CN103805584B - A kind of recombinant trypsin purification process - Google Patents
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Abstract
Invention relates to biological technical field, provide the purification process of a kind of recombinant trypsin, the recombinant trypsin that first culture propagation is produced by the method former activates, then use macroporous absorbent resin and cation-exchange chromatography to purify and obtain pure trypsase, avoid and directly zymotic fluid need to be diluted or the process of ultrafiltration during purified trypsin proenzyme from yeast fermentation broth, and need not individually proenzyme be digested pre-treatment, save purification step, simple to operate, yield is higher；It addition, the chromatographic stuffing low price that the present invention uses, it is suitable for large-scale production.Experiment shows, recombinant trypsin purity and activity that the present invention provides are the highest, can be as industrial production raw material.
Description
This application claims and submit Patent Office of the People's Republic of China, Application No. on November 12nd, 2012
201210450131.0, the Chinese patent application of invention entitled " a kind of recombinant trypsin purification process "
Priority, entire contents is hereby incorporated by the application.
Technical field
The present invention relates to biological technical field, particularly relate to the purification process of a kind of recombinant trypsin.
Background technology
Trypsase (Trypsin, EC3.4.21.4) belongs to serine protease, can specifically cut alkalescence
Amino acid, such as lysine and arginic carboxy-terminal peptide bond.In vertebrate, rise as digestive ferment
Effect.Its precursor trypsinogen synthesizes in pancreas and composition as pancreatic juice is secreted, through enterokinase or
Tryptic activation, becomes activated trypsase.Trypsase can also limit the rotten albumen of decomposition
The precursor of other enzymes such as proenzyme, procarboxypeptidase, phosphatide proenzyme, plays activation, is the strongest
Protease.Tryptic primary commercial utility is for the industry of rh-insulin as industrial enzymes
Produce.
Initially, tryptic production is extracting directly in the pancreas by animals such as pig, ox, sheep, is carrying
When taking, first trypsinogen in pancreas is activated, then utilize 70% ethanol precipitation trypsase
Extract thick enzyme.But, use 70% ethanol precipitation not only can waste a large amount of organic solvent, and extraction efficiency
Relatively low；It addition, be often mixed with other enzymes from the trypsase that pancreas extracting directly obtains, cause
Specific decline, thus since the eighties in last century, recombinant production trypsase from different biologies
Method be widely used.
In early days, people use Escherichia coli as the expression vector of recombinant trypsin, and such as, the world is specially
Profit WO99/10503 just have employed this method, but, due to the high table of prokaryotes foreign gene
Reach characteristic, cause the generation of inclusion body, so that the purge process of this product needing add sex change with multiple
Property process, adds the complexity of purifying, is not suitable for large-scale industrial production.
Subsequently, in the method that European patent EP 1399568B1 provides, eucaryote Pichia pastoris conduct
The use of expression vector effectively prevent the generation of inclusion body, and the method passes through cationic ion-exchange resin to sending out
Ferment liquid is purified and obtains trypsinogen, and then proenzyme is activated by regulation pH value.But finish red ferment
Female fermented liquid supernatant electrical conductivity is higher, generally 15ms/cm～30ms/cm, and ion-exchange chromatography filler
Generally only < just can capture under the conditions of 5ms/cm in feed liquid electrical conductivity.Therefore Pichia pastoris zymotic fluid
Needs are diluted or cationic ion-exchange resin could be used after ultrafiltration to capture proenzyme, but use the side of ultrafiltration
Method is not appropriate for large-scale production, and dilution before purification can cause pole when mass producing
Big water resource waste, and after diluting, volume increases, and causes the prolongation of purification time.
Macouzet M et al. the C end that recombinant trypsin is former merged one section histidine-tagged, and lead to
Cross and restructuring trypsinogen is purified by histidine-tagged identification, simplify purge process, and can
Avoid zymotic fluid being diluted or ultrafiltration in purge process, but the histidine-tagged of introducing cannot be thorough
Removing, the histidine-tagged of residual easily produces impact to later stage commercial Application.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is to provide a kind of recombinant trypsin purifying side
Method, the method yield that the present invention provides is high, and the trypsase produced is mainly used in industrial use, the party
Method is easy and simple to handle, and simplifies purification step, is suitable for large-scale production.
The invention provides the purification process of a kind of recombinant trypsin, the method comprises the following steps:
Step 1: obtain the zymotic fluid former containing recombinant trypsin；
Step 2: take the zymotic fluid former containing recombinant trypsin, isolated supernatant, activation, it is thus achieved that
Solution containing recombinant trypsin；
Step 3: take the solution containing recombinant trypsin, purified, dry, to obtain final product.
As preferably, to the activation of trypsinogen particularly as follows: take supernatant, add CaCl2And regulate
PH to 7.0～8.5, activates 8 hours～24 hours under room temperature.Or addition pancreatin and/or intestines swash in supernatant
Enzyme activates.The present invention selects to be added without pancreatin and/or enterokinase activates.
Preferably, CaCl2Final concentration of 10mmol/L～100 in the solution of recombinant trypsin
mmol/L。
First zymotic fluid is separated and activation process by the present invention, it is not necessary to purification of Recombinant from zymotic fluid
Zymotic fluid need to be diluted or ultrafiltration during trypsinogen, it also avoid from zymotic fluid, first purify pancreas egg
The process again purified after white proenzyme reactivation pancreatin, simplifies purification step.
Preferably, the purifying to the solution containing recombinant trypsin includes macroporous adsorption resin chromatography and sun
The step of ion-exchange chromatography.
Preferably, macroporous adsorption resin chromatography use filler be Rohmhaas XAD4, Rohmhaas
XAD1180, Rohmhaas XAD16 or Rohmhaas XAD7HP.
Preferably, the mixed liquor that eluent is ethanol, acetic acid and water that macroporous adsorption resin chromatography uses.
Preferably, the compound method of macroporous adsorption resin chromatography eluent is: taking volume fraction respectively is 10%
Acetic acid aqueous solution and volume fraction be the ethanol water of 90%, mix with the volume ratio of 1:0.7～1.2,
Obtain.
Preferably, the filler that cation-exchange chromatography uses is: GE SP-sepharose ff, GE
SP-sepharose HP、GE SP-sepharose XL、GE SP-sephadex C-25、GE SP-sephadex
C-50, tosoh SP550, Bio-Rad UNOsphere s, Bio-Rad rapid s or Bio-Rad high s.
Preferably, the aqueous solution that eluent is acetic acid and NaCl that cation-exchange chromatography uses.
Preferably, the compound method of cation-exchange chromatography eluent is: taking volume fraction respectively is 4%
Acetic acid aqueous solution and molar concentration are the NaCl solution of 1mol/L, mix with the volume ratio of 1:0.8～1.2 respectively
Close, to obtain final product.
Preferably, the step 1 in the purification process of recombinant trypsin particularly as follows:
Step a: the encoding trypsin protogene of acquisition；
Step b: take encoding trypsin protogene, construction recombination plasmid；
Step c: take recombinant plasmid, builds recombinant pichia yeast strain；
Step d: take recombinant pichia yeast strain, to restructuring trypsinogen abduction delivering, it is thus achieved that described contain
There is the yeast fermentation broth that recombinant trypsin is former.
Compared with prior art, the restructuring pancreas that first culture propagation is produced by the purification process that the present invention provides
Pepsinogen uses macroporous absorbent resin and cation-exchange chromatography to be purified and obtain after carrying out activation process
Pure trypsase.Avoid and directly zymotic fluid need to be entered during purified trypsin proenzyme from yeast fermentation broth
Row dilution or the process of ultrafiltration, and individually proenzyme need not be digested pre-treatment, save purification step,
Simple to operate, and yield is higher；It addition, the chromatographic stuffing low price that the present invention uses, it is suitable for big rule
Mould produces.Testing result shows, recombinant trypsin purity and activity that the present invention provides are the highest, can
Meet the demand as industrial production raw material.
Accompanying drawing explanation
Fig. 1 show the SDS-PAGE of the recombinant trypsin that the embodiment of the present invention 5 provides,
Wherein, swimming lane M is protein low-molecular-weight Marker；Swimming lane 1 is recombinant trypsin mark product, swimming lane
2 recombinant trypsin provided for the embodiment of the present invention 5.
Detailed description of the invention
The invention provides the purification process of a kind of recombinant trypsin, those skilled in the art can use for reference
Present disclosure, is suitably modified technological parameter and realizes.Special needs to be pointed out is, all similar replacements and
Changing apparent to those skilled in the art, they are considered as being included in the present invention.This
Method and the application of invention are described by preferred embodiment, and related personnel substantially can not take off
In present invention, spirit and scope, method described herein and application it is modified or suitably changes
With combination, realize and apply the technology of the present invention.
The invention provides the purification process of a kind of recombinant trypsin, the method comprises the following steps: first
First, it is thus achieved that containing the zymotic fluid that recombinant trypsin is former；Then, take containing former the sending out of recombinant trypsin
Ferment liquid, isolated supernatant, activation, it is thus achieved that containing the solution of recombinant trypsin；Then, take and contain
There is the solution of recombinant trypsin, purified, dry, to obtain final product.
Wherein, to the activation of trypsinogen particularly as follows: take supernatant, add CaCl2And regulate pH extremely
7.0～8.5, activate 8 hours～24 hours under room temperature.
The mode obtaining supernatant is micro-filtration, centrifugal or filtration.
For improving activation effect, CaCl2In the solution of recombinant trypsin final concentration of 10
Mmol/L～100mmol/L.
For improving activation effect further, pancreatin and/or enterokinase can be added in supernatant.
First zymotic fluid is separated and activation process by the present invention, it is not necessary to purification of Recombinant from zymotic fluid
Zymotic fluid need to be diluted or ultrafiltration during trypsinogen, it also avoid from zymotic fluid, first purify pancreas egg
The process again purified after white proenzyme reactivation pancreatin, simplifies purification step.
In order to simplify operating procedure while improving purity, to containing in the purification process that the present invention provides
The purifying of the solution of recombinant trypsin includes the step of macroporous adsorption resin chromatography and cation-exchange chromatography
Suddenly.
Specifically, the step of macroporous adsorption resin chromatography is: first, and take containing recombinant trypsin is molten
Liquid loading is to large pore resin absorption column；Then, large pore resin absorption column is rinsed extremely with the Tris-HCl aqueous solution
Baseline；Then, with the mixed liquor of ethanol, acetic acid and water, large pore resin absorption column is eluted, it is thus achieved that
Recombinant trypsin crude product solution.
For reaching more preferable purification effect, what macroporous adsorption resin chromatography used filler is Rohmhaas
XAD4, Rohmhaas XAD1180, Rohmhaas XAD16 or Rohmhaas XAD7HP.
Wherein, the mixed liquor that eluent is ethanol, acetic acid and water that macroporous adsorption resin chromatography uses.
The compound method of macroporous adsorption resin chromatography eluent is: take the ethanol that volume fraction is 15% respectively
The aqueous solution and volume fraction are the acetic acid aqueous solution of 20%, mix with the volume ratio of 1:0.7～1.2, to obtain final product.
It addition, the concretely comprising the following steps of cation-exchange chromatography: first, take recombinant trypsin crude product solution
Loading is to cation-exchange chromatography resin column；Then, cation-exchange chromatography tree is rinsed with acetic acid aqueous solution
Fat post is to baseline；Then, gradient elution is carried out with the aqueous solution of acetic acid and NaCl, it is thus achieved that restructuring tryptose
Enzyme solutions.
Cation-exchange chromatography use filler be: GE SP-sepharose ff, GE SP-sepharose HP,
GE SP-sepharose XL、GE SP-sephadex C-25、GE SP-sephadex C-50、tosoh
SP550, Bio-Rad UNOsphere s, Bio-Rad rapid s or Bio-Rad high s.
Wherein, the aqueous solution that eluent is acetic acid and NaCl that cation-exchange chromatography uses.
The compound method of cation-exchange chromatography eluent is: take the acetic acid that volume fraction is 4% respectively water-soluble
Liquid and molar concentration are the NaCl solution of 1mol/L, mix with the volume ratio of 1:0.8～1.2, to obtain final product.
Acquisition pattern containing the former zymotic fluid of recombinant trypsin is: first, it is thus achieved that coding tryptose
Prochymosin gene；Then, encoding trypsin protogene, construction recombination plasmid are taken；Then, restructuring matter is taken
Grain, builds recombinant pichia yeast strain；Then, take recombinant pichia yeast strain, to recombinant trypsin
Former abduction delivering, it is thus achieved that containing the yeast fermentation broth that recombinant trypsin is former.
Particularly as follows: first acquisition pattern containing the former zymotic fluid of recombinant trypsin, uses linking PCR
The encoding trypsin protogene from people, pig or ox that synthesizes of mode；Then, this gene is connected
Enter pPIC9K plasmid vector, be built into recombinant plasmid；Then, this recombinant plasmid transformed is entered Pichia pastoris
Competent cell, is built into recombinant pichia yeast strain；Then, take positive recombinant pichia yeast strain,
Use BMMY fluid nutrient medium to carry out mass propgation, and use methyl alcohol as derivant induction restructuring pancreas egg
The expression of white proenzyme, it is thus achieved that containing the yeast fermentation broth that recombinant trypsin is former.
The recombinant trypsin that first culture propagation is produced by the purification process that the present invention provides former activates
Use macroporous absorbent resin and cation-exchange chromatography to be purified after process, purge process has been carried out letter
Change, and higher yield can be ensured；It addition, the chromatographic stuffing low price that the present invention uses, it is suitable for big
Large-scale production.Products obtained therefrom is suitable for the use of industrialized production.
Solvent of the present invention and reagent are common commercially available product, all can be buied by market.
Embodiment 1 contains the preparation of the former zymotic fluid of recombinant trypsin
By being connected the nucleotide sequence of PCR composite coding Porcine trypsin protogene, PCR segment is used
Xho I and EcoR I carries out double digestion, inserts Expression vector pPIC9K plasmid, construction recombination plasmid
pPIC9K-trypsin.In this recombinant plasmid, destination gene expression is to enter under AOX1 promoter controls
Row, there is alpha signal peptide before genes of interest, recombinant expressed trypsinogen can be made to be secreted into culture medium
In.
After being digested by the recombinant plasmid Sac I built, use the mode that electricity converts by recombinant plasmid transformed
Enter in competent yeast cells, build recombinant bacterial strain.The recombinant bacterial strain built is inoculated in containing G418
YPD solid medium on, carry out positive strain screening, the positive strain that screening obtains is for tryptose
Zymogen expression.
Picking positive strain, is seeded in 10ml liquid YPD medium, 29 DEG C～30 DEG C, 250r/min
Overnight incubation.In the ratio of 1:100, the bacterium solution of incubated overnight is seeded to the BMGY liquid training of 100ml
Supporting base, in 29 DEG C～30 DEG C, 250r/min cultivates, and monitors OD with spectrophotometer600Value.Work as OD600
Stop when value reaches between 2～6 cultivating.Under room temperature, 1500g is centrifuged 5min and collects thalline, then uses
100ml pH is the BMMY fluid nutrient medium re-suspended cell of 3～5, and being subsequently placed in volume is 1L shaking flask
In, 29 DEG C, the former expression of 250r/min methanol induction recombinant trypsin.Add every 24h afterwards
The methyl alcohol of 500 μ l, Fiber differentiation stops induction, takes zymotic fluid and be analyzed after 96 hours.
The activation that embodiment 2 recombinant trypsin is former
The zymotic fluid that taking the embodiment of the present invention 1 provides carries out micro-filtration and takes supernatant, adds in supernatant
CaCl2To its final concentration of 10mmol/L, and regulate zymotic fluid pH to 7.0, under room temperature, stirring at low speed
Activate 24 hours, it is thus achieved that containing the solution of recombinant trypsin.
The activation that embodiment 3 recombinant trypsin is former
The zymotic fluid that taking the embodiment of the present invention 1 provides carries out micro-filtration and takes supernatant, adds in supernatant
CaCl2To its final concentration of 50mmol/L, and regulate zymotic fluid pH to 8.0, under room temperature, stirring at low speed
Activate 8 hours, it is thus achieved that containing the solution of recombinant trypsin.
The activation that embodiment 4 recombinant trypsin is former
The zymotic fluid that taking the embodiment of the present invention 1 provides carries out micro-filtration and takes supernatant, adds in supernatant
CaCl2To its final concentration of 100mmol/L, and regulating zymotic fluid pH to 8.5, under room temperature, low speed stirs
Mix activation 6 hours, it is thus achieved that containing the solution of recombinant trypsin.
The purifying of embodiment 5 recombinant trypsin
The solution containing recombinant trypsin that taking the embodiment of the present invention 2～4 provides filters, and then goes up
Sample uses Rohmhaas XAD4 to large pore resin absorption column, filler, and loading flow velocity is not more than 300cm/h,
Large pore resin absorption column is rinsed extremely with the Tris-HCl aqueous solution of the 20mmol/L of pH8.0 after end of the sample
Baseline, then, elutes with eluent, it is thus achieved that recombinant trypsin crude product solution, chromatographs the rate of recovery
91.5%.Wherein, the compound method of eluent is: take respectively acetic acid aqueous solution that volume fraction is 10% and
Volume fraction is the ethanol water of 90%, mixes with the volume ratio of 1:0.7.
Take recombinant trypsin crude product solution loading and use GE to cation-exchange chromatography resin column, filler
SP-sepharose ff, rinses to baseline with the acetic acid aqueous solution that volume fraction is 2%, carries out gradient elution,
Obtain recombinant trypsin solution, chromatograph the rate of recovery 92.9%.Wherein, the compound method of eluent is: point
Do not take acetic acid aqueous solution that volume fraction is 4% and molar concentration is the NaCl solution of 1mol/L, with 1:0.8
Volume ratio mixing.
The purifying of embodiment 6 recombinant trypsin
The solution containing recombinant trypsin that taking the embodiment of the present invention 2～4 provides filters, and then goes up
Sample uses Rohmhaas XAD1180 to large pore resin absorption column, filler, and loading flow velocity is not more than 300
Cm/h, rinses macroporous absorption tree with the Tris-HCl aqueous solution of the 20mmol/L of pH8.0 after end of the sample
Fat post is to baseline, then, elutes with eluent, it is thus achieved that recombinant trypsin crude product solution, chromatography
The rate of recovery 90.5%.Wherein, the compound method of eluent is: take the acetic acid that volume fraction is 10% respectively
The aqueous solution and volume fraction are the ethanol water of 90%, mix with the volume ratio of 1:1.
Take recombinant trypsin crude product solution loading to use to cation-exchange chromatography resin column, filler
SP-sepharose HP, rinses to baseline with the acetic acid aqueous solution that volume fraction is 2%, carries out gradient and wash
De-, it is thus achieved that recombinant trypsin solution, chromatograph the rate of recovery 94.5%.Wherein, the compound method of eluent is:
Take acetic acid aqueous solution that volume fraction is 4% respectively and molar concentration is the NaCl solution of 1mol/L, with
1:0.9 volume ratio mixing.
The purifying of embodiment 7 recombinant trypsin
The solution containing recombinant trypsin that taking the embodiment of the present invention 2～4 provides filters, and then goes up
Sample uses Rohmhaas XAD16 to large pore resin absorption column, filler, and loading flow velocity is not more than 300cm/h,
Large pore resin absorption column is rinsed extremely with the Tris-HCl aqueous solution of the 20mmol/L of pH8.0 after end of the sample
Baseline, then, elutes with eluent, it is thus achieved that recombinant trypsin crude product solution, chromatographs the rate of recovery
88.6%.Wherein, the compound method of eluent is: take respectively acetic acid aqueous solution that volume fraction is 10% and
Volume fraction is the ethanol water of 90%, mixes with the volume ratio of 1:1.2.
Take recombinant trypsin crude product solution loading to use to cation-exchange chromatography resin column, filler
SP-sepharose XL, rinses to baseline with the acetic acid aqueous solution that volume fraction is 2%, carries out gradient and wash
De-, it is thus achieved that recombinant trypsin solution, chromatograph the rate of recovery 90.4%.Wherein, the compound method of eluent
For: take acetic acid aqueous solution that volume fraction is 4% respectively and molar concentration is the NaCl solution of 1mol/L,
Mix with the volume ratio of 1:1.2.
The purifying of embodiment 8 recombinant trypsin
The solution containing recombinant trypsin that taking the embodiment of the present invention 2～4 provides filters, and then goes up
Sample uses Rohmhaas XAD7HP to large pore resin absorption column, filler, and loading flow velocity is not more than 300
Cm/h, rinses macroporous absorption tree with the Tris-HCl aqueous solution of the 20mmol/L of pH8.0 after end of the sample
Fat post is to baseline, then, elutes with eluent, it is thus achieved that recombinant trypsin crude product solution, chromatography
The rate of recovery 90.7%.Wherein, the compound method of eluent is: take the acetic acid that volume fraction is 10% respectively
The aqueous solution and volume fraction are the ethanol water of 90%, mix with the volume ratio of 1:1.
Take recombinant trypsin crude product solution loading and use SP to cation-exchange chromatography resin column, filler
Sephadex C-25, rinses to baseline with the acetic acid aqueous solution that volume fraction is 2%, carries out gradient elution,
Obtain recombinant trypsin solution, chromatograph the rate of recovery 91.1%.Wherein, the compound method of eluent is: point
Do not take acetic acid aqueous solution that volume fraction is 4% and molar concentration is the NaCl solution of 1mol/L, with 1:0.9
Volume ratio mixing.
The purifying of embodiment 9 recombinant trypsin
The solution containing recombinant trypsin that taking the embodiment of the present invention 2～4 provides filters, and then goes up
Sample uses Rohmhaas XAD7HP to large pore resin absorption column, filler, and loading flow velocity is not more than 300
Cm/h, rinses macroporous absorption tree with the Tris-HCl aqueous solution of the 20mmol/L of pH8.0 after end of the sample
Fat post is to baseline, then, elutes with eluent, it is thus achieved that recombinant trypsin crude product solution, chromatography
The rate of recovery 90.3%.Wherein, the compound method of eluent is: take the acetic acid that volume fraction is 10% respectively
The aqueous solution and volume fraction are the ethanol water of 90%, mix with the volume ratio of 1:1.
Take recombinant trypsin crude product solution loading and use SP to cation-exchange chromatography resin column, filler
Sephadex C-50, rinses to baseline with the acetic acid aqueous solution that volume fraction is 2%, carries out gradient elution,
Obtain recombinant trypsin solution, chromatograph the rate of recovery 90.7%.Wherein, the compound method of eluent is: point
Do not take acetic acid aqueous solution that volume fraction is 4% and molar concentration is the NaCl solution of 1mol/L, with 1:1
Volume ratio mixing.
The purifying of embodiment 10 recombinant trypsin
The solution containing recombinant trypsin that taking the embodiment of the present invention 2～4 provides filters, and then goes up
Sample uses Rohmhaas XAD16 to large pore resin absorption column, filler, and loading flow velocity is not more than 300cm/h,
Large pore resin absorption column is rinsed extremely with the Tris-HCl aqueous solution of the 20mmol/L of pH8.0 after end of the sample
Baseline, then, elutes with eluent, it is thus achieved that recombinant trypsin crude product solution, chromatographs the rate of recovery
90.0%.Wherein, the compound method of eluent is: take respectively acetic acid aqueous solution that volume fraction is 10% and
Volume fraction is the ethanol water of 90%, mixes with the volume ratio of 1:1.1.
Take recombinant trypsin crude product solution loading and use tosoh to cation-exchange chromatography resin column, filler
SP550, rinses to baseline with the acetic acid aqueous solution that volume fraction is 2%, carries out gradient elution, it is thus achieved that
Recombinant trypsin solution, chromatographs the rate of recovery 93.0%.Wherein, the compound method of eluent is: take respectively
Volume fraction be 4% acetic acid aqueous solution and molar concentration be the NaCl solution of 1mol/L, with 1:0.9's
Volume ratio mixes.
The purifying of embodiment 11 recombinant trypsin
The solution containing recombinant trypsin that taking the embodiment of the present invention 2～4 provides filters, and then goes up
Sample uses Rohmhaas XAD1180 to large pore resin absorption column, filler, and loading flow velocity is not more than 300
Cm/h, rinses macroporous absorption tree with the Tris-HCl aqueous solution of the 20mmol/L of pH8.0 after end of the sample
Fat post is to baseline, then, elutes with eluent, it is thus achieved that recombinant trypsin crude product solution, chromatography
The rate of recovery 89.6%.Wherein, the compound method of eluent is: take the acetic acid that volume fraction is 10% respectively
The aqueous solution and volume fraction are the ethanol water of 90%, mix with the volume ratio of 1:1.
Take recombinant trypsin crude product solution loading to use to cation-exchange chromatography resin column, filler
Bio-Rad UNOsphere s, rinses to baseline with the acetic acid aqueous solution that volume fraction is 2%, carries out ladder
Degree wash-out, it is thus achieved that recombinant trypsin solution, chromatographs the rate of recovery 92.3%.Wherein, the preparation side of eluent
Method is: take acetic acid aqueous solution that volume fraction is 4% respectively and molar concentration is the NaCl solution of 1mol/L,
Mix with the volume ratio of 1:0.9 respectively.
The purifying of embodiment 12 recombinant trypsin
The solution containing recombinant trypsin that taking the embodiment of the present invention 2～4 provides filters, and then goes up
Sample uses Rohmhaas XAD1180 to large pore resin absorption column, filler, and loading flow velocity is not more than 300
Cm/h, rinses macroporous absorption tree with the Tris-HCl aqueous solution of the 20mmol/L of pH8.0 after end of the sample
Fat post is to baseline, then, elutes with eluent, it is thus achieved that recombinant trypsin crude product solution, chromatography
The rate of recovery 89.0%.Wherein, the compound method of eluent is: take the acetic acid that volume fraction is 10% respectively
The aqueous solution and volume fraction are the ethanol water of 90%, mix with the volume ratio of 1:1.
Take recombinant trypsin crude product solution loading to use to cation-exchange chromatography resin column, filler
Bio-Rad rapid s, rinses to baseline with the acetic acid aqueous solution that volume fraction is 2%, carries out gradient and wash
De-, it is thus achieved that recombinant trypsin solution, chromatograph the rate of recovery 92.0%.Wherein, the compound method of eluent is:
Take acetic acid aqueous solution that volume fraction is 4% respectively and molar concentration is the NaCl solution of 1mol/L, with
1:0.9 volume ratio mixing.
The purifying of embodiment 13 recombinant trypsin
The solution containing recombinant trypsin that taking the embodiment of the present invention 2～4 provides filters, and then goes up
Sample uses Rohmhaas XAD1180 to large pore resin absorption column, filler, and loading flow velocity is not more than 300
Cm/h, rinses macroporous absorption tree with the Tris-HCl aqueous solution of the 20mmol/L of pH8.0 after end of the sample
Fat post is to baseline, then, elutes with eluent, it is thus achieved that recombinant trypsin crude product solution, chromatography
The rate of recovery 88.5%.Wherein, the compound method of eluent is: take the acetic acid that volume fraction is 10% respectively
The aqueous solution and volume fraction are the ethanol water of 90%, mix with the volume ratio of 1:1.
Take recombinant trypsin crude product solution loading to use to cation-exchange chromatography resin column, filler
Bio-Rad high s, rinses to baseline with the acetic acid aqueous solution that volume fraction is 2%, carries out gradient elution,
Obtain recombinant trypsin solution, chromatograph the rate of recovery 91.5%.Wherein, the compound method of eluent is: point
Do not take acetic acid aqueous solution that volume fraction is 4% and molar concentration is the NaCl solution of 1mol/L, with 1:0.8
Volume ratio mixing.
The recombinant trypsin purity detecting that embodiment 14 present invention provides
The recombinant trypsin purity providing the embodiment of the present invention 5～13 detects, and detection uses
SDS-PAGE electrophoresis, wherein, embodiment 5 provide recombinant trypsin electrophoresis result as it is shown in figure 1,
Wherein, swimming lane M is protein low-molecular-weight Marker；Swimming lane 1 is recombinant trypsin mark product, swimming lane
2 recombinant trypsin provided for the embodiment of the present invention 5.The restructuring tryptose that embodiment 6 to 13 provides
The electrophoresis result of enzyme is consistent with the above results.
Result shows, the recombinant trypsin stripe size that the embodiment of the present invention 5～13 provides is consistent with mark product,
Meeting desired value, band is clear, bright, other bands does not occur, indicate with the present invention in swimming lane
Recombinant trypsin purity prepared by the method that embodiment provides is good.
The recombinant trypsin Activity determination that embodiment 15 present invention provides
The recombinant trypsin activity providing the embodiment of the present invention 5～13 detects, and takes commercially available heavy simultaneously
Group trypsase sterling carries out Enzyme assay, using result as comparison.
Detect by making recombinant trypsin and benzoyl L-arginine ethyl ester (benzoyl L-arginine
Ethylester, BAEE) react, ultraviolet specrophotometer is measured OD253Value increasing in time
Dosage also calculates.
Result shows, the activity of recombinant trypsin is 12000BAEEU/ (mg pro), commercially available restructuring pancreas
The activity of protease sterling is 7500BAEEU/ (mg pro), it was demonstrated that the method provided with the present invention purifies
Recombinant trypsin has higher activity.
The above is only the preferred embodiment of the present invention, it is noted that general for the art
For logical technical staff, under the premise without departing from the principles of the invention, it is also possible to make some improvement and profit
Decorations, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (7)
1. the purification process of a recombinant trypsin, it is characterised in that comprise the following steps:
Step 1: obtain the zymotic fluid former containing recombinant trypsin；
Step 2: take the described zymotic fluid former containing recombinant trypsin, micro-filtration, centrifugal or be filtrated to get
Supernatant, activation, it is thus achieved that containing the solution of recombinant trypsin；
Step 3: take the described solution containing recombinant trypsin, through macroporous adsorption resin chromatography and sun from
Sub-displacement chromatography, be dried, to obtain final product.
Method the most according to claim 1, it is characterised in that described in step 2 activation particularly as follows:
Take described supernatant, add CaCl2And regulate pH to 7.0～8.5, activate 8 hours～24 little under room temperature
Time.
Method the most according to claim 2, it is characterised in that described CaCl2At described restructuring pancreas
Final concentration of 10mmol/L～100mmol/L in the solution of protease.
Method the most according to claim 1, it is characterised in that described macroporous adsorption resin chromatography is adopted
Filler be Rohmhaas XAD4, Rohmhaas XAD1180, Rohmhaas XAD16 or
Rohmhaas XAD7HP。
Method the most according to claim 1, it is characterised in that described macroporous adsorption resin chromatography is adopted
The mixed liquor that eluent is ethanol, acetic acid and water.
Method the most according to claim 1, it is characterised in that described cation-exchange chromatography uses
Filler be: GE SP-sepharose ff, GE SP-sepharose HP, GE SP-sepharose XL,
GE SP-sephadex C-25、GE SP-sephadex C-50、tosoh SP550、Bio-Rad UNOsphere
S, Bio-Rad rapid s or Bio-Rad high s.
Method the most according to claim 1, it is characterised in that described cation-exchange chromatography uses
The aqueous solution that eluent is acetic acid and NaCl.
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| 双柱吸附法对莲子心总碱的分离纯化研究;张吉祥等;《离子交换与吸附》;20081231;第24卷(第6期);512-517 * |
| 汤伟等.大孔吸附树脂法及离子交换层析柱法精制纯化丹参多糖的研究.《中药材》.2010,第33卷(第12期),1937-1941. * |
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