CN114058636A - Method for cloning, expressing and purifying transthyretin gene - Google Patents
Method for cloning, expressing and purifying transthyretin gene Download PDFInfo
- Publication number
- CN114058636A CN114058636A CN202111358279.7A CN202111358279A CN114058636A CN 114058636 A CN114058636 A CN 114058636A CN 202111358279 A CN202111358279 A CN 202111358279A CN 114058636 A CN114058636 A CN 114058636A
- Authority
- CN
- China
- Prior art keywords
- ttr
- protein
- buffer
- anion exchange
- purified
- 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
- 238000000034 method Methods 0.000 title claims abstract description 26
- 238000010367 cloning Methods 0.000 title claims abstract description 10
- 108010071690 Prealbumin Proteins 0.000 title claims description 65
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 26
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 23
- 238000000746 purification Methods 0.000 claims abstract description 20
- 238000001641 gel filtration chromatography Methods 0.000 claims abstract description 12
- 101150091380 TTR gene Proteins 0.000 claims abstract description 8
- 238000001742 protein purification Methods 0.000 claims abstract description 8
- 238000005185 salting out Methods 0.000 claims abstract description 8
- 238000005406 washing Methods 0.000 claims abstract description 7
- 238000005349 anion exchange Methods 0.000 claims abstract description 6
- 238000000502 dialysis Methods 0.000 claims abstract description 6
- 239000007853 buffer solution Substances 0.000 claims abstract description 5
- 239000000872 buffer Substances 0.000 claims description 17
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 13
- 230000014509 gene expression Effects 0.000 claims description 12
- 238000002305 strong-anion-exchange chromatography Methods 0.000 claims description 10
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 9
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 8
- 239000007983 Tris buffer Substances 0.000 claims description 8
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 7
- 239000013613 expression plasmid Substances 0.000 claims description 7
- 238000003259 recombinant expression Methods 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 239000013598 vector Substances 0.000 claims description 6
- 230000001580 bacterial effect Effects 0.000 claims description 5
- 238000010276 construction Methods 0.000 claims description 4
- 239000012505 Superdex™ Substances 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical group N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- 239000013604 expression vector Substances 0.000 claims description 3
- 239000006166 lysate Substances 0.000 claims description 3
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 3
- 239000001488 sodium phosphate Substances 0.000 claims description 3
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 3
- 108091026890 Coding region Proteins 0.000 claims description 2
- 241000588724 Escherichia coli Species 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 239000012149 elution buffer Substances 0.000 claims description 2
- 238000002523 gelfiltration Methods 0.000 claims description 2
- 108091008146 restriction endonucleases Proteins 0.000 claims description 2
- 239000013612 plasmid Substances 0.000 abstract description 13
- 239000000523 sample Substances 0.000 abstract description 8
- 238000011160 research Methods 0.000 abstract description 7
- 230000001939 inductive effect Effects 0.000 abstract description 4
- 238000012544 monitoring process Methods 0.000 abstract description 3
- 238000004440 column chromatography Methods 0.000 abstract description 2
- 238000001976 enzyme digestion Methods 0.000 abstract description 2
- 239000012521 purified sample Substances 0.000 abstract description 2
- 238000003786 synthesis reaction Methods 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract 1
- 102000009190 Transthyretin Human genes 0.000 description 57
- 102100029290 Transthyretin Human genes 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000012258 culturing Methods 0.000 description 5
- 229930027917 kanamycin Natural products 0.000 description 4
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- 229930182823 kanamycin A Natural products 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 108010042407 Endonucleases Proteins 0.000 description 2
- 102000004533 Endonucleases Human genes 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000003941 amyloidogenesis Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000012474 protein marker Substances 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 description 1
- 125000001255 4-fluorophenyl group Chemical group [H]C1=C([H])C(*)=C([H])C([H])=C1F 0.000 description 1
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 108010088350 Lactate Dehydrogenase 5 Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 description 1
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 210000002987 choroid plexus Anatomy 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 125000002147 dimethylamino group Chemical group [H]C([H])([H])N(*)C([H])([H])[H] 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 230000005713 exacerbation Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 210000001508 eye Anatomy 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 230000000951 immunodiffusion Effects 0.000 description 1
- 229940125721 immunosuppressive agent Drugs 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000009384 kangtai Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 210000002418 meninge Anatomy 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 210000001428 peripheral nervous system Anatomy 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 238000013094 purity test Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000002207 retinal effect Effects 0.000 description 1
- 102000029752 retinol binding Human genes 0.000 description 1
- 108091000053 retinol binding Proteins 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 229940034208 thyroxine Drugs 0.000 description 1
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 201000007905 transthyretin amyloidosis Diseases 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 235000019155 vitamin A Nutrition 0.000 description 1
- 239000011719 vitamin A Substances 0.000 description 1
- 229940045997 vitamin a Drugs 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Toxicology (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for cloning, expressing and purifying TTR gene, which comprises the steps of obtaining TTR recombinant plasmid by chemical synthesis and double enzyme digestion, inducing protein to express in large quantity, salting out, buffer solution replacement dialysis, anion exchange column and gel filtration chromatography column to obtain TTR protein with purified precision. The protein purification method is based on an AKTA protein purification system, is simple and convenient to operate, can simultaneously realize automatic washing, online monitoring, real-time observation and the like of a sample, can adjust the purification process in time according to the condition of the sample and the subsequent requirement on the purified sample, obtains the TTR protein with high purity, can save precious plasma resources to the maximum extent, and simultaneously lays a foundation for the in vitro research of TTR genes in future.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for cloning, prokaryotic expression and protein purification of a transthyretin (TTR) gene.
Technical Field
Transthyretin (TTR), also known as Prealbumin (PA), is a tetrameric protein formed by 4 identical subunits connected by non-covalent bonds, each subunit consisting of 127 amino acids and having a relative molecular mass of 55 kDa. In vivo, TTR is produced primarily by the choroid plexus of the liver and brain, and also is synthesized in small amounts by retinal epithelial cells, primarily in blood and cerebrospinal fluid, at levels of about 3.4-5. mu. mol/L in blood and about 0.04-0.4. mu. mol/L in cerebrospinal fluid. The protein has the main physiological functions of transferring thyroxine and transferring vitamin A by combining with retinol binding protein.
Under normal physiological conditions, TTR functions as a stable tetramer, but when a gene coding for TTR is mutated or grows with age, the protein tetramer structure of TTR is unstable, TTR monomer pathologically aggregates in tissues such as peripheral nervous system, heart, eye, kidney, meninges and the like to form insoluble amyloid deposition, and transthyretin Amyloidosis (ATTR) disease is caused. Research reports that ATTR diseases have the characteristics of low morbidity, progressive exacerbation, various clinical manifestations, great difficulty in clinical diagnosis and differential diagnosis and the like, the survival period of patients after confirmed diagnosis is only 2-12 years generally, and most of patients can die from heart diseases.
Currently, in clinical treatment of ATTR disease, liver transplantation has become the first line of treatment, and not only can the variant TTR be reduced, but also the overall survival rate can be improved. However, liver transplantation has the effects of donor shortage, operation risk, acute rejection, long-term use of immunosuppressive agents, patient condition limitation and the like. In recent years, given the increasing maturity of protein expression and purification techniques and protein stability research techniques, more and more research teams have focused therapeutic strategies for ATTR disease on inhibiting the dissociation of TTR tetramers, i.e. slowing the rate of amyloid formation. Therefore, the efficient purification scheme of the TTR protein has significant practical significance and application value for the research and treatment of diseases.
At present, the reported TTR protein extraction and purification methods (EP0711786a2, US5310878a1) disclose a method for purifying human plasma prealbumin, which requires 11 extraction steps including ion exchange, immunodiffusion, affinity chromatography, size exclusion chromatography, and multiple buffer replacement and concentration steps under specific conditions. In addition, CN103833843A discloses a method for extracting and purifying prealbumin PA from normal human plasma, which requires solution extraction, buffer solution replacement dialysis, and purification of three kinds of chromatographic columns in AKTA system. The methods directly extract and purify TTR protein from human plasma, and have the disadvantages of complicated steps and complex operation, thus being difficult to meet the requirements of industrial production or clinical research.
Disclosure of Invention
In view of the limitation of the existing TTR protein purification, the invention provides a brand new TTR purification method, which comprises the steps of obtaining TTR recombinant plasmids by chemical synthesis and double enzyme digestion, inducing protein to express in large quantity, primarily purifying TTR protein by salting out, buffer solution replacement dialysis, anion exchange column and other steps, and finally obtaining highly purified TTR protein by matching with a gel filtration chromatographic column. The establishment of the method not only greatly saves precious plasma resources, but also greatly promotes the development of relevant research on pathogenesis and treatment of ATTR diseases.
In order to achieve the technical effects, the invention specifically provides the following technical scheme:
in a first aspect of the present invention, there is provided a method for cloning, expressing, isolating and purifying transthyretin (TTR), the method comprising the steps of:
(1) cloning of TTR gene:
NdeI-TTR-Xho I sequence is designed and PCR amplified through splicing chemically synthesized and purified TTR gene vector. Preferably, the nucleotide sequence of the Nde I-TTR-Xho I sequence is shown in SEQ ID NO. 1.
(2) Construction of recombinant expression plasmids:
digesting the expression vector by using restriction enzyme, inserting the TTR coding sequence into the corresponding site of the vector to obtain the recombinant expression plasmid.
(3) Expression and purification of TTR protein:
culturing competent BL21(DE3) transferred into recombinant expression plasmid at 37 deg.C, adding IPTG to induce protein expression, performing ultrasonic cell disruption, salting out and dialysis, and purifying TTR protein with strong anion exchange chromatography column and gel filtration chromatography column.
In one embodiment, in step (1), the cleavage sites at the N-terminus and C-terminus are NdeI and Xho I, respectively.
In one embodiment, the chemically synthesized TTR gene fragment is 396bp in length.
In one embodiment, in step (2), the expression vector is pET-29b (+).
In one embodiment, in step (3), the concentration of IPTG is 0.2-2 mM.
In one embodiment, in step (3), the conditions for ultrasonic cell disruption are 100-200W, 1s, and 5 s; the bacterial lysate was 50mM Tris, 150mM NaCl, pH 7.5.
In one embodiment, in step (3), the agent used for salting out is ammonium sulfate.
In one embodiment, in step (3), the conditions of the strong anion exchange chromatography column and the buffer are as follows:
a chromatographic column: 50mL Source 15Q.
Anion exchange buffer a: 25mM Tris, 1mM EDTA, pH 8.0.
Elution buffer B: 350mM NaCl.
In one embodiment, in step (3), the conditions for purifying TTR protein by strong anion exchange chromatography are: washing the chromatographic column by 50mL of solution A; solution B was eluted with a linear gradient (160mL, 50-350mM) followed by washing with solution B.
In one embodiment, in step (3), the gel filtration chromatography column and the buffer are under the following conditions:
a chromatographic column: 120mL Superdex 200.
SEC buffer: 10mM sodium phosphate, 100mM KCl, 1mM EDTA, pH 7.4.
In one embodiment, in step (3), the conditions for purifying TTR protein by gel filtration chromatography are: elution flow rate was 1mL/min, SEC buffer elution volume 120 mL.
Compared with the prior art, the invention has the following beneficial technical effects:
(1) establishing a prokaryotic gene expression system capable of being used for expressing TTR protein in a large quantity;
(2) by utilizing the salting-out characteristic of the protein, the foreign protein is greatly reduced through twice precipitation purification;
(3) based on the AKTA protein purification system, the method is simple and convenient to operate, simultaneously realizes multiple steps of automatic washing, online monitoring, real-time observation and the like of the sample, and can adjust the purification process in time according to the sample condition and the subsequent requirement on the purified sample.
Drawings
FIG. 1 Strong anion exchange chromatography purification of TTR profile;
FIG. 2 gel filtration chromatography purification of TTR profile;
FIG. 3 is a graph showing the electrophoresis results of purified TTR protein; wherein, from left to right, the channel 1 is a protein Marker; the channel 2 is a sample after strong anion exchange chromatography purification; the channel 3 is a sample after gel filtration chromatography purification;
FIG. 4UPLC purity test;
FIG. 5 molecular weight size of TTR by mass spectrometry;
FIG. 6 is a graphical representation of the results of tetrameric TTR protein activity.
Detailed Description
The invention will now be further described with reference to the following examples, which are intended to be illustrative of the invention and are not to be construed as limiting the invention.
The experimental procedures used in the following examples are conventional ones without specific mention.
The test consumables used in the examples described below were commercially available without specific reference.
Example 1: construction of recombinant cloning vector Nde I-TTR-Xho I
Primer design for TTR
Using pET-29b (+) as a template, designing an NdeI-TTR-Xho I sequence by splicing a chemically synthesized and purified TTR gene vector, and performing PCR amplification, wherein the nucleotide sequence of the NdeI-TTR-Xho I sequence is shown in SEQ ID NO:1 and consists of an endonuclease site NdeI-TTR protein coding sequence-an endonuclease site Xho I.
NdeI-TTR-Xho I is cloned and connected with digested pET-29b (+) plasmid template
mu.L of Nde I-TTR-Xho I was taken, 2. mu.L of digested pET-29b (+) plasmid template was added, 5. mu.L of ligation buffer and 2. mu.L of sterile water were added to 10. mu.L of the system, and ligation was performed at 16 ℃ for 2 hours.
Cloning and transformation of pET-29b (+) -TTR plasmid template
Transferring 2 mu L of the ligation product to 50 mu LDH5 alpha competent cells, and standing for 30min on ice; heat shock is carried out for 45s at 42 ℃; standing on ice for 5 min; adding 450 μ L LB (Tryptone 10g/L, Yeast extract 5g/L, NaCl 10g/L, pH 7.5), shaking at 37 deg.C and 220rpm for 1 h; centrifuging at 4000rpm for 3 min; most of the supernatant was removed in a clean bench, the pellet was gently suspended, and plated on LB plate containing 50. mu.g/mL kanamycin (Kan); the LB plate was placed upside down in a 37 ℃ incubator overnight.
Identification of clone pET-29b (+) -TTR
Randomly selecting 4 positive clones, and inoculating the positive clones into 4mL LB (Kan +); shaking overnight at 220rpm with a shaker at 37 ℃; plasmid pET-29b (+) -TTR was extracted, stored at-80 ℃ and plasmid sequencing was performed with primer T7.
Example 2: construction of recombinant expression plasmid pET-29b (+) -TTR
1. Digestion and recovery
Nde I and Xho I restriction sites are added into the TTR gene sequence, Nde I-TTR-Xho I and pET-29b (+) plasmid are subjected to double digestion by using Nde I and Xho I, electrophoresis is carried out by using 1% agarose gel, and the target fragment is recovered by using gel.
T4 DNA ligase ligation and transformation
The connecting system is as follows: mu.L of 10 Xligase buffer, 1. mu.L of 10-double-digested pET-29b (+) plasmid, 7. mu.L of Nde I-TTR-Xho I fragment, 1. mu. L T4 DNA ligase, were ligated overnight at 16 ℃.
The ligation products were transformed into DH 5. alpha. competence, transformed, plated on LB plates containing 50. mu.g/mL kanamycin (Kan), and cultured overnight at 37 ℃ in an inverted state.
3. Positive clone identification
Randomly selecting 4 positive clones, and inoculating the positive clones into 4mL LB (Kan +); shaking overnight at 220rpm with a shaker at 37 ℃; the plasmids were extracted for T7 primer-directed plasmid sequencing.
Example 3: inducible expression of TTR protein
1. Materials and reagents
Kanamycin: biyun Tian, China
Isopropyl thiogalactoside: biyun Tian, China
2. Instruments and devices:
an incubator: thermo, USA
UV-5100 ultraviolet visible light breadth meter: shanghai non-analytic Instrument Co., Ltd, China
3. The experimental steps are as follows:
(1) recombinant plasmid transformed Escherichia coli
The recombinant plasmid pET-29b (+) -TTR was transformed into competent BL21(DE3), spread on LB plate containing kanamycin (Kan), and cultured overnight at 37 ℃ in an inverted state.
(2) Isopropyl thiogalactoside (IPTG) induced protein expression
Selecting a single colony of the recombinant strain, inoculating the single colony in LB (Kan +), culturing overnight by a shaking table at 37 ℃ and at 220 rpm; inoculating 2L LB (Kan +) at a ratio of 1:20-40, shaking at 37 deg.C and culturing at 220rpm to OD600nmWhen 0.6-0.8 was reached, IPTG (0.2-2mM) was added to induce protein expression.
Example 4: purification and identification of TTR protein
1. Materials and reagents
Tris (Tris): biyun Tian, China
EDTA: biyun Tian, China
NaCl: xiong science Inc., China
Coomassie brilliant blue G250: biyun Tian, China
2. Instruments and devices:
a pH meter: MettlerToledo, Switzerland
Low-temperature freezing centrifuge: thermo, USA
Ultrasonic cell crusher: ningbo Xinzhi Biotech GmbH, China
A magnetic stirrer: darongxing laboratory instruments Ltd, China
Protein purification instrument: AKTA, USA
Electrophoresis apparatus: beijing, six Biotechnology Ltd, China
AB Sciex 5800MALDI-TOF/TOFTMAppearance: AB SCIEX, USA
A multifunctional microplate reader: tecan, Switzerland
Inducible expression of TTR protein
Selecting a single colony of the recombinant strain, inoculating the single colony in LB (Kan +), culturing overnight by a shaking table at 37 ℃ and at 220 rpm; inoculating 2L LB (Kan +) at a ratio of 1:20-40, shaking at 37 deg.C and culturing at 220rpm to OD600nmWhen 0.6-0.8 was reached, IPTG (0.2-2mM) was added to induce protein expression.
Centrifugation is carried out for 30min at 4 ℃ and 4000g, the supernatant is discarded, and the precipitate is suspended by bacterial lysate.
4. Cell disruption, salting out and dialysis
And (3) fully cracking the bacterial liquid by ultrasonic waves, and then centrifuging the crushed bacterial liquid in a centrifuge at the rotation speed of 16000g for 30min at 4 ℃. The precipitate was discarded and the supernatant was salted out with ammonium sulfate. Finally, the pellet after centrifugation was suspended in 40mL of anion exchange buffer A (25mM Tris, 1mM EDTA, pH 8.0) and dialyzed against buffer A overnight at 4 ℃.
5. Purification of TTR by strong anion exchange chromatography
50mL Source 15Q Strong anion exchange chromatography: solution A (25mM Tris, 1mM EDTA, pH 8.0), solution B (350mM NaCl); after the purified fraction is pumped into the column, it is first subjected to AKTATMThe column was washed off non-specifically adsorbed contaminating proteins by washing the column with 50mL of solution A on a fast protein chromatograph purifactor 100 system, which was then programmed to perform a linear gradient elution with solution B (160mL, 50-350mM) followed by a wash with solution B (50mL, 350 mM). The eluting peak contains TTR, collecting absorption peak II, i.e. about 25mL of TTR eluent, discarding peak I, and the map result is shown in figure 1. Samples were taken for SDS-PAGE and the results are shown in FIG. 3.
6. Purification of TTR by gel filtration chromatography
7. Identification of purity of purified TTR
SDS-PAGE gel electrophoresis through laser gray scanner determination of its purity to reach 99% and above, and show that purified TTR protein monomer molecular mass is about 14kDa, the result is shown in figure 3. In addition, the purity identification result of UPLC is also shown as a single peak, the result is shown in fig. 4, and the purity of TTR protein can be up to 99.5% by calculating the percentage of peak area.
Molecular weight size analysis of TTR
The TTR protein end product obtained in this example was desalted and then passed through AB Sciex 5800MALDI-TOF/TOFTMMass spectrometer detection to determine the molecular weight showed that the molecular weight of the purified TTR protein was about 14kDa as shown in FIG. 5. Consistent with the results of the molecular weight of the monomeric TTR on SDS-PAGE.
9. Activity assay of purified TTR protein
To perform activity analysis on purified TTR protein, the final product TTR protein was first prepared as solutions of different concentration gradients, i.e. 0, 0.5, 1, 2.5, 5, 10 μ M, to which a quantitative amount of fluorescent small molecule a2(S- (4-fluorophenyl) (E) -3- (dimethylamino) -5- (4-hydroxy-3, 5-dimethyltryptyl) thionate) bound only to tetrameric TTR was added, respectively [ Choi S, one DS, Kelly JW. "organic thin selective to transthytin in cells and remains dark units a chemical reaction to yield fluorescent antibody fluorescent protein complex" j.am. chem.2010; 132:16043-16051]Incubation was performed followed by monitoring the fluorescence of the a2 modified TTR conjugate using excitation light at 328nm and emission light at 430nm, with 3 duplicate wells per concentration. And finally, establishing a linear regression model by taking the tetramer TTR concentration as an abscissa and the TTR conjugate fluorescence intensity as an ordinate. The results show that all points fall on the linear regression trend line, and R20.9989, and the two have higher linear relation, which indicates that the purified tetrameric TTR protein has extremely high activity.
It should be understood that the above-mentioned embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQ ID NO:1
CATATGGGTCCTACGGGCACCGGTGAATCCAAGTGTCCTCTGATGGTCAAAGTTCTAGATGCTGTCCGAGGCAGTCCTGCCATCAATGTGGCCGTGCATGTGTTCAGAAAGGCTGCTGATGACACCTGGGAGCCATTTGCCTCTGGGAAAACCAGTGAGTCTGGAGAGCTGCATGGGCTCACAACTGAGGAGGAATTTGTAGAAGGGATATACAAAGTGGAAATAGACACCAAATCTTACTGGAAGGCACTTGGCATCTCCCCATTCCATGAGCATGCAGAGGTGGTATTCACAGCCAACGACTCCGGCCCCCGCCGCTACACCATTGCCGCCCTGCTGAGCCCCTACTCCTATTCCACCACGGCTGTCGTCACCAATCCCAAGGAATGACTCGAG
Sequence listing
<110> Dalian run-on Kangtai medical laboratory Co., Ltd
<120> method for cloning, expressing and purifying transthyretin gene
<130> CP211042
<141> 2021-11-16
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 396
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
catatgggtc ctacgggcac cggtgaatcc aagtgtcctc tgatggtcaa agttctagat 60
gctgtccgag gcagtcctgc catcaatgtg gccgtgcatg tgttcagaaa ggctgctgat 120
gacacctggg agccatttgc ctctgggaaa accagtgagt ctggagagct gcatgggctc 180
acaactgagg aggaatttgt agaagggata tacaaagtgg aaatagacac caaatcttac 240
tggaaggcac ttggcatctc cccattccat gagcatgcag aggtggtatt cacagccaac 300
gactccggcc cccgccgcta caccattgcc gccctgctga gcccctactc ctattccacc 360
acggctgtcg tcaccaatcc caaggaatga ctcgag 396
Claims (10)
1. A transthyretin gene (TTR) cloning, expression and protein purification method is characterized by comprising the following steps:
(1) cloning of TTR gene:
NdeI-TTR-Xho I sequence is designed and PCR amplified through splicing chemically synthesized and purified TTR gene vector.
(2) Construction of recombinant expression plasmids:
digesting the expression vector by using restriction enzyme, inserting the TTR coding sequence into the corresponding site of the vector to obtain the recombinant expression plasmid.
(3) Expression and purification of TTR protein:
BL21(DE3) competent Escherichia coli transferred with the recombinant expression plasmid is cultured at 37 ℃, IPTG is added to induce protein expression, then ultrasonic cell disruption, salting out and dialysis are carried out, and TTR protein is purified by a strong anion exchange chromatographic column and a gel filtration chromatographic column.
2. The method according to claim 1, wherein the Nde I-TTR-Xho I sequence is as shown in SEQ ID NO 1.
3. The method of claim 1, wherein in step (2), the vector is pET-29b (+).
4. The method of claim 1, wherein in step (3), the concentration of IPTG is 0.2-2 mM.
5. The method as claimed in claim 1, wherein in the step (3), the conditions for the ultrasonic cell disruption are 100-200W, 1s and 5 s; the bacterial lysate was 50mM Tris, 150mM NaCl, pH 7.5.
6. The method according to claim 1, wherein in the step (3), the agent for salting out is ammonium sulfate.
7. The method of claim 1, wherein the strong anion exchange chromatography column and the buffer are under the following conditions:
a chromatographic column: 50mL Source 15Q;
anion exchange buffer a: 25mM Tris, 1mM EDTA, pH 8.0.
Elution buffer B: 350mM NaCl.
8. The method of claim 1, wherein in step (3), the conditions for purifying TTR protein by strong anion exchange chromatography are as follows: washing the chromatographic column by 50mL of solution A; and (3) carrying out linear gradient elution on the solution B under the elution condition of 160mL and 50-350mM, and then washing with the solution B.
9. The method of claim 1, wherein the gel filtration chromatography column and buffer conditions are as follows:
a chromatographic column: 120mL Superdex 200.
Buffer solution: 10mM sodium phosphate, 100mM KCl, 1mM EDTA, pH 7.4, the buffer is SEC buffer.
10. The method of claim 1, wherein in step (3), the conditions for purifying TTR protein by gel filtration chromatography are as follows: elution flow rate was 1mL/min, SEC buffer elution volume 120 mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111358279.7A CN114058636A (en) | 2021-11-16 | 2021-11-16 | Method for cloning, expressing and purifying transthyretin gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111358279.7A CN114058636A (en) | 2021-11-16 | 2021-11-16 | Method for cloning, expressing and purifying transthyretin gene |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114058636A true CN114058636A (en) | 2022-02-18 |
Family
ID=80272808
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111358279.7A Pending CN114058636A (en) | 2021-11-16 | 2021-11-16 | Method for cloning, expressing and purifying transthyretin gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114058636A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116286927A (en) * | 2023-03-17 | 2023-06-23 | 桂林英美特生物技术有限公司 | Preparation method and application of recombinant Prealbumin (PA) protein |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5310878A (en) * | 1992-10-23 | 1994-05-10 | Baxter Diagnostics Inc. | Biosynthetic cerebrospinal fluid control and method of use |
| EP0711786A2 (en) * | 1991-03-07 | 1996-05-15 | Dade International Inc. | Purifying prealbumin |
| WO1999016889A1 (en) * | 1997-10-01 | 1999-04-08 | G.D. Searle & Co. | Fusion proteins comprising an angiostatin moiety and their use in anti-tumor treatment |
| US20100120893A1 (en) * | 2008-10-20 | 2010-05-13 | Dinah Wen-Yee Sah | Compositions and Methods for Inhibiting Expression of Transthyretin |
| US20110237646A1 (en) * | 2008-08-07 | 2011-09-29 | Isis Pharmaceuticals, Inc. | Modulation of transthyretin expression for the treatment of cns related disorders |
| CN103038345A (en) * | 2010-04-29 | 2013-04-10 | Isis制药公司 | Modulation of transthyretin expression |
| CN103833843A (en) * | 2012-11-27 | 2014-06-04 | 上海复星医药（集团）股份有限公司 | Method for extracting and purifying prealbumin PA from plasma of normal people |
| CN106255702A (en) * | 2013-12-20 | 2016-12-21 | 生物控股有限公司 | Antibody-based therapy for transthyretin (TTR) amyloidosis and its human antibody |
| CN110960687A (en) * | 2019-12-17 | 2020-04-07 | 上海卡序生物医药科技有限公司 | Application of transthyretin for transferring fusion protein into eyes |
| CN111875692A (en) * | 2019-10-30 | 2020-11-03 | 南京理工大学 | Rare crucian transthyretin protein gene, protein sequence and application thereof |
-
2021
- 2021-11-16 CN CN202111358279.7A patent/CN114058636A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0711786A2 (en) * | 1991-03-07 | 1996-05-15 | Dade International Inc. | Purifying prealbumin |
| US5310878A (en) * | 1992-10-23 | 1994-05-10 | Baxter Diagnostics Inc. | Biosynthetic cerebrospinal fluid control and method of use |
| WO1999016889A1 (en) * | 1997-10-01 | 1999-04-08 | G.D. Searle & Co. | Fusion proteins comprising an angiostatin moiety and their use in anti-tumor treatment |
| US20110237646A1 (en) * | 2008-08-07 | 2011-09-29 | Isis Pharmaceuticals, Inc. | Modulation of transthyretin expression for the treatment of cns related disorders |
| US20100120893A1 (en) * | 2008-10-20 | 2010-05-13 | Dinah Wen-Yee Sah | Compositions and Methods for Inhibiting Expression of Transthyretin |
| CN103038345A (en) * | 2010-04-29 | 2013-04-10 | Isis制药公司 | Modulation of transthyretin expression |
| CN103833843A (en) * | 2012-11-27 | 2014-06-04 | 上海复星医药（集团）股份有限公司 | Method for extracting and purifying prealbumin PA from plasma of normal people |
| CN106255702A (en) * | 2013-12-20 | 2016-12-21 | 生物控股有限公司 | Antibody-based therapy for transthyretin (TTR) amyloidosis and its human antibody |
| CN111875692A (en) * | 2019-10-30 | 2020-11-03 | 南京理工大学 | Rare crucian transthyretin protein gene, protein sequence and application thereof |
| CN110960687A (en) * | 2019-12-17 | 2020-04-07 | 上海卡序生物医药科技有限公司 | Application of transthyretin for transferring fusion protein into eyes |
Non-Patent Citations (2)
| Title |
|---|
| 李钧敏等: "人血浆转甲状腺素蛋白的提纯及其对肝癌细胞生长的抑制作用", 《中国肿瘤生物治疗杂志》, vol. 6, no. 2, 30 June 1999 (1999-06-30), pages 97 - 101 * |
| 胡城: "转甲状腺素蛋白野生型及其V30A、V30M突变体的表达、纯化和稳定性研究", 《中国优秀博硕士学位论文全文数据库(硕士)基础科学辑》, no. 09, 15 September 2013 (2013-09-15), pages 006 - 29 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116286927A (en) * | 2023-03-17 | 2023-06-23 | 桂林英美特生物技术有限公司 | Preparation method and application of recombinant Prealbumin (PA) protein |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5909172B2 (en) | Production of recombinant protein by self-proteolytic cleavage of fusion protein | |
| CN116333097A (en) | High-activity recombinant human fibronectin and preparation method and application thereof | |
| CN107245494A (en) | Efficient Soluble Expression and Purification of Aβ42 in Escherichia coli | |
| CN114058636A (en) | Method for cloning, expressing and purifying transthyretin gene | |
| WO2022253266A1 (en) | Recombinant protein purification method | |
| CN110305224B (en) | An Aβ42-modified protein with the function of impeding protein aggregation and its expression and purification method | |
| JP5550797B2 (en) | Mutant protein capable of specifically and rapidly binding to human cardiac troponin I | |
| CN111116757A (en) | Ferritin fusion protein with galactose-binding lectin EW29 label, protein cage nanoparticles and preparation method thereof | |
| CN114908113A (en) | Preparation method of human interleukin-5 recombinant protein | |
| CN102250938B (en) | A kind of preparation method of sea anemone cardiotonic peptide | |
| CN117229381A (en) | Recombinant ginseng hypoglycemic peptide and preparation method and application thereof | |
| TWI531578B (en) | Solubility enhancing peptide and use thereof | |
| CN102127156A (en) | Fusion tagging protein for nonchromatographic sepration of target protein and coding gene and preparation method thereof | |
| CN116041478A (en) | Preparation method of human recombinant thermostable protein FGF2 based on Escherichia coli in vitro purification system | |
| CN116254283A (en) | A method for preparing the transmembrane domain of a single transmembrane protein | |
| CN108841846B (en) | A recombinant fluorescent protein with large Stokes shift and preparation method thereof | |
| CN107746432A (en) | A kind of modified proteins of A β 42 and its expression and purification method | |
| CN120230739B (en) | A LbuCas13a modified protein and its editing system | |
| CN115873811A (en) | PBCV-1 ligase mutant, expression and purification method and application | |
| WO2008089690A1 (en) | Preparation process of recombinant human p43 protein | |
| US11248217B2 (en) | Engineered carbohydrate-active enzymes for glycan polymers synthesis | |
| CN115851748A (en) | Preparation method and application of β2-MG truncated body | |
| CN108103083A (en) | Nucleotide sequence encoding recombinant human aldolase C, expression vector, engineering bacteria and method for producing recombinant human aldolase C | |
| CN116042588A (en) | Preparation method and application of recombinant creatinase | |
| Gibson | The Biophysical Characterization of a Novel Amyloidogenic BETA2-Microglobulin Variant, P32L |
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 | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20221111 Address after: 116001 No. 1 (- 4), Floor 4, No. 11A, Yuguang Street, Zhongshan District, Dalian, Liaoning Applicant after: Dalian Boyuan Medical Technology Co.,Ltd. Address before: 3 / F, No. 300-8, jinlongsi Road, Ganjingzi District, Dalian City, Liaoning Province, 116033 Applicant before: Dalian Runsheng Kangtai Medical Laboratory Co.,Ltd. |
|
| TA01 | Transfer of patent application right |