CN112481225A - Purification method of heterologous expression halogenase - Google Patents
Purification method of heterologous expression halogenase Download PDFInfo
- Publication number
- CN112481225A CN112481225A CN202110017687.XA CN202110017687A CN112481225A CN 112481225 A CN112481225 A CN 112481225A CN 202110017687 A CN202110017687 A CN 202110017687A CN 112481225 A CN112481225 A CN 112481225A
- Authority
- CN
- China
- Prior art keywords
- nta
- halogenase
- protein
- collecting
- ice
- 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
- 238000000746 purification Methods 0.000 title claims abstract description 15
- 238000000034 method Methods 0.000 title claims abstract description 12
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 51
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 46
- 239000011347 resin Substances 0.000 claims abstract description 6
- 229920005989 resin Polymers 0.000 claims abstract description 6
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 claims description 12
- 239000000872 buffer Substances 0.000 claims description 9
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 9
- 238000001962 electrophoresis Methods 0.000 claims description 8
- 230000001939 inductive effect Effects 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 4
- 102000016943 Muramidase Human genes 0.000 claims description 3
- 108010014251 Muramidase Proteins 0.000 claims description 3
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims description 3
- 241001655322 Streptomycetales Species 0.000 claims description 3
- 241001052560 Thallis Species 0.000 claims description 3
- 229920004890 Triton X-100 Polymers 0.000 claims description 3
- 239000013504 Triton X-100 Substances 0.000 claims description 3
- 230000010261 cell growth Effects 0.000 claims description 3
- 229960000274 lysozyme Drugs 0.000 claims description 3
- 239000004325 lysozyme Substances 0.000 claims description 3
- 235000010335 lysozyme Nutrition 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 239000013604 expression vector Substances 0.000 abstract description 5
- 241000588724 Escherichia coli Species 0.000 abstract description 3
- 108091005804 Peptidases Proteins 0.000 abstract description 3
- 239000004365 Protease Substances 0.000 abstract description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract description 3
- 241000187747 Streptomyces Species 0.000 abstract description 3
- 239000007853 buffer solution Substances 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 150000004820 halides Chemical class 0.000 description 4
- 239000000243 solution Substances 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000187180 Streptomyces sp. Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].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=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 230000002140 halogenating effect Effects 0.000 description 2
- 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 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 1
- 108091006054 His-tagged proteins Proteins 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 108010059993 Vancomycin Proteins 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 1
- 229910052794 bromium Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 125000005843 halogen group Chemical group 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 229940056137 sivextro Drugs 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- XFALPSLJIHVRKE-GFCCVEGCSA-N tedizolid Chemical compound CN1N=NC(C=2N=CC(=CC=2)C=2C(=CC(=CC=2)N2C(O[C@@H](CO)C2)=O)F)=N1 XFALPSLJIHVRKE-GFCCVEGCSA-N 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- 239000002699 waste material 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
-
- 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
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a purification method of heterologous expression halogenase, belonging to the technical field of genetic engineering. According to the invention, a high-efficiency expression vector of the halogenase is constructed, the protein is enriched, the HIS tag purification resin is used for purifying pET-abxH R halogenase, the target protein is eluted under the optimal buffer solution concentration (NTA-60 and NTA-80), the BCA kit determines the protein concentration, and the purified protease is stored in a refrigerator at the temperature of-20 ℃ for later use. The invention provides a streptomyces-derived halogenase expression and purification method, which has good universality and is particularly suitable for halogenase expressed in escherichia coli.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a purification method of heterologous expression halogenase.
Background
Halides are a class of organic compounds containing the halogen atom fluorine, chlorine, bromine or iodine. Most halides have important biological activity, and many clinically used antibiotics and antitumor drugs are halides derived from microorganisms, such as chloramphenicol and vancomycin with antibacterial activity, and butterfly mycin, C-1027 with antitumor activity. 3 of the 4 new antibiotics approved for marketing in 2014 were halides, dalvanine, Oritavanci, and Sivextro, respectively.
The general strategy for expressing the foreign protein is to connect a target gene to a proper vector, then transfer the vector into an expression strain to express the target protein, and finally, crush the expression strain, enrich and purify the required target protein. The process needs to solve two problems of expression and purification of target protein: expressing a sufficient amount of protein in the expression strain by the target gene; 2. and selecting a proper means to purify the target protein. In addition, sufficient expression is also advantageous for protein purification.
For the convenience of subsequent purification, the skilled artisan often uses tags such as 6 × His, MBP, etc. to purify the protein of interest by affinity chromatography. In addition, it is also a problem how to identify whether a gene of interest is expressed in an expression strain. The general method is to break and enrich the expression strain and then judge whether the target gene is expressed or not through SDS-PAGE gel, and the construction of an expression vector is often not done all at once and needs to be continuously optimized, which causes much time waste and reagent loss.
In conclusion, it is desired to develop a novel method for purifying protein expression, which can purify and enrich target protein with high efficiency and high purity.
Disclosure of Invention
The present invention aims at providing one kind of heterologous expression vector of halogenase and its construction based on the background technology.
In order to achieve the purpose, the technical scheme is as follows:
a purification method for heterologously expressed halogenating enzyme, wherein the sequence of the halogenating enzyme is shown as SEQ ID NO.1, comprises the following steps:
a) constructing a streptomycete halogenase heterologous expression strain pET-abxH R, culturing, inducing and collecting thalli;
b) extracting the His-tag protein under non-denaturing conditions:
(1) preparing cells, inoculating, inducing expression, and collecting cells;
(2) adding 1/20 NTA-0 Buffer and PMSF with cell growth volume, wherein the working concentration of PMSF is 1mM and is added at present;
(3) suspending cells, adding appropriate amount of lysozyme, mixing, standing on ice for 30min, and ultrasonically treating or homogenizing on ice to break cells;
(4) adding 10% Triton X-100 to final concentration of 0.05%, mixing, and standing on ice for 15 min;
(5) centrifuging at 12000rpm and 4 deg.C for 15min, and collecting supernatant and placing on ice for use or storing at-20 deg.C;
(6) loading NTA resin into appropriate chromatographic column, and washing with NTA-0 buffer in 10 times NTA volume;
(7) adding the sample into NTA chromatographic column, controlling the flow rate at about 15mL/h, eluting with NTA-20, NTA-40, NTA-60, NTA-80, NTA-100, NTA-200, NTA-300 and NTA-400 buffer respectively, controlling the flow rate at about 15mL/h, collecting eluate, and collecting an NTA volume per tube;
(8) the collected eluate is subjected to SDS-PAGE protein electrophoresis to analyze the binding condition of the protein, and the storage condition of the purified target protein is determined according to the property and the application of the protein.
In order to solve the problems in the prior art, the invention constructs a high-efficiency expression vector of the halogenase, enriches the protein, purifies pET-abxH R halogenase by using HIS label purification resin, elutes the target protein under the optimal buffer solution concentration (NTA-60 and NTA-80), the BCA kit determines the protein concentration, and the purified protease is stored in a refrigerator at the temperature of-20 ℃ for later use. The invention provides a streptomyces-derived halogenase expression and purification method, which has good universality and is particularly suitable for halogenase expressed in escherichia coli.
Drawings
FIG. 1 shows the SDS-PAGE confirmation of the gradient elution proteins of the present application.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the specific embodiments of the present invention. Referring to the drawings, like numbers indicate like or similar elements throughout the views. The described embodiments are only some, not all embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
As shown in FIG. 1, SDS-PAGE was performed to verify the gradient elution of protein for the present application. BSA is bovine serum albumin, has molecular weight of 66.430 kDa, has a size which is not greatly different from that of the identified halogenase gene of 65 kDa, and is taken as marker. 1 is pET-abxH R strain without IPTG, and 2 and 3 elute the target protein under NTA-60 and NTA-80 gradient respectively.
In order to solve the problems in the prior art, the invention constructs a high-efficiency expression vector of the halogenase, enriches the protein, purifies pET-abxH R halogenase by using HIS label purification resin, elutes the target protein under the optimal buffer solution concentration (NTA-60 and NTA-80), the BCA kit determines the protein concentration, and the purified protease is stored in a refrigerator at the temperature of-20 ℃ for later use.
The invention provides a streptomyces-derived halogenase expression and purification method, wherein the sequence of the halogenase is shown as SEQ ID NO.1, and the halogenase has good universality and is particularly suitable for the halogenase expressed in escherichia coli.
Constructing streptomycete halogenase heterologous expression strain pET-abxH R, culturing in large quantity, inducing and collecting thalli, extracting His label protein under non-denaturing condition, gradient eluting target protein, carrying out SDS-PAGE protein electrophoresis analysis on the collected eluent to analyze the combination condition of the protein, and determining the storage condition of the purified target protein according to the property and the application of the protein.
Inducing and expressing the positive clone bacteria:
(1) 100 muL of positive clonal bacteria are inoculated in 5 100mL LB liquid culture medium containing 100 ng/muL of ampicillin at final concentration;
(2) shaking culture at 37 deg.C and 220 rpm, and culturing to OD600=0.3-0.5；
(3) IPTG was added to the mixture at a final concentration of 1.0 mM/L, and the mixture was subjected to induction culture at 28 ℃.
Bulk collection of cells step:
(1) centrifuging the induced thallus at 12000rpm for 3min, and discarding the supernatant;
(2) adding 5mL of deionized water into each tube, blowing, uniformly mixing, centrifuging at 12000rpm for 3min, and removing the supernatant; repeat 3 times. The final pellet was collected as cells.
Extraction of His-tagged protein under non-denaturing conditions
(1) Preparing cells, inoculating, inducing expression, and collecting cells;
(2) adding 1/20 NTA-0 Buffer and PMSF with cell growth volume, wherein the working concentration of PMSF is 1mM and is added at present;
(3) suspending the cells, adding a proper amount of lysozyme, mixing uniformly, standing on ice for 30min, and ultrasonically or homogenously crushing the cells on ice;
(4) adding 10% Triton X-100 to final concentration of 0.05%, mixing, and standing on ice for 15 min;
(5) centrifuging at 12000rpm and 4 deg.C for 15min, and collecting supernatant and placing on ice for use or storing at-20 deg.C;
(6) loading NTA resin into appropriate chromatographic column, and washing with NTA-0 buffer in 10 times NTA volume;
(7) adding the sample into NTA chromatographic column, controlling the flow rate at about 15mL/h, eluting with NTA-20, NTA-40, NTA-60, NTA-80, NTA-100, NTA-200, NTA-300 and NTA-400 buffer respectively, controlling the flow rate at about 15mL/h, collecting eluate, and collecting an NTA volume per tube;
(8) the collected eluate is subjected to SDS-PAGE protein electrophoresis to analyze the binding condition of the protein, and the storage condition of the purified target protein is determined according to the property and the application of the protein.
SDS-PAGE protein electrophoretic analysis step
(1) Respectively taking 10 muL of eluent, adding 20 muL of 5 xSDS-PAGE electrophoresis sample loading buffer solution, and carrying out cracking for 10min at 99 ℃ in a metal bath; carrying out 10 muL protein molecule standard sample application, carrying out 10% SDS-PAGE gel electrophoresis analysis, carrying out electrophoresis at 100V for 2h after electrophoresis at 50V for 30min, and adjusting time according to actual conditions;
(2) stripping the gel, placing the gel in a protein staining solution containing Coomassie brilliant blue R250, and carrying out shaking staining on a decoloring shaker for 2-6 h;
(3) and decoloring the Coomassie brilliant blue protein decoloring solution for 6h, exposing and developing by a two-color infrared laser imaging system, and judging whether the protein is expressed or not.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Sequence listing
<110> Zunyi university of medical science
<120> purification method of heterologous expression halogenase
<141> 2021-01-07
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3
<212> PRT
<213> Streptomyces sp.FJ31-2
<400> 1
<210> 2
<211> 1251
<212> DNA
<213> Streptomyces sp.FJ31-2
<400> 2
gtgacacact caatggacac gacagacaca ggcgaccccg gtacctccta cgacgtcgtt 60
gtgatcggcg gcggccccgc cggctcgagc accgcggccc agctggcccg ggccggccac 120
cgggtgctgg tgctggagcg ggagaagttc ccccgctacc acatcggcga gtccctcatc 180
cccggctgcc tcgacctggt cagggacctg ggcctgtggg accggctgga aggcctcggc 240
ttcaccaaga agtacggcgg caccctgctg tggggcgcca ccgagggcac ctgggacttc 300
cggttcgccg acggcagcga ctacgagtac tccttccagg tgcgccgcgc cgacttcgac 360
aaggacgtgg agttcgacgg cgaccgcgcc accggcctgg tctacaccgt caagggctcg 420
gacgagcccg tacgggtctc ctcccgcttc ctcgtcgacg cctccggaca gcagcggctc 480
ctcggccgcc ggctcgggct cgtcgactgg cacgaggacc tgcgcaatgt cgcggtgtgg 540
agctacttcc agggctgcga cttcatcgag ggcgagaacc gggccggcga cgtggtcgtg 600
gagaacatgt ggcaggagga ccagcctccc ggctggttct ggttcatccc gctgcacgac 660
ggcacggtca gcatcggcta tgtcacccgc accgctctgc tgcagcgctc cggtctgacc 720
ccggaccagc tgttcgaaca gcagctggcc gcctcggacg aggtcaagcg gctcacccgg 780
aacgccacca ggaccagcgc ctaccgcacc ctgcgcgact ggagctatga gtgcacccgc 840
ttccatggcc ccggctgggc cgtggtcggc gacgccgcgg cctttgtcga cccgctgttc 900
tccaccggcg tcaccctggc catgcgcggt gcgcgggacc tggcccccgc ggtcgaccag 960
gcgctgcgcg acccgggcca ggaggccgat ctgctcaagg cgtacgagga cgggtaccgc 1020
gaattcctcg gccacgtcct gaccttcgtg cgcttcttct acaacatcaa gatgcacaag 1080
gaacagtact gggagggcgc ccagaccatc gtcgacccgc agaagctcca ggcgccgaag 1140
atcgacttcg cggtcctgct ggccggcatg accggcatcc ggcccacgcc cgagatgcag 1200
gcggacgcca gggccaagac caacgacatc cactcccgct tcgcgacatg a 1251
Claims (1)
1. A purification method of heterologous expression halogenase, which is characterized in that: the sequence of the halogenase is shown as SEQ ID NO.1, and the halogenase comprises the following steps:
a) constructing a streptomycete halogenase heterologous expression strain pET-abxH R, culturing, inducing and collecting thalli;
b) extracting the His-tag protein under non-denaturing conditions:
(1) preparing cells, inoculating, inducing expression, and collecting cells;
(2) adding 1/20 NTA-0 Buffer and PMSF with cell growth volume, wherein the working concentration of PMSF is 1mM and is added at present;
(3) suspending cells, adding appropriate amount of lysozyme, mixing, standing on ice for 30min, and ultrasonically treating or homogenizing on ice to break cells;
(4) adding 10% Triton X-100 to final concentration of 0.05%, mixing, and standing on ice for 15 min;
(5) centrifuging at 12000rpm and 4 deg.C for 15min, and collecting supernatant and placing on ice for use or storing at-20 deg.C;
(6) loading NTA resin into appropriate chromatographic column, and washing with NTA-0 buffer in 10 times NTA volume;
(7) adding the sample into NTA chromatographic column, controlling the flow rate at about 15mL/h, eluting with NTA-20, NTA-40, NTA-60, NTA-80, NTA-100, NTA-200, NTA-300 and NTA-400 buffer respectively, controlling the flow rate at about 15mL/h, collecting eluate, and collecting an NTA volume per tube;
(8) the collected eluate is subjected to SDS-PAGE protein electrophoresis to analyze the binding condition of the protein, and the storage condition of the purified target protein is determined according to the property and the application of the protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110017687.XA CN112481225A (en) | 2021-01-07 | 2021-01-07 | Purification method of heterologous expression halogenase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110017687.XA CN112481225A (en) | 2021-01-07 | 2021-01-07 | Purification method of heterologous expression halogenase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112481225A true CN112481225A (en) | 2021-03-12 |
Family
ID=74916102
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110017687.XA Pending CN112481225A (en) | 2021-01-07 | 2021-01-07 | Purification method of heterologous expression halogenase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112481225A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112592929A (en) * | 2021-01-07 | 2021-04-02 | 遵义医科大学 | Preparation method and application of halogenase recombinant expression vector |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130244296A1 (en) * | 2011-09-01 | 2013-09-19 | Jixun Zhan | Halogenation enzymes |
| US20160138066A1 (en) * | 2013-04-17 | 2016-05-19 | Heinrich-Heine-Universität Düsseldorf | Methods for the expression of peptides and proteins |
-
2021
- 2021-01-07 CN CN202110017687.XA patent/CN112481225A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130244296A1 (en) * | 2011-09-01 | 2013-09-19 | Jixun Zhan | Halogenation enzymes |
| US20160138066A1 (en) * | 2013-04-17 | 2016-05-19 | Heinrich-Heine-Universität Düsseldorf | Methods for the expression of peptides and proteins |
Non-Patent Citations (4)
| Title |
|---|
| YUE,C.等: "UNVERIFIED: Streptomyces sp. FJS31-2 anthrabenzoxocinones biosynthetic gene cluster,complete sequence", 《GENBANK: KU243130.1》 * |
| YUHONG LÜ等: "Molecular Genetic Characterization of an Anthrabenzoxocinones Gene Cluster in Streptomyces Sp. FJS31-2 for the Biosynthesis of BE-24566B and Zunyimycin Ale", 《MOLECULES》 * |
| 杨婷等: "新型核苷碱基卤化酶AcmX的原核表达和结晶", 《四川大学学报(自然科学版)》 * |
| 肖琳琳: "表达融合蛋白APB-DOCK8转基因番茄防龋疫苗免疫SD大鼠的实验研究", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112592929A (en) * | 2021-01-07 | 2021-04-02 | 遵义医科大学 | Preparation method and application of halogenase recombinant expression vector |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hall et al. | Identification and characterization of the Escherichia coli RecT protein, a protein encoded by the recE region that promotes renaturation of homologous single-stranded DNA | |
| KR20120125569A (en) | Bacterial host strain comprising a mutant spr gene and a wild-type tsp gene | |
| CN102140476B (en) | Gene of recombinant staphylococal protein A, expression vector containing gene and application thereof | |
| TWI660042B (en) | Expression construct and method for producing proteins of interest | |
| CN112481225A (en) | Purification method of heterologous expression halogenase | |
| CN108003243B (en) | Method for purifying protein | |
| CN115109782A (en) | Expression and renaturation method of recombinant human CXCL16 protein | |
| CN113832127A (en) | Mutant of restriction enzyme BamH I and application thereof | |
| CN106318922A (en) | Preparation method of Pfu DNA polymerase | |
| AU2002308753B2 (en) | Acetate-free purification of plasmid DNA on hydroxyapatite | |
| CN109609539B (en) | Method for preparing ELABELA polypeptide from recombinant pichia pastoris | |
| CN113430220A (en) | Synthesis method, construction method and application of genetic engineering bacteria for expressing soluble feline omega interferon | |
| CN105602977A (en) | Preparation method and application of high-activity human cytokine Eotaxin-2 | |
| CN102140444A (en) | Low temperature alkaline phosphatase and preparation method thereof | |
| CN112689674B (en) | Dextran affinity tag and application thereof | |
| RU2618850C2 (en) | pET-mChBac75Na PLASMID VECTOR, ESCHRICHIA COLI BL21(DE3/ pET-mChBac75Na BACTERIA STRAINS FOR CHBAC7NA MINIBACTENECIN ANTIMICROBIAL PEPTIDE EXPRESSION AND METHODS FOR PRODUCTION OF THESE PEPTIDES | |
| CN104480081A (en) | Sso7d-Sau recombinant DNA polymerase | |
| KR101775790B1 (en) | Cell lysis composition for nucleic acid separating and refining | |
| CN115216463B (en) | Recombinant trypsin with stability and preparation method and application thereof | |
| TWI712691B (en) | Dextran affinity tag and application thereof | |
| CN117187210B (en) | Mutant Bst DNA polymerase large fragment and preparation method thereof | |
| CN115747187B (en) | Recombinase UvsX and expression gene and application thereof | |
| Watrud | The Biological Revolution: Tools and Products of Biotechnology | |
| CN109134637B (en) | Method for purifying fluorescent protein | |
| WO2022166572A1 (en) | Method and system for continuous cloning of long dna fragment |
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: 20210312 |