CN109503704A - A kind of recombinant human iron separation and purification of protein method - Google Patents
A kind of recombinant human iron separation and purification of protein method Download PDFInfo
- Publication number
- CN109503704A CN109503704A CN201811622011.8A CN201811622011A CN109503704A CN 109503704 A CN109503704 A CN 109503704A CN 201811622011 A CN201811622011 A CN 201811622011A CN 109503704 A CN109503704 A CN 109503704A
- Authority
- CN
- China
- Prior art keywords
- buffer
- chromatographic column
- elution buffer
- bestarose
- purification
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 title claims abstract description 66
- 238000000746 purification Methods 0.000 title claims abstract description 34
- 238000000926 separation method Methods 0.000 title claims abstract description 34
- 229910052742 iron Inorganic materials 0.000 title claims abstract description 33
- 238000001814 protein method Methods 0.000 title claims abstract description 29
- 239000012149 elution buffer Substances 0.000 claims abstract description 60
- 238000008416 Ferritin Methods 0.000 claims abstract description 47
- 102000008857 Ferritin Human genes 0.000 claims abstract description 47
- 108050000784 Ferritin Proteins 0.000 claims abstract description 47
- 239000000872 buffer Substances 0.000 claims abstract description 45
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 24
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 20
- 238000005571 anion exchange chromatography Methods 0.000 claims abstract description 16
- 239000000706 filtrate Substances 0.000 claims abstract description 15
- 241000894006 Bacteria Species 0.000 claims abstract description 12
- 238000005277 cation exchange chromatography Methods 0.000 claims abstract description 9
- 241000588724 Escherichia coli Species 0.000 claims abstract description 6
- 238000005119 centrifugation Methods 0.000 claims abstract description 5
- 239000007788 liquid Substances 0.000 claims abstract description 5
- 238000001914 filtration Methods 0.000 claims abstract description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 96
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 40
- 238000001514 detection method Methods 0.000 claims description 28
- 238000002523 gelfiltration Methods 0.000 claims description 27
- 229910003460 diamond Inorganic materials 0.000 claims description 24
- 239000010432 diamond Substances 0.000 claims description 24
- 239000007983 Tris buffer Substances 0.000 claims description 21
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 21
- 239000011780 sodium chloride Substances 0.000 claims description 20
- 239000012501 chromatography medium Substances 0.000 claims description 19
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 17
- 229920002684 Sepharose Polymers 0.000 claims description 16
- 239000012535 impurity Substances 0.000 claims description 15
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 14
- 238000004255 ion exchange chromatography Methods 0.000 claims description 13
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 claims description 12
- 238000011068 loading method Methods 0.000 claims description 12
- 238000012545 processing Methods 0.000 claims description 9
- 239000011324 bead Substances 0.000 claims description 8
- 125000002091 cationic group Chemical group 0.000 claims description 7
- 239000002609 medium Substances 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000004140 cleaning Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 229920002271 DEAE-Sepharose Polymers 0.000 claims description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 4
- 239000013622 capto Q Substances 0.000 claims description 4
- 238000005374 membrane filtration Methods 0.000 claims description 4
- 239000012619 Butyl Sepharose® Substances 0.000 claims description 2
- 239000012564 Q sepharose fast flow resin Substances 0.000 claims description 2
- 150000001450 anions Chemical class 0.000 claims description 2
- 239000012618 butyl sepharose high performance Substances 0.000 claims description 2
- 235000012489 doughnuts Nutrition 0.000 claims description 2
- 230000005611 electricity Effects 0.000 claims 3
- 102000004169 proteins and genes Human genes 0.000 abstract description 19
- 108090000623 proteins and genes Proteins 0.000 abstract description 19
- 238000011084 recovery Methods 0.000 abstract description 11
- 238000000855 fermentation Methods 0.000 abstract 1
- 230000004151 fermentation Effects 0.000 abstract 1
- 238000000034 method Methods 0.000 description 21
- 238000010828 elution Methods 0.000 description 5
- 150000002500 ions Chemical class 0.000 description 5
- 238000007689 inspection Methods 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 238000010612 desalination reaction Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 2
- 238000000265 homogenisation Methods 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- IYLLULUTZPKQBW-UHFFFAOYSA-N Acrinol Chemical compound CC(O)C(O)=O.C1=C(N)C=CC2=C(N)C3=CC(OCC)=CC=C3N=C21 IYLLULUTZPKQBW-UHFFFAOYSA-N 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 239000007832 Na2SO4 Substances 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- VEQOALNAAJBPNY-UHFFFAOYSA-N antipyrine Chemical compound CN1C(C)=CC(=O)N1C1=CC=CC=C1 VEQOALNAAJBPNY-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000002059 diagnostic imaging Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002539 nanocarrier Substances 0.000 description 1
- 229960005222 phenazone Drugs 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- JBJWASZNUJCEKT-UHFFFAOYSA-M sodium;hydroxide;hydrate Chemical compound O.[OH-].[Na+] JBJWASZNUJCEKT-UHFFFAOYSA-M 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of recombinant human iron separation and purification of protein methods, comprising the following steps: obtains bacterium mud after the centrifugation of Escherichia coli bacteria liquid after fermentation, bacterium mud heated after being dissolved in buffer, is centrifuged or filtration treatment, and filtrate is obtained；The filtrate that step 1) obtains is separated using anion chromatography, collects the elution buffer containing destination protein；The elution buffer containing destination protein is obtained to step 2) using cation chromatography and carries out secondary separation, collects the elution buffer containing destination protein；It is separated three times using elution buffer of the hydrophobic chromatography to the ferritin containing purpose that step 3) obtains, collects the elution buffer containing destination protein.The present invention has the advantages that easy to operate, destination protein rate of recovery height, with high purity.
Description
[technical field]
The present invention relates to the technical field of protein separation, especially a kind of recombinant human iron separation and purification of protein method
Technical field.
[background technique]
Ferritin (Ferritin) is widely present in various organisms, is formed by 24 subunits are spontaneous, and caged albumen is multiple
Whole body structure is a kind of natural polymeric biomaterial.It has the function of storage ferro element, anti-oxidant etc. in vivo,
But as a kind of natural high molecular material, it also can be used as nano-carrier and be widely used in drug conveying, medical imaging etc., while iron
Albumen can be used for the Clinics and Practices of tumour in conjunction with the TfR of high efficient expression in tumour cell.Ferritin can
High efficient expression is obtained in Escherichia coli with transgenic technology, then carries out isolating and purifying for the broken realization albumen of somatic cells.
Traditional ferritin isolation and purification method mostly uses greatly the precipitation method, including ethanol precipitation, rivanol precipitating and ammonium sulfate precipitation, but
It is all lower using ferritin relative purity and the rate of recovery made from the precipitation method, after gradually adopt chromatography replace the precipitation method, pass through
The chromatography schemes such as ion-exchange chromatography and hydrophobic chromatography isolate and purify ferritin.But existing some chromatography exist back
Yield is low, and every step chromatography requires to carry out that desalination dilution operation is relatively complicated to be unfavorable for being mass produced, and the purity of ferritin is also
The rank of medical supplies cannot be reached.
[summary of the invention]
The object of the invention is to solve the problems of the prior art, a kind of recombinant human iron separation and purification of protein side is proposed
Method can make ferritin isolation and purification method is easy to operate to be suitble to be mass produced, and protein recovery is high, purity is high.
To achieve the above object, the invention proposes a kind of recombinant human iron separation and purification of protein methods, comprising the following steps:
1) bacterium mud is obtained after the Escherichia coli bacteria liquid centrifugation after fermenting, bacterium mud is heated after being dissolved in buffer, is centrifuged
Or filtration treatment, obtain filtrate；
2) filtrate that step 1) obtains is separated using anion chromatography, collects the elution buffer of ferritin；
3) secondary separation is carried out using the elution buffer that cation chromatography obtains ferritin to step 2), collects iron content
The elution buffer of albumen；
4) it is separated three times using elution buffer of the hydrophobic chromatography to the ferritin that step 3) obtains, collects iron content
The elution buffer of albumen.
Preferably, the buffer of dissolution bacterium mud is 5-100mM PB or Tris pH7.0, the step in the step 1)
Rapid 1) the middle temperature range heated and time are respectively 70-75 DEG C, 5-15min, and filtration treatment uses in the step 1)
0.22um doughnut or membrane filtration processing mode.
Preferably, the step 2) specifically: take the chromatographic column equipped with anion chromatography medium, handled with 0.5MNaOH
Chromatographic column balances loading after chromatographic column with combination buffer later, then with cleaning buffer solution cleans impurity, with elution buffer into
Row elution, collects the elution buffer of ferritin, cleans the stronger impurity of binding ability with 0.5M NaOH, use 10mM
NaOH saves chromatographic column.
Preferably, anion chromatography medium is Q Bestarose High Performance, Q in the step 2)
Sepharose High Performance、DEAE Bestarose High Performance、DEAE Sepharose
High Performance、SOURCE Q、Q Bestarose Fast Flow、Q Sepharose Fast Flow、DEAE
Bestarose Fast Flow、DEAE Sepharose Fast Flow、Q Bestarose Big Beads、Q
sepharose Big Beads、Q Bestarose Big Beads、Q sepharose Big Beads、Diamond Q、
Capto Q, Diamond Q mustang, Capto Q mustang, Capto DEAE, Diamond DEAE or Diamond
One of DEAE mustang；Combination buffer is 5-100mM PB or Tris pH7.0 in the step 2)；The step
2) cleaning buffer solution is one in 5-100mM PB or MES pH6.0 or 5-100mM PB or Tris 0.1M NaCl pH7.0 in
Kind；Elution buffer is 5-100mM NaAC pH4.5 or 5-100mMPB 0.25MNaCl pH7.0 in the step 2)；It is described
The PH and conductance that combination buffer balances chromatographic column to efflux in step 2) keep one with the PH of combination buffer and conductance
It causes.
Preferably, the step 3) is specially；The chromatographic column equipped with cationic chromatography media is taken, is handled with 0.5MNaOH
Chromatographic column balances loading after chromatographic column with combination buffer later, then is eluted with elution buffer, collects ferritin
Elution buffer cleans the stronger impurity of binding ability with 0.5M NaOH, saves chromatographic column with 10mM NaOH.
Preferably, step 3) the middle-jiao yang, function of the spleen and stomach ion chromatography medium is SP Bestarose High Performance, SP
Sepharose High Performance、Diamond MMC、Capto MMC、Diamond MMC mustang、Capto
MMC impres、SP Bestarose Fast Flow、SP Sepharose Fast Flow、Capto SP impres、
One of Diamond SP mustang, CM Bestarose Fast Flow or CM Sepharose Fast Flow；Institute
Stating combination buffer in step 3) is 5-100mM NaAc pH5.0 or 5-100mM NaAc 0.2M NaCl pH5.0；The step
It is rapid 3) in elution buffer be 5-100mM PB 0.01M NaCl pH6.0 or 5-100mM PB pH6.0；In the step 3)
The PH and conductance of combination buffer balance chromatographic column to efflux are consistent with the PH of combination buffer and conductance.
Preferably, the step 4) is specially；The chromatographic column equipped with hydrophobic chromatoghaphy medium is taken, is handled with 0.5M NaOH
Chromatographic column balances loading after chromatographic column with combination buffer later, then is eluted with elution buffer, collects ferritin
Elution buffer, elutes impurity with pure water, cleans chromatographic column with 0.5M NaOH, saves chromatographic column with 10mM NaOH.
Preferably, in the step 4) hydrophobic chromatoghaphy medium be Butyl Bestarose High Performance,
Butyl Sepharose High Performance、Diamond Butyl mustang、Capto Butyl impres、
Butyl Bestarose Fast Flow、Phenyl Sepharose Fast Flow HS、Phenyl Sepharose Fast
Flow LS、Phenyl Bestarose Fast Flow HS、Phenyl Bestarose Fast Flow LS、Butyl
Sepharose Fast Flow、Diamond butyl、Capto butyl、Capto Phenyl HS、Capto Phenyl
One of impres, Diamond Phenyl mustang or Diamond Phenyl；Combination buffer in the step 4)
For 5-100mM PB or Tris0.8-2M Na2SO4pH7.0,5-100mM PB or Tris 1.5-3M NaCl pH7.0,5-
100mM PB or Tris 0.8-2.5M (NH4)2SO4PH7.0 or 5-100mM PB or Tris 0.8-2M K2In HPO4pH7.0
It is a kind of；Elution buffer is 5-100mM PB or Tris 0.2-0.5M Na2SO4pH7.0,5-100mM PB in the step 4)
Or Tris 0.8-1.2M NaCl pH7.0,5-100mM PB or Tris 0.4-0.8M (NH4)2SO4pH7.0、5-100mM PB
Or Tris 0.4-0.8M K2One of HPO4pH7.0；Combination buffer balances chromatographic column to efflux in the step 4)
PH and conductance be consistent with the PH of combination buffer and conductance.
Preferably, the step 1), step 2), step 3) and step 4) are both needed to carry out albumen Indexs measure.
Preferably, the albumen index Indexs measure uses gel filtration detection and ion chromatography detection mode.
Beneficial effects of the present invention: the present invention is using anion chromatography, cation chromatography and three step chromatography side of hydrophobic chromatography
Ferritin purity made from formula and the rate of recovery are high, purity >=97%, can reach medical supplies rank, do not needed between every step chromatography into
The cumbersome desalination dilution operation of row, method is easy to operate, is suitble to large-scale production, by using gel filtration detection and ion color
Spectrum detection detects every step chromatographic elution sample, increases the reliability of ferritin isolation and purification method.
Feature and advantage of the invention will be described in detail by embodiment combination attached drawing.
[Detailed description of the invention]
Fig. 1 is a kind of gel mistake of the step 1) process of the embodiment 1 of recombinant human iron separation and purification of protein method of the present invention
Filter detection figure；
Fig. 2 is at a kind of step 2) anion chromatography of the embodiment 1 of recombinant human iron separation and purification of protein method of the present invention
Reason process gel filtration detection figure；
Fig. 3 is at a kind of cationic chromatography of step 3) of the embodiment 1 of recombinant human iron separation and purification of protein method of the present invention
Reason process gel filtration detection figure；
Fig. 4 is a kind of step 4) the hydrophobic chromatography processing of embodiment 1 of recombinant human iron separation and purification of protein method of the present invention
Process gel filtration detection figure；
Fig. 5 is a kind of step 1) process gel filtration of the embodiment 2 of recombinant human iron separation and purification of protein method of the present invention
Detection figure；
Fig. 6 is at a kind of step 2) anion chromatography of the embodiment 2 of recombinant human iron separation and purification of protein method of the present invention
Reason process gel filtration detection figure；
Fig. 7 is at a kind of cationic chromatography of step 3) of the embodiment 2 of recombinant human iron separation and purification of protein method of the present invention
Reason process gel filtration detection figure；
Fig. 8 is a kind of step 4) the hydrophobic chromatography processing of embodiment 2 of recombinant human iron separation and purification of protein method of the present invention
Process gel filtration detection figure；
Fig. 9 is the ion of filtrate sample before a kind of three step Image processings of recombinant human iron separation and purification of protein method of the present invention
Detection figure；
Figure 10 is the ion inspection of sample after a kind of three step Image processings of recombinant human iron separation and purification of protein method of the present invention
Mapping；
Figure 11 is a kind of blank ion detection comparative diagram of recombinant human iron separation and purification of protein method of the present invention.
[specific embodiment]
Embodiment 1, a kind of recombinant human iron separation and purification of protein method of the present invention, comprising the following steps:
1) bacterium mud will be obtained after the centrifugation of 10g escherichia coli fermented broth, bacterium mud is that 20mM PB is added in 1:10 by mass volume ratio
PH7.0, after stirring evenly, high-pressure homogenization is twice broken under 800bar environment, is heated to 72 DEG C and constant heating 5min, 12000g are centrifuged
Lower centrifugal treating 10min is acted on, supernatant is removed later, with 0.22um membrane filtration, obtains filtrate, gel filtration inspection is carried out to filtrate
It surveys and ion chromatography detects；
2) anion chromatography is handled: being taken 30ml anion chromatography MEDIUM Q Bestarose High Performance, is filled
Into BXK16/20 chromatographic column, following chromatography process is carried out on AKTA protein purification instrument, handles anion with 0.5M NaOH
Chromatography media, then maintained an equal level position with 120ml combination buffer 50mM PBpH7.0 balance chromatographic column to baseline, take 100ml's
The filtrate tune PH to 7.0 obtained by step 1), loading clean impurity with 100ml cleaning buffer solution 25mM PB pH6.0, use
100ml elution buffer 50mM PB 0.25M NaCl pH7.0 elution purpose ferritin and the elution buffer for collecting ferritin
Liquid cleans impurity with 40ml 25mM PB 1M NaCl pH7.0, cleans pillar with 40ml 0.5M NaOH later, wash away and do not wash
De- combination foreign protein is balanced with 10mM NaOH and saves chromatographic column, carries out gel filtration inspection to the elution buffer of ferritin
It surveys and ion chromatography detects, it is 97% which, which obtains destination protein purity in the elution buffer of ferritin,
The rate of recovery is 76.8%, carrying capacity 30mg/ml；
3) cationic Image processing: appropriate cation chromatography media SP Bestarose High Performance dress is taken
Enter BXK16/20, column bed height is 11.3cm, chromatographic column is handled with 40ml 0.5M NaOH, with combination buffer 50mM NaAc
PH5.0 balances chromatographic column, takes the elution buffer sample 145ml of the ferritin obtained by step 2), adjusts pH to 5.0, loading,
It is cleaned with combination buffer, elutes destination protein with 60ml elution buffer 20mM PB 0.01MNaCl pH6.0, it is pure with 3CV
Pillar is cleaned with 3CV NaOH after water elution impurity, 10mM NaOH saves chromatographic column, carries out to the elution buffer of ferritin
Gel filtration detection and ion chromatography detection, it is pure which chromatographs to obtain destination protein in the elution buffer of ferritin
Degree is 97.7%, the rate of recovery 99.3%, carrying capacity 10mg/ml；
4) hydrophobic chromatography is handled: appropriate hydrophobic chromatoghaphy medium Butyl Bestarose High Performance being taken to be packed into
BXK16/20, column bed height are 11cm, chromatographic column are handled with 40ml 0.5M NaOH, with combination buffer 20mM PB 1.0M
Na2SO4pH7.0 balances chromatographic column, takes the elution buffer sample 10ml of the ferritin obtained by step 3), sample adds
Na2SO4 tune conductance is extremely identical as the conductance of combination buffer, adjusts pH7.0, and loading is cleaned with combination buffer, uses elution buffer
Liquid 20mM PB 0.5M Na2SO4pH7.0 elutes destination protein, cleans column with 60ml NaOH after eluting impurity with 60ml pure water
Son, 10mM NaOH save chromatographic column, carry out gel filtration detection to the elution buffer of ferritin and ion chromatography detects,
It is 98.7% that the step hydrophobic chromatography, which obtains destination protein purity in the elution buffer of ferritin, and the rate of recovery 68.2% carries
Amount is 20mg/ml；
It is 27.89min that step 1) can get filtrate sample gel filtration testing goal albumen appearance time refering to fig. 1；
Step 2) can get washing using anion chromatography MEDIUM Q Bestarose High Performance refering to Fig. 2
De- buffer sample gel filtration testing goal albumen appearance time is 27.92min；
Step 3) can get refering to Fig. 3 using cation chromatography media SP Bestarose High Performance's
Elution buffer sample gel filtration testing goal albumen appearance time is 27.85min；
Step 4) can get refering to Fig. 4 using hydrophobic chromatoghaphy medium Butyl Bestarose High Performance's
Elution buffer sample gel filtration testing goal albumen appearance time is 27.96min.
Embodiment 2, a kind of recombinant human iron separation and purification of protein method of the present invention, comprising the following steps:
1) bacterium mud will be obtained after the centrifugation of 10g escherichia coli fermented broth, bacterium mud is that 20mM PB is added in 1:10 by mass volume ratio
PH7.0, after stirring evenly, high-pressure homogenization is twice broken under 800bar environment, is heated to 72 DEG C and constant heating 5min, 12000g are centrifuged
Lower centrifugal treating 10min is acted on, supernatant is removed later, with 0.22um membrane filtration, obtains filtrate, gel filtration inspection is carried out to filtrate
It surveys and ion chromatography detects；
2) anion chromatography is handled: 40ml anion chromatography medium DEAE Bestarose High Performance is taken,
Be attached in BXK16/20 chromatographic column, following chromatography process carries out on AKTA protein purification instrument, with 0.5M NaOH handle yin from
Sub- chromatography media, then maintained an equal level position with 120ml combination buffer 50mM PB pH7.0 balance chromatographic column to baseline, take 100ml
The filtrate tune PH to 7.0 obtained by step 1), loading is clear with 100ml cleaning buffer solution 50mM PB 0.1M NaCl pH7.0
Impurity is washed, elute purpose ferritin with 120ml elution buffer 50mM PB 0.25M NaCl pH7.0 and collects ferritin
Elution buffer, clean impurity with the 50mM PB 1M NaCl pH7.0 of 40ml, clean column with 40ml 0.5MNaOH later
Son washes away the binding protein not eluted, is balanced with 10mM NaOH and saves chromatographic column, carries out to the elution buffer of ferritin
Gel filtration detection and ion chromatography detection, it is pure which obtains destination protein in the elution buffer of ferritin
Degree is 76.48%, the rate of recovery 80%, carrying capacity 30mg/ml；
3) cationic Image processing: taking appropriate cation chromatography media Diamond MMC mustang to be packed into BXK16/20,
Column bed height is 13cm, chromatographic column is handled with 40ml 0.5M NaOH, with combination buffer 20mMNaAc 0.2M NaCl
PH5.0 balances chromatographic column, takes the elution buffer sample 145ml of the ferritin obtained by step 2), sample is diluted with pure water
It is extremely identical with combination buffer conductance, PH to 7.0 is adjusted, loading is cleaned with combination buffer, with 40ml elution buffer 20mM
PB pH6.0 elutes destination protein, cleans pillar with the NaOH of 40ml, 10mM NaOH saves chromatographic column, washes to ferritin
De- buffer carries out gel filtration detection and ion chromatography detection, which chromatographs to obtain the elution buffer of ferritin
Middle destination protein purity is 94%, the rate of recovery 88.6%, carrying capacity 20mg/ml；
4) hydrophobic chromatography is handled: appropriate hydrophobic chromatoghaphy medium Phenyl Bestarose Fast Flow HS being taken to be packed into
BXK16/20, column bed height are 10.5cm, chromatographic column are handled with 40ml 0.5M NaOH, with combination buffer 20mM PB 2M
NaCl pH7.0 balances chromatographic column, takes the elution buffer sample 10ml of the ferritin obtained by step 3), sample adds NaCl
It adjusts conductance extremely identical as the conductance of combination buffer, adjusts pH7.0, loading is cleaned with combination buffer, with elution buffer 20mM
PB 1M NaCl pH7.0 elutes destination protein, cleans pillar, 10mM with 60ml NaOH after eluting impurity with 60ml pure water
NaOH saves chromatographic column, carries out gel filtration detection to the elution buffer of ferritin and ion chromatography detects, the step is hydrophobic
It is 98.47% that chromatography, which obtains destination protein purity in the elution buffer of ferritin, the rate of recovery 61%, and carrying capacity is
17.76mg/ml；
It is 27.89min that step 1), which can get filtrate sample gel filtration testing goal albumen appearance time refering to Fig. 5,；
Step 2) can get refering to Fig. 6 using anion chromatography medium DEAE Bestarose HighPerformance's
Elution buffer sample gel filtration testing goal albumen appearance time is 27.86min；
Step 3) can get the elution buffer using cation chromatography media Diamond MMC mustang refering to Fig. 7
Sample gel filtration testing goal albumen appearance time is 27.91min；
Step 4) can get washing using hydrophobic chromatoghaphy medium Phenyl Bestarose Fast Flow HS refering to Fig. 8
De- buffer sample gel filtration testing goal albumen appearance time is 28.04min.
A kind of recombinant human iron separation and purification of protein method of the present invention, the present invention using anion chromatography, cation chromatography and
Ferritin purity made from three step chromatography scheme of hydrophobic chromatography and the rate of recovery are high, and refering to Fig. 9 to 11, main peak becomes in ion detection figure
Greatly, miscellaneous peak is smaller and smaller, purity >=97%, can reach medical supplies rank, does not need to carry out cumbersome desalination between every step chromatography
Dilution operation, method is easy to operate, is suitble to large-scale production, by using gel filtration detection and ion chromatography detection to every step
Chromatographic elution sample is detected, and the reliability of ferritin isolation and purification method is increased.
Above-described embodiment is the description of the invention, is not limitation of the invention, after any pair of simple transformation of the present invention
Scheme all belong to the scope of protection of the present invention.
Claims (10)
1. a kind of recombinant human iron separation and purification of protein method, it is characterised in that: the following steps are included:
1) bacterium mud is obtained after the Escherichia coli bacteria liquid centrifugation after fermenting, and bacterium mud heated after being dissolved in buffer, is centrifuged or mistake
Filter processing, obtains filtrate；
2) filtrate that step 1) obtains is separated using anion chromatography, collects the elution buffer of ferritin；
3) secondary separation is carried out using the elution buffer that cation chromatography obtains ferritin to step 2), collects ferritin
Elution buffer；
4) it is separated three times using elution buffer of the hydrophobic chromatography to the ferritin that step 3) obtains, collects ferritin
Elution buffer.
2. recombinant human iron separation and purification of protein method as described in claim 1, it is characterised in that: dissolve bacterium in the step 1)
The buffer of mud is 5-100mM PB or Tris pH7.0, and the temperature range and time heated in the step 1) is respectively 70-
75 DEG C, 5-15min, filtration treatment uses 0.22um doughnut or membrane filtration processing mode in the step 1).
3. recombinant human iron separation and purification of protein method as described in claim 1, it is characterised in that: the step 2) specifically:
The chromatographic column equipped with anion chromatography medium is taken, chromatographic column is handled with 0.5M NaOH, balances chromatographic column with combination buffer later
Loading afterwards, then impurity is cleaned with cleaning buffer solution, it is eluted with elution buffer, collects the elution buffer of ferritin,
The stronger impurity of binding ability is cleaned with 0.5M NaOH, saves chromatographic column with 10mM NaOH.
4. recombinant human iron separation and purification of protein method as claimed in claim 3, it is characterised in that: anion in the step 2)
Chromatography media is Q Bestarose High Performance, Q Sepharose High Performance, DEAE
Bestarose High Performance、DEAE Sepharose High Performance、SOURCE Q、Q
Bestarose Fast Flow、Q Sepharose Fast Flow、DEAE Bestarose Fast Flow、DEAE
Sepharose Fast Flow、Q Bestarose Big Beads、Q sepharose Big Beads、Q Bestarose
Big Beads、Q sepharose Big Beads、Diamond Q、Capto Q、Diamond Q mustang、Capto Q
One of mustang, Capto DEAE, Diamond DEAE or Diamond DEAE mustang；
Combination buffer is 5-100mM PB or Tris pH7.0 in the step 2)；
Cleaning buffer solution is 5-100mM PB or MES pH6.0 or 5-100mM PB or Tris 0.1M NaCl in the step 2)
One of pH7.0；
Elution buffer is 5-100mM NaAC pH4.5 or 5-100mMPB 0.25M NaCl pH7.0 in the step 2)；
In the step 2) combination buffer balance chromatographic column to efflux PH and conductance with the PH of combination buffer and electricity
It leads and is consistent.
5. recombinant human iron separation and purification of protein method as described in claim 1, it is characterised in that: the step 3) is specially；
The chromatographic column equipped with cationic chromatography media is taken, chromatographic column is handled with 0.5M NaOH, balances chromatographic column with combination buffer later
Loading afterwards, elution buffer are eluted, and the elution buffer of ferritin is collected, and clean binding ability ratio with 0.5M NaOH
Stronger impurity saves chromatographic column with 10mM NaOH.
6. recombinant human iron separation and purification of protein method as claimed in claim 5, it is characterised in that: cationic in the step 3)
Chromatography media is SP Bestarose High Performance, SP Sepharose High Performance, Diamond
MMC、Capto MMC、Diamond MMC mustang、Capto MMC impres、SP Bestarose Fast Flow、SP
Sepharose Fast Flow、Capto SP impres、Diamond SP mustang、CM Bestarose Fast Flow
Or one of CM Sepharose Fast Flow；
Combination buffer is 5-100mM NaAc pH5.0 or 5-100mM NaAc 0.2M NaCl pH5.0 in the step 3)；
Elution buffer is 5-100mM PB 0.01M NaCl pH6.0 or 5-100mM PB pH6.0 in the step 3)；
In the step 3) combination buffer balance chromatographic column to efflux PH and conductance with the PH of combination buffer and electricity
It leads and is consistent.
7. recombinant human iron separation and purification of protein method as described in claim 1, it is characterised in that: the step 4) is specific
For；The chromatographic column equipped with hydrophobic chromatoghaphy medium is taken, chromatographic column is handled with 0.5M NaOH, is balanced chromatograph with combination buffer later
Loading after column, then eluted with elution buffer, the elution buffer of ferritin is collected, impurity is eluted with pure water, uses
0.5M NaOH cleans chromatographic column, saves chromatographic column with 10mM NaOH.
8. recombinant human iron separation and purification of protein method as described in claim 7, it is characterised in that: hydrophobic in the step 4)
Chromatography media be Butyl Bestarose High Performance, Butyl Sepharose High Performance,
Diamond Butyl mustang、Capto Butyl impres、Butyl Bestarose Fast Flow、Phenyl
Sepharose Fast Flow HS、Phenyl Sepharose Fast Flow LS、Phenyl Bestarose Fast
Flow HS、Phenyl Bestarose Fast Flow LS、Butyl Sepharose Fast Flow、Diamond
butyl、Capto butyl、Capto Phenyl HS、Capto Phenyl impres、Diamond Phenyl mustang
Or one of Diamond Phenyl；
Combination buffer is 5-100mM PB or Tris 0.8-2M Na in the step 4)2SO4pH7.0,5-100mM PB or
Tris 1.5-3M NaCl pH7.0,5-100mM PB or Tris 0.8-2.5M (NH4)2SO4PH7.0 or 5-100mM PB or
Tris 0.8-2M K2One of HPO4pH7.0；
Elution buffer is 5-100mM PB or Tris 0.2-0.5M Na2SO4pH7.0,5-100mM PB in the step 4)
Or Tris 0.8-1.2M NaCl pH7.0,5-100mM PB or Tris 0.4-0.8M (NH4)2SO4pH7.0、5-100mM PB
Or Tris 0.4-0.8M K2One of HPO4pH7.0.
In the step 4) combination buffer balance chromatographic column to efflux PH and conductance with the PH of combination buffer and electricity
It leads and is consistent.
9. recombinant human iron separation and purification of protein method as described in claim 1, it is characterised in that: the step 1), step 2),
Step 3) and step 4) are both needed to carry out albumen Indexs measure.
10. recombinant human iron separation and purification of protein method as described in claim 1, it is characterised in that: the albumen index index
Detection uses gel filtration detection and ion chromatography detection mode.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811622011.8A CN109503704A (en) | 2018-12-28 | 2018-12-28 | A kind of recombinant human iron separation and purification of protein method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811622011.8A CN109503704A (en) | 2018-12-28 | 2018-12-28 | A kind of recombinant human iron separation and purification of protein method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109503704A true CN109503704A (en) | 2019-03-22 |
Family
ID=65756534
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811622011.8A Pending CN109503704A (en) | 2018-12-28 | 2018-12-28 | A kind of recombinant human iron separation and purification of protein method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109503704A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110759986A (en) * | 2019-10-18 | 2020-02-07 | 大连工业大学 | An efficient method for the preparation of reversible self-assembled proteins |
| CN111875696A (en) * | 2020-08-17 | 2020-11-03 | 郑州伊美诺生物技术有限公司 | Method for purifying Ferr |
| CN112646045A (en) * | 2021-01-06 | 2021-04-13 | 海默斯（重庆）医学生物技术有限公司 | Recombinant keratin separation and purification method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20020092089A (en) * | 2001-06-02 | 2002-12-11 | 한국생명공학연구원 | Expression vector containing gene of human ferritin, transformant thereof and method for preparing human ferritin using the same |
| KR20040007892A (en) * | 2002-07-11 | 2004-01-28 | 주식회사 바이오프로젠 | Recombinant Human Ferritin Protein And A Process for Producing the Same |
| CN102127166A (en) * | 2010-01-14 | 2011-07-20 | 中国科学院地质与地球物理研究所 | Method for separating purified ferritins from biological tissues or engineering bacteria |
| CN107033237A (en) * | 2017-05-11 | 2017-08-11 | 深圳市卫光生物制品股份有限公司 | A kind of Human Plasma Apolipoprotein A I isolation and purification method |
-
2018
- 2018-12-28 CN CN201811622011.8A patent/CN109503704A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20020092089A (en) * | 2001-06-02 | 2002-12-11 | 한국생명공학연구원 | Expression vector containing gene of human ferritin, transformant thereof and method for preparing human ferritin using the same |
| KR20040007892A (en) * | 2002-07-11 | 2004-01-28 | 주식회사 바이오프로젠 | Recombinant Human Ferritin Protein And A Process for Producing the Same |
| CN102127166A (en) * | 2010-01-14 | 2011-07-20 | 中国科学院地质与地球物理研究所 | Method for separating purified ferritins from biological tissues or engineering bacteria |
| CN107033237A (en) * | 2017-05-11 | 2017-08-11 | 深圳市卫光生物制品股份有限公司 | A kind of Human Plasma Apolipoprotein A I isolation and purification method |
Non-Patent Citations (1)
| Title |
|---|
| 李志鹏等: "铁蛋白Ferritin原核表达和纯化及纳米颗粒胞外自组装", 《畜牧兽医学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110759986A (en) * | 2019-10-18 | 2020-02-07 | 大连工业大学 | An efficient method for the preparation of reversible self-assembled proteins |
| CN110759986B (en) * | 2019-10-18 | 2023-01-31 | 大连工业大学 | Efficient preparation method of reversible self-assembled protein |
| CN111875696A (en) * | 2020-08-17 | 2020-11-03 | 郑州伊美诺生物技术有限公司 | Method for purifying Ferr |
| CN112646045A (en) * | 2021-01-06 | 2021-04-13 | 海默斯（重庆）医学生物技术有限公司 | Recombinant keratin separation and purification method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102977182B (en) | The method of one or more impurity levels in sample is reduced in protein purification | |
| AU2014264697B2 (en) | Continuous multistep process for purifying antibodies | |
| KR101569783B1 (en) | A Method of Antibody Purification | |
| CN104672328B (en) | A kind of production method of Human Antithrombin Ⅲ | |
| CN102492040B (en) | Method for purifying anti-HER2 or/and anti-HER3 antibody proteins | |
| CN109503704A (en) | A kind of recombinant human iron separation and purification of protein method | |
| CN107383161A (en) | The purifying of biomolecule | |
| CN109929027B (en) | Method for purifying recombinant fusion protein by linear elution step | |
| JP2017060488A (en) | Method for purifying human granulocyte-colony stimulating factor from recombinant e. coli | |
| CN103497248B (en) | A method for isolating and purifying antibody from cell culture supernatant | |
| JP7430012B2 (en) | Adalimumab NON-PROTEIN A purification method | |
| CN109641969A (en) | The method of isotype for separated monoclonal antibody | |
| CN112409477A (en) | IgM purification method | |
| CN107964044B (en) | Method for purifying anti-CD 20 monoclonal antibody from milk sample | |
| CN109320611B (en) | Purification method of human-mouse chimeric monoclonal antibody biological similar drug | |
| US20220267411A1 (en) | Refining method of ophthalmic protein pharmaceutical | |
| CN112898387B (en) | Purification method of recombinant staphylococcus aureus vaccine antigen protein | |
| CN113929733A (en) | Monoclonal antibody purification method of mixed ion exchange filler | |
| CN115925890A (en) | Method for purifying anti-new coronavirus neutralizing antibody | |
| CN109929038B (en) | Purification method of VEGF (vascular endothelial growth factor) capture agent fusion protein | |
| JP7445332B2 (en) | Optimized method for bevacizumab purification | |
| WO1995029187A1 (en) | Chromatographic process for the copurification of chondroitinase i and ii proteins | |
| CN106519029A (en) | Preparation process for A beta oligomer antibody | |
| CN111521813A (en) | Preparation method of green fluorescent protein fusion protein immunoaffinity column, immunoaffinity column and application thereof | |
| CN108101987A (en) | A kind of purification process for recombinating the full human monoclonal antibody of anti-tnf-alpha |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190322 |