 flow through liquid repeat loading it is primary, retention time 12min, loading temperature be 25 DEG C
Collection flows through, and electrophoresis detection is as shown in Figure 5.
From electrophoresis result as can be seen that digestion condition is 50mM Tris, pH8.0；50mM NaCl is in room temperature condition
Fusion protease can be cut away 90% or more by lower digestion 10-12 minutes.
It can be seen that no matter immobilization digestion accounts in cost or all on the time and has great advantage than traditional digestion, in detail
It is shown in Table 2.
Immobilization enterokinase column is in 50mM Tris, pH8.0；50mM NaCl is at room temperature 10 points in the digestion time
Clock carries out digestion to GH fusion protein, and 90% or more fusion protein is cut open, and immobilization digestion comparison tradition of the invention is quiet
State digestion data are as follows:
Table 2: traditional digestion and the immobilization enterokinase digestion fusion protein table of comparisons
As seen from the above table, for enterokinase, in the usage amount of time cost and enzyme, immobilization digestion of the invention
Method is also all significantly better than static digestion.
The series connection of immobilized enzyme column and chromatographic column
When obtaining target protein using the digestion of fusion protein, usually insertion is suitable between fusion head and destination protein
Label, such as 6His, GST, BMP etc. can more convenient purifying fusion protein, while can also easily be removed after digestion
Merge head.The affinity chromatography of series connection label after immobilised enzymes, it may be convenient to the fusion protein that removal is merged head and do not cut, directly
It connects to obtain destination protein.For example, for the fusion protein with 6His label by containing fusion head, mesh after immobilization digestion
Albumen and the fusion protein do not cut, digested liquid directly pass through concatenated metal chelate chromatography column, merge head and do not cut
Fusion protein is attracted on chromatographic column, and destination protein flows through.It can be obtained by the purpose egg of high-purity by a step
It is white, the time is saved, efficiency is improved.
The fusion protein of tape label can also not connect chromatographic column according to the property of destination protein, reach a step digestion and pure
The purpose of change.It, can when the isoelectric point difference of destination protein and fusion head is larger, and the isoelectric point of destination protein is higher than digestion pH
With with anion chromatography column and immobilized enzyme column series connection, the destination protein etc. after digestion is not adsorbed on anionic exchange medium
On, and slant acidity can be adsorbed on pillar, while the enzyme being detached from can be also adsorbed on anion medium.
Embodiment 3: the series connection of immobilization fibrin ferment and anion-exchange column
It will solidification fibrin ferment column XK16/20 column (5ml) and Q sepharose Big Bead column XK16/20 (5ml) series connection.
Chromatographic runs are carried out with following program:
Equilibrium liquid: 25mM Tris-HCl (pH8.0)+50mM NaCl balances 50ml
Loading: Trx-PTH is added by concatenated chromatographic column with 2ml/min flow velocity, the digestion time is 2.5 minutes
Receive peak: collection penetrates peak.
The peak that penetrates being collected into carries out electrophoresis.Reached by series connection digestion and the purity for purifying a step destination protein
90% or more.
As a result referring to Fig. 6.
As can be seen from the above results not only saving time and economic cost using method of the invention, program is simplified,
Avoid the possibility of pollution, moreover it is possible to keep even obtaining higher destination protein purity.
Embodiment 4: the series connection of immobilization enterokinase and NI affinity column
Chromatographic column: the Sepharose 4B XK16/20 column of immobilization enterokinase, 5ml series connection NI FF chromatographic column 5ml
Equilibrium liquid: 25mM Tris, pH8.0；50mM NaCl
Sample: GH fusion protein PA precipitates 1.2g, (H401-2), with Buffer:50mM Tris, pH8.0；50mM NaCl
Sufficiently dissolution, centrifugation removal precipitating, OD 7.5OD/ml
Loading flow velocity 1ml/min, retention time 4min, loading temperature is 25 DEG C
Collection penetrates peak, electrophoresis detection purity.
Referring to Fig. 7 as it can be seen that obtained purity of protein is higher
As can be seen from the above results not only saving time and economic cost using method of the invention, program is simplified,
Avoid the possibility of pollution, moreover it is possible to keep even obtaining higher destination protein purity.
After use, two columnss in series are dismantled, is respectively washed, saves backup.
In addition, inventor has found through repetition test, the digestion column in the present invention can be stored in 20% alcohol, not influenced
Its digesting efficiency.Therefore, kit of the invention can save for a long time, and not influence its effect, and commercialization is suitble to sell.
Although illustrating the present invention above in association with specific embodiment, in the feelings for not departing from spirit of the invention
Under condition, those skilled in the art can carry out modification appropriate to it according to ability domain knowledge, these modifications should also fall into this hair
In the range of bright protection.The scope of the present invention is defined by the claims.
Claims (3)
1. a kind of method of the purifying protein from the fusion protein containing restriction enzyme site, includes the following steps:
The enzyme of the restriction enzyme site specificity digestion will be fixed on the medium in the first chromatographic column；
It will be fixed with the first chromatographic column and the second chromatography column in series of enzyme, second chromatographic column can adsorb the fusion protein
Non- destination protein part after digestion, and destination protein will not be adsorbed；
By the sample containing the fusion protein followed by the first chromatographic column and the second chromatographic column, wherein the destination protein is
PTH, the enzyme are fibrin ferment,
Wherein, the fusion protein is thioredoxin-PTH fusion protein, and the medium in first chromatographic column is handed over using high
The agarose of connection, greater than 9.0, the isoelectric point of the fusion protein and non-destination protein is lower than the isoelectric point of the destination protein
7.5, second chromatographic column uses anionic exchange medium.
2. it is small to carry out 12-20 wherein the enzyme is fixed on 4 ± 2 DEG C for the method as described in claim 1 under the conditions of pH8-8.5
When.
3. a kind of kit using the method as described in claim 1 purifying protein, including the first chromatographic column and the second chromatography
Column, wherein the medium in the first chromatographic column is fixed with the enzyme that can obtain destination protein from fusion protein digestion, the second chromatographic column
In medium can adsorb the non-destination protein part after the fusion protein is digested, and the purpose egg will not be adsorbed
White, the kit is sequentially connected in series the first chromatographic column and the second chromatographic column when in use, wherein the fusion protein is sulphur
Oxygen also albumen-PTH fusion protein, the enzyme is fibrin ferment, wherein the destination protein is PTH, in first chromatographic column
For medium using the agarose of high crosslinking, the isoelectric point of the destination protein is greater than 9.0, the fusion protein and non-destination protein
Isoelectric point is lower than 7.5, and second chromatographic column uses anionic exchange medium.
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| DK3500586T3 (en) * | 2016-10-17 | 2021-05-31 | Enzene Biosciences Ltd | CONTINUOUS PROCEDURE TO REDUCE HETEROGENITY OF THERAPEUTIC PROTEIN |
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