CN116693638B - Application of PG1-LC protein as hydrolase of SNAP-25 - Google Patents
Application of PG1-LC protein as hydrolase of SNAP-25 Download PDFInfo
- Publication number
- CN116693638B CN116693638B CN202310545267.8A CN202310545267A CN116693638B CN 116693638 B CN116693638 B CN 116693638B CN 202310545267 A CN202310545267 A CN 202310545267A CN 116693638 B CN116693638 B CN 116693638B
- Authority
- CN
- China
- Prior art keywords
- snap
- protein
- drosophila
- hydrolase
- amino acid
- 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.)
- Active
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 86
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 77
- 108010057722 Synaptosomal-Associated Protein 25 Proteins 0.000 title claims abstract description 48
- 102000004183 Synaptosomal-Associated Protein 25 Human genes 0.000 title claims abstract description 48
- 235000018102 proteins Nutrition 0.000 claims abstract description 76
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 15
- 238000003776 cleavage reaction Methods 0.000 claims abstract description 8
- 230000007017 scission Effects 0.000 claims abstract description 6
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims abstract description 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims abstract description 3
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims abstract description 3
- 229960001230 asparagine Drugs 0.000 claims abstract description 3
- 235000009582 asparagine Nutrition 0.000 claims abstract description 3
- 235000013922 glutamic acid Nutrition 0.000 claims abstract description 3
- 239000004220 glutamic acid Substances 0.000 claims abstract description 3
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims abstract description 3
- 108700009372 Drosophila Snap25 Proteins 0.000 claims description 39
- 238000001976 enzyme digestion Methods 0.000 claims description 20
- 239000013604 expression vector Substances 0.000 claims description 20
- 238000006243 chemical reaction Methods 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 13
- 210000004027 cell Anatomy 0.000 claims description 12
- 238000000338 in vitro Methods 0.000 claims description 11
- 210000004962 mammalian cell Anatomy 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 239000000413 hydrolysate Substances 0.000 claims description 8
- 239000011535 reaction buffer Substances 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 5
- 230000007062 hydrolysis Effects 0.000 claims description 5
- 238000006460 hydrolysis reaction Methods 0.000 claims description 5
- 239000007983 Tris buffer Substances 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 4
- 230000003834 intracellular effect Effects 0.000 claims description 3
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 2
- 230000001225 therapeutic effect Effects 0.000 claims 2
- 102000004190 Enzymes Human genes 0.000 abstract description 3
- 108090000790 Enzymes Proteins 0.000 abstract description 3
- 230000002255 enzymatic effect Effects 0.000 abstract description 2
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 abstract 1
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 abstract 1
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 abstract 1
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 abstract 1
- 241000193403 Clostridium Species 0.000 description 11
- 108090000604 Hydrolases Proteins 0.000 description 10
- 102000004157 Hydrolases Human genes 0.000 description 10
- 239000012634 fragment Substances 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 4
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 108010041948 SNARE Proteins Proteins 0.000 description 3
- 102000000583 SNARE Proteins Human genes 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 108010042653 IgA receptor Proteins 0.000 description 2
- 102000018697 Membrane Proteins Human genes 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- 241001147789 Paeniclostridium ghonii Species 0.000 description 2
- 102100034014 Prolyl 3-hydroxylase 3 Human genes 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 101710117021 Tyrosine-protein phosphatase YopH Proteins 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 229910052759 nickel Inorganic materials 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000012474 protein marker Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- HVLSXIKZNLPZJJ-TXZCQADKSA-N HA peptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HVLSXIKZNLPZJJ-TXZCQADKSA-N 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 241000193465 Paeniclostridium sordellii Species 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 102000014384 Type C Phospholipases Human genes 0.000 description 1
- 108010079194 Type C Phospholipases Proteins 0.000 description 1
- 208000009443 Vascular Malformations Diseases 0.000 description 1
- 241000269370 Xenopus <genus> Species 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 210000001097 facial muscle Anatomy 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000001660 hyperkinetic effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- -1 media Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000003957 neurotransmitter release Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000000946 synaptic effect Effects 0.000 description 1
- 230000021966 synaptic vesicle transport Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
- 239000011592 zinc chloride Substances 0.000 description 1
- 235000005074 zinc chloride Nutrition 0.000 description 1
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 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
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- 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
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明提供了PG1‑LC蛋白作为SNAP‑25的水解酶的应用；PG1‑LC蛋白的酶切位点位于SNAP‑25氨基酸序列的第196位的天冬酰胺与197位的谷氨酸之间，以及SNAP‑25氨基酸序列的第209位的谷氨酰胺与210位的亮氨酸之间。本发明首次给出了PG1‑LC蛋白作为SNAP‑25的水解酶的应用，PG1‑LC蛋白具有极高的酶活性，仅需0.1μM就能在三分钟之内将0.3mg/mL的SNAP‑25完全水解。
The invention provides the application of PG1-LC protein as a hydrolase of SNAP-25; the enzyme cleavage site of PG1-LC protein is located between asparagine at position 196 and glutamic acid at position 197 of the SNAP-25 amino acid sequence. , and between glutamine at position 209 and leucine at position 210 of the SNAP-25 amino acid sequence. The present invention provides for the first time the application of PG1-LC protein as a hydrolase of SNAP-25. PG1-LC protein has extremely high enzymatic activity and can convert 0.3 mg/mL SNAP-25 within three minutes with only 0.1 μM. 25 Completely hydrolyzed.
Description
Technical Field
The invention belongs to the technical field of molecular biology and biochemistry, relates to a protein function, and in particular relates to an application of PG1-LC protein as hydrolase of SNAP-25.
Background
Clostridium gossypii (Paeniclostridium ghonii) is a gram-positive bacterium having a rod-like form and mesophilic properties. The bacteria can move and proliferate in a binary division mode, belong to obligate anaerobic bacteria, and can grow by utilizing various organic substances. Under the adverse environment, the bacteria can generate the endophyte, so that the bacteria can resist high temperature and dehydration environment. Clostridium gossypii was previously known for a long time as clostridium gossypii (Clostridium ghonii), and was classified as clostridium (paeniclothridium) by sequencing its 16S RNA and performing phylogenetic analysis, and then combining it with clostridium bifidum (Clostridium sordellii) of the same genus. Clostridium gossypii belongs to non-pathogenic bacteria, and previous studies have shown that this type of bacteria has a positive role in the treatment of solid tumor cancers. This is mainly because vascular malformations inside solid tumors limit the supply of oxygen to create a local anoxic environment, providing a suitable environment for the growth of clostridium gossypii. On one hand, germination and growth of clostridium gossypii in solid tumors can effectively excite immune response, so that anti-tumor treatment is facilitated; on the other hand, clostridium gossypii can secrete collagenase IV and phospholipase C, so that apoptosis and necrosis of tumor cells are promoted.
The biosafety level of clostridium gossypii is classified into one class, and no reports of toxin genes and virulence proteins are seen. In 2022, a certain subject group in Canada finds a possible virulence protein in a clostridium gossypii strain NCTR 3900 by means of bioinformatics, and the protein is named as PG-toxin1-LC (namely PG1-LC described in the invention), so that the function of PG-toxin1-LC is not reported, and the potential application value of the protein is yet to be developed.
Chain-cleaving synaptogenesis associated membrane protein 25 (snap-25, synaptogenesis associated membrane-associated protein), an important member of the SNARE complex, which has its homologous genes in human, rat, drosophila, xenopus, etc. organisms, plays a key role in calcium-triggered synaptic vesicle fusion and neurotransmitter release into synaptic clefts, and if the SNARE complex fails to form or is destroyed by hydrolysis, it blocks acetylcholine release, rendering the tissue unacceptable, and by using the above principles, certain muscle hyperfunctional diseases can be treated, or the hyperkinetic state of facial muscles weakened, leading to improved or eliminated wrinkles.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide the application of PG1-LC protein as the hydrolase of SNAP-25, and solve the technical problems that the function of PG-toxin1-LC protein in the prior art is unknown and the application is limited.
In order to solve the technical problems, the invention adopts the following technical scheme:
use of the PG1-LC protein as a hydrolase for SNAP-25.
The invention also has the following technical characteristics:
specifically, the cleavage site of PG1-LC protein comprises: between asparagine 196 and glutamic acid 197 of the SNAP-25 amino acid sequence, and between glutamine 209 and leucine 210 of the SNAP-25 amino acid sequence.
Specifically, the amino acid sequence of the PG1-LC protein is as follows: MIPINIKDFNYSDPVNNQ DIILVKNEKGSFDKGFFVADKILLVPARYGNISTDEGGITSKKEKAHVDKKIYLETDSEKNEYLKNMTTLLKRMNSYSTGNKLLNLIIKGEPIYSKDLQGKFIEQTPSRYLDTNTGKRRVNVMITGPGSNVLTKKCTHNGMGLENDPNGKHSNGTGILSTIEFSPNYLIAYNKCVADPVLTLFHELVHSMHNLYGIAFPDNVKVPYNALKDKNLVSGEEALSEILTFGGKDLTTEHLETLWKKLAETVIIVKDFVKTDTQAKDVFLNNLRFLSKNENIKIDTIEDIVNGTLKIKNNISNLTECEFCKEIGDVRIRTRYAVHSEDVTPVEVVDFKNNYKLNSGFLEGQDISKKYFITNPPKMRRRALRNFKCTIQ.
Specifically, the SNAP-25 is Drosophila SNAP-25.
Alternatively and in particular, the method for the use of the PG1-LC protein as an intracellular hydrolase comprises the steps of:
step one, constructing a mammalian cell expression vector;
step two, PG1-LC protein and SNAP-25 are co-expressed:
and (3) transfecting the PG1-LC protein obtained in the step (I) and the mammalian cell expression vector of the SNAP-25 into mammalian cells, culturing for 24-72 hours, collecting cells and extracting the protein to obtain a hydrolysate of the SNAP-25.
Alternatively and in particular, the method for the use of the PG1-LC protein as an in vitro hydrolase comprises the steps of:
step one, constructing an in vitro expression vector;
step two, preparing PG1-LC protein and SNAP-25;
step three, enzyme digestion reaction is carried out:
and (3) adding the PG1-LC protein obtained in the step (II) and SNAP-25 into a reaction buffer solution together, uniformly mixing to obtain an enzyme digestion reaction solution, and then carrying out enzyme digestion reaction on the enzyme digestion reaction solution at the temperature of 37 ℃ for 1-30 min to obtain a hydrolysis product of the SNAP-25.
Preferably, in the third step, the final concentration of PG1-LC protein in the cleavage reaction solution is 0.1. Mu.M, and the final concentration of SNAP-25 is 0.3mg/mL.
Preferably, in the third step, the pH value of the reaction buffer solution is 7.5, and the reaction buffer solution comprises the following components: 50mM Tris,150mM NaCl and 0.1mM ZnCl 2 。
Compared with the prior art, the invention has the following technical effects:
the invention provides the application of PG1-LC protein as the hydrolase of SNAP-25 for the first time, the PG1-LC protein has extremely high enzyme activity, and 0.3mg/mL of SNAP-25 can be completely hydrolyzed within three minutes only by 0.1 mu M.
Drawings
FIG. 1 is a western blot of SNAP-25 hydrolysate from example 1. In fig. 1: "-" indicates a negative control group (i.e., total protein of HEK293T cells transfected with SNAP-25 expression vector), "a" indicates a control protein a group, "B" indicates a control protein B group, "PG1" indicates a PG1-LC protein group, and "PG2" indicates a PG2-LC protein group; "HA-SNAP25 (fly)" indicates the detection result using the HA antibody, "FLAG" indicates the detection result using the FLAG tag antibody, and "beta-actin" indicates the detection result using the actin antibody.
FIG. 2 is a polyacrylamide gel electrophoresis of the SNAP-25 hydrolysate of example 1. In fig. 2: "M" represents a protein Marker, "SNAP25 (fly)" represents unhydrolyzed Drosophila SNAP-25, and "hydroylate" represents a Hydrolysate of SNAP-25.
FIG. 3 is a mass spectrum of SNAP-25 hydrolysate in example 2.
FIG. 4 is a schematic diagram showing the position of the cleavage site of PG1-LC protein in example 2.
FIG. 5 is a polyacrylamide gel electrophoresis of the SNAP-25 hydrolysate of comparative example 1. In fig. 5: "M" represents a protein Marker, "SNAP25 (fly)" represents unhydrolyzed Drosophila SNAP-25, and "hydroylate" represents a Hydrolysate of SNAP-25.
The following examples illustrate the invention in further detail.
Detailed Description
All reagents, media, carriers and competence used in the present invention are those known in the art, for example:
coli competent cells were obtained from Transetta (DE 3) Chemically Competent Cell, beijing full gold Biotechnology Co., ltd.
The following specific embodiments of the present invention are provided, and it should be noted that the present invention is not limited to the following specific embodiments, and all equivalent changes made on the basis of the technical solutions of the present application fall within the protection scope of the present invention.
Example 1:
this example shows the use of PG1-LC protein as an intracellular hydrolase for Drosophila SNAP-25.
The amino acid sequence of the PG1-LC protein is as follows: MIPINIKDFNYSDPVNNQDIILVKNEKGS FDKGFFVADKILLVPARYGNISTDEGGITSKKEKAHVDKKIYLETDSEKNEYLKNMTTLLKRMNSYSTGNKLLNLIIKGEPIYSKDLQGKFIEQTPSRYLDTNTGKRRVNVMITGPGSNVLTKKCTHNGMGLENDPNGKHSNGTGILSTIEFSPNYLIAYNKCVADPVLTLFHELVHSMHNLYGIAFPDNVKVPYNALKDKNLVSGEEALSEILTFGGKDLTTEHLETLWKKLAETVIIVKDFVKTDTQAKDVFLNNLRFLSKNENIKIDTIEDIVNGTLKIKNNISNLTECEFCKEIGDVRIRTRYAVHSEDVTPVEVVDFKNNYKLNSGFLEGQDISKKYFITNPPKMRRRALRNFKCTIQ.
The amino acid sequence of Drosophila SNAP-25 is as follows: MPADPSEEVAPQVPKTELEELQINAQG VADESLESTRRMLALCEESKEAGIRTLVALDDQGEQLDRIEEGMDQINADMREAEKNLSGMEKCCGICVLPCNKSQSFKEDDGTWKGNDDGKVVNNQPQRVMDDRNGMMAQAGYIGRITNDAREDEMEENMGQVNTMIGNLRNMALDMGSELENQNRQIDRINRKGESNEARIAVANQRAHQLLK.
The application method specifically comprises the following steps:
step one, constructing a mammalian cell expression vector:
PCR primers were designed based on the nucleotide sequences of PG1-LC protein and Drosophila SNAP-25, and are shown in Table 1. Then, PCR was performed to obtain gene fragments of PG1-LC protein and SNAP-25. And (3) carrying out enzyme digestion connection on the gene fragment and the expression vector pcDNA3.1-myc/His, and obtaining the PG1-LC protein and the mammalian cell expression vector of Drosophila SNAP-25 after transformation, screening and identification. FLAG tag is fused at the N end of PG1-LC, and HA tag is fused at the N end of Drosophila SNAP-25, so that immunoblotting detection of later protein is facilitated.
The nucleotide sequence of the PG1-LC protein is as follows: 5'-atgattccaattaacattaaggactttaattactccgatccag tgaacaaccaggatatcatccttgtaaaaaacgaaaaggggtcgtttgacaagggattctttgttgcagacaaaatcttacttgtccccgcgcgctacgggaacatctccactgatgaaggaggaattacgtcgaagaaggagaaagctcacgtagataaaaagatctacttggagacagattccgaaaagaacgagtaccttaagaatatgaccactttgttaaagcgtatgaattcatactctacgggaaataaacttttaaacttaatcattaagggcgagccaatttactcaaaagatttgcaaggaaaattcattgaacagacccccagtcgctaccttgacactaatacagggaaacgccgtgttaatgttatgattaccggaccgggctccaacgttttaacaaagaaatgcacccataacggcatgggcttagaaaacgacccgaatggaaagcattctaacggtacaggcatcctttcaactatcgagttcagtccgaattaccttattgcttacaataaatgtgtagctgatccggtgttaacgttattccatgaattagtgcattccatgcataacctgtacggcatcgcgttcccggataatgttaaagtcccctataatgccttaaaggacaagaatctggtgtcaggtgaggaagcattgtcagagattttgactttcggtggtaaagacttaaccactgaacatctggagacgttgtggaagaagctggcggagactgtgatcatcgttaaggattttgtaaagactgatacccaggctaaggatgtgtttttgaataatttgcgctttttaagcaagaacgagaacatcaagatcgacactatcgaggatatcgttaatgggacattaaagatcaaaaataacatctctaatttgacggagtgcgagttttgtaaggagattggcgatgtacgcatccgcacgcgttatgcggttcattccgaagacgtcactcccgtagaggtagtggacttcaaaaacaactataagctgaactcgggatttttggagggacaggatatctcaaaaaaatacttcattactaatccaccgaagatgcgtcgtcgcgcgttacgcaactttaaatgcacaattcaa-3'.
The nucleotide sequence of Drosophila SNAP-25 is as follows: 5'-atgccagcggatccatctgaagaagttgcccctcaggt cccgaagaccgagctagaagagctgcaaattaatgcgcaaggagtagccgatgagtccctggaaagtacgcgacgtatgcttgctctgtgtgaggagagcaaggaggcagggattcgaacacttgtagcccttgatgatcaaggagaacaactggatcgtattgaagaaggaatggatcaaattaatgcagacatgagagaagcagaaaaaaatttaagtggaatggaaaaatgttgcggcatttgtgttcttccgtgcaataaaagtcaatcattcaaagaagatgatgggacctggaaaggaaatgatgacggaaaagttgtaaataatcagccacagagagtgatggatgatagaaatggcatgatggcgcaagcgggttatattggcaggataacgaacgacgctagagaagatgaaatggaagaaaatatgggccaggtaaacactatgataggcaatctccgaaatatggcattggatatgggctctgagctggaaaatcaaaatcgtcaaattgatagaataaaccgaaagggtgaatctaatgaagcgcggatagcagttgctaatcaaagggcacatcaactattaaag-3'.
In this example, mammalian cell expression vectors were also constructed for control protein a and control protein B, two proteins known in the art to be associated with SNARE complex hydrolysis, as subsequent controls.
Step two, PG1-LC protein and SNAP-25 are co-expressed:
the PG1-LC protein obtained in the step one and the mammalian cell expression vector of Drosophila SNAP-25 are transfected into HEK293T cells together, after 24 hours of culture, the cells are collected and the protein is extracted, and then Western-blot is adopted for identification, and the result is shown in figure 1. As can be seen from FIG. 1, PG1-LC protein is capable of hydrolyzing Drosophila SNAP-25 after co-expressing PG1-LC protein and Drosophila SNAP-25 in cells.
Example 2:
the example shows the use of PG1-LC protein as an in vitro hydrolase of Drosophila SNAP-25, and the method of use comprises the following steps:
step one, constructing an in vitro expression vector:
PCR primers were designed based on the nucleotide sequences of PG1-LC protein and Drosophila SNAP-25, and are shown in Table 1. Then, PCR was performed to obtain gene fragments of PG1-LC protein and SNAP-25. The gene fragment and the expression vector pET28a are subjected to enzyme digestion connection, and the gene fragment is directly placed after a DNA sequence for encoding 6 XHis and in the same reading frame, and after transformation, screening and identification, the in vitro expression vector of PG1-LC protein and Drosophila SNAP-25 is obtained.
Step two, preparing PG1-LC protein and SNAP-25:
the PG1-LC protein and the Drosophila SNAP-25 expression vector obtained in the step one are respectively transformed into competent cells of escherichia coli, positive clones capable of expressing the PG1-LC protein and the Drosophila SNAP-25 are obtained after culturing, screening and identifying, the positive clones are cultured at 20 ℃ for 16 hours, thalli are collected, cells are lysed, and supernatant is obtained after centrifugation and purified by using a nickel column and a desalting column.
Step three, enzyme digestion reaction is carried out:
the PG1-LC protein purified in the second step was added to a reaction buffer (50mM Tris,150mM NaCl,0.1mM ZnCl) together with SNAP-25 2 The method comprises the steps of carrying out a first treatment on the surface of the pH 7.5), and then the enzyme digestion reaction liquid is divided into six groups, and enzyme digestion reaction products are identified by SDS-PAGE after enzyme digestion reaction for 1, 3, 5, 10, 15 and 30min at 37 ℃ respectively, and the result is shown in figure 2.
As can be seen from FIG. 2, the PG1-LC protein is capable of hydrolyzing Drosophila SNAP-25 in vitro. The PG1-LC protein Drosophila SNAP-25 has extremely high enzyme activity, and 0.1 mu M PG1-LC can completely hydrolyze 0.3mg/mL Drosophila SNAP-25 in three minutes.
In this example, in order to identify the cleavage site of PG1-LC in Drosophila SNAP-25, the hydrolyzed product was analyzed by mass spectrometry using the following method: after enrichment of the hydrolyzed small peptides, peptide fragments were analyzed by shotgun method and PRM method, and the results are shown in FIG. 3. As can be seen from FIG. 3, in the hydrolysis product of PG1-LC against Drosophila SNAP-25, a larger number of peptides were detected as EARIAVANQRAHQLLK, and peptides such as VANQRAHQLLK, QRAHQLLK, NQRAHQLLK were also detected (not shown).
As shown in FIG. 4, it was inferred that the cleavage site of PG1-LC protein for Drosophila SNAP-25 was located between N at position 196 and E at position 197 of the amino acid sequence of Drosophila SNAP-25, and between Q at position 209 and L at position 210.
Comparative example 1:
this comparative example shows the use of PG2-LC protein as an in vitro hydrolase of Drosophila SNAP-25.
The amino acid sequence of the PG2-LC protein is as follows: MAPITIKDFKYNTLPNGNDVVLVKNEK GTADKGFFVADNILVVPERYGEIQGEEGGTAPKEATEVRDKQYLQSEEHKDEFLKMITVLLKRINSKPEGSEFLNIMTKAEPLYDINNGEFKERCTSRYVETTTGQRRVNVIITGPGTNLTEGITIPYITENTRALESNGFGSASTISITPFYELGYIKDHKNPEEKYCADPAMSLYHELVHAFHNLYGINFNHTKIEETSLEEYVTFGRYNEKSCVEMNKVGMYAGTSLLKSFDEINTLKEADKSKINNIFENKLTLLFGEKTSANNESDIAKIALKIDKKIHAFTECEFAKAFNTKTGGIYVRDDYNYTSATPLKFEKLLDNYHPQDGFIKYGKPMYEGQHIKNKYIATNPAITSKKARFRFLLKK.
The application method specifically comprises the following steps:
step one, constructing an in vitro expression vector:
PCR primers were designed based on the nucleotide sequences of PG2-LC protein and Drosophila SNAP-25, and are shown in Table 1. Then, PCR was performed to obtain gene fragments of PG2-LC protein and SNAP-25. The gene fragment and the expression vector pET28a are subjected to enzyme digestion connection, and the gene fragment is directly placed after a DNA sequence for encoding 6 XHis and in the same reading frame, and after transformation, screening and identification, the PG2-LC protein and the in vitro expression vector of Drosophila SNAP-25 are obtained.
The nucleotide sequence of the PG2-LC protein is as follows: 5'-atggcaccgatcacgatcaaggattttaagtataatacctt accgaatgggaatgacgttgtccttgttaagaatgagaaggggacggctgacaaaggattctttgtggccgataatatcttggtcgttcctgaacgctatggggagattcaaggggaagaaggaggaacggcacccaaggaagcgaccgaagtacgtgacaaacagtacttacagtccgaagagcataaagacgagttcttaaaaatgattacggtgcttttaaaacgtatcaatagtaagcctgagggttcggagtttttaaatatcatgactaaagctgaacccttgtatgatattaataacggcgagttcaaggagcgttgtacctcacgctatgtagaaactacaacaggtcagcgccgtgttaacgtaatcattacggggccaggaacgaaccttactgaaggtattacgattccctatatcacagaaaatacgcgcgctttggagtctaatggcttcggttccgcttccacgatctcaatcacccctttttacgagctgggatatatcaaagatcataaaaatccagaagaaaaatactgcgccgaccccgcaatgagcctgtatcacgaacttgttcatgccttccacaacctttacggcattaacttcaaccacactaagatcgaagagacttcgctggaggagtatgtcacgtttggtcgttataatgagaaaagttgtgtcgaaatgaataaagttggaatgtatgctggtacatctttattgaagtccttcgacgaaatcaatacgctgaaggaggcggataagagtaaaatcaacaatatctttgagaataaattgacgttgttgtttggcgaaaagacctcagcgaataacgagtcggatattgctaagattgcattaaagattgacaagaaaatccatgcctttacggaatgcgagttcgctaaagcgttcaacaccaaaactggagggatctatgtacgcgacgactacaattacacgtccgccacccctttgaagtttgagaaacttttagataattatcacccccaagacggatttattaagtacggaaaaccgatgtacgagggtcaacacattaaaaacaaatatatcgccactaatccagccatcacgtctaagaaggcacgctttcgtttcttattatgcaagaaa-3'.
Step two, preparing PG2-LC protein and SNAP-25:
and (3) respectively converting the PG2-LC protein and the Drosophila SNAP-25 expression vector obtained in the step (A) into competent cells of escherichia coli, culturing, screening and identifying to obtain positive clones capable of expressing the PG2-LC protein and the Drosophila SNAP-25, culturing the positive clones at 20 ℃ for 16 hours, collecting thalli, lysing the cells, centrifuging to obtain supernatant, and purifying the protein by using a nickel column and a desalting column.
Step three, enzyme digestion reaction is carried out:
and (3) adding the PG2-LC protein purified in the second step and SNAP-25 into a reaction buffer (50mM Tris,150mM NaCl,0.1mM ZnCl2;pH 7.5) together, uniformly mixing to obtain an enzyme digestion reaction solution, dividing the enzyme digestion reaction solution into six groups, wherein one group is left as a blank control, and the remaining five groups are subjected to enzyme digestion reaction at 37 ℃ for 0.5, 1, 2, 3, 4 and 6 hours respectively, and then identifying enzyme digestion reaction products by adopting SDS-PAGE, wherein the result is shown in figure 5.
As can be seen from FIG. 5, the PG2-LC protein has relatively weak enzymatic activity against Drosophila SNAP-25, compared with the PG1-LC protein, and the same amount of Drosophila SNAP-25 cannot be completely hydrolyzed in three hours.
TABLE 1 primer sequences
Claims (7)
- The use of pg1-LC protein as a hydrolase for SNAP-25, wherein said hydrolase is an intracellular hydrolase; the SNAP-25 is Drosophila SNAP-25;the amino acid sequence of the PG1-LC protein is as follows: MIPINIKDFNYSDPVNNQDIILVKNEKGSFDKGFFVADKILLVPARYGNISTDEGGITSKKEKAHVDKKIYLETDSEKNEYLKNMTTLLKRMNSYSTGNKLLNLIIKGEPIYSKDLQGKFIEQTPSRYLDTNTGKRRVNVMITGPGSNVLTKKCTHNGMGLENDPNGKHSNGTGILSTIEFSPNYLIAYNKCVADPVLTLFHELVHSMHNLYGIAFPDNVKVPYNALKDKNLVSGEEALSEILTFGGKDLTTEHLETLWKKLAETVIIVKDFVKTDTQAKDVFLNNLRFLSKNENIKIDTIEDIVNGTLKIKNNISNLTECEFCKEIGDVRIRTRYAVHSEDVTPVEVVDFKNNYKLNSGFLEGQDISKKYFITNPPKMRRRALRNFKCTIQ;the amino acid sequence of the Drosophila SNAP-25 is as follows: MPADPSEEVAPQVPKTELEELQINAQGVADESLESTRRMLALCEESKEAGIRTLVALDDQGEQLDRIEEGMDQINADMREAEKNLSGMEKCCGICVLPCNKSQSFKEDDGTWKGNDDGKVVNNQPQRVMDDRNGMMAQAGYIGRITNDAREDEMEENMGQVNTMIGNLRNMALDMGSELENQNRQIDRINRKGESNEARIAVANQRAHQLLK;the application is for non-therapeutic purposes.
- 2. The application according to claim 1, characterized in that the method of application comprises the steps of:step one, constructing a mammalian cell expression vector;step two, PG1-LC protein and SNAP-25 are co-expressed:and (3) transfecting the PG1-LC protein obtained in the step (A) and the mammalian cell expression vector of the Drosophila SNAP-25 into mammalian cells, culturing for 24-72 hours, collecting cells and extracting protein to obtain a hydrolysate of the SNAP-25.
- The use of pg1-LC protein as a hydrolase for SNAP-25, wherein the hydrolase is an in vitro hydrolase; the SNAP-25 is Drosophila SNAP-25;the amino acid sequence of the PG1-LC protein is as follows: MIPINIKDFNYSDPVNNQDIILVKNEKGSFDKGFFVADKILLVPARYGNISTDEGGITSKKEKAHVDKKIYLETDSEKNEYLKNMTTLLKRMNSYSTGNKLLNLIIKGEPIYSKDLQGKFIEQTPSRYLDTNTGKRRVNVMITGPGSNVLTKKCTHNGMGLENDPNGKHSNGTGILSTIEFSPNYLIAYNKCVADPVLTLFHELVHSMHNLYGIAFPDNVKVPYNALKDKNLVSGEEALSEILTFGGKDLTTEHLETLWKKLAETVIIVKDFVKTDTQAKDVFLNNLRFLSKNENIKIDTIEDIVNGTLKIKNNISNLTECEFCKEIGDVRIRTRYAVHSEDVTPVEVVDFKNNYKLNSGFLEGQDISKKYFITNPPKMRRRALRNFKCTIQ;the amino acid sequence of the Drosophila SNAP-25 is as follows: MPADPSEEVAPQVPKTELEELQINAQGVADESLESTRRMLALCEESKEAGIRTLVALDDQGEQLDRIEEGMDQINADMREAEKNLSGMEKCCGICVLPCNKSQSFKEDDGTWKGNDDGKVVNNQPQRVMDDRNGMMAQAGYIGRITNDAREDEMEENMGQVNTMIGNLRNMALDMGSELENQNRQIDRINRKGESNEARIAVANQRAHQLLK;the application is for non-therapeutic purposes.
- 4. The use according to claim 3, wherein the cleavage site of the PG1-LC protein comprises: between asparagine 196 and glutamic acid 197 of the amino acid sequence of Drosophila SNAP-25, and between glutamine 209 and leucine 210 of the amino acid sequence of Drosophila SNAP-25.
- 5. A use according to claim 3, characterized in that the method of the use comprises the steps of:step one, constructing an in vitro expression vector;step two, preparing PG1-LC protein and SNAP-25;step three, enzyme digestion reaction is carried out:and (3) adding the PG1-LC protein obtained in the step (II) and the Drosophila SNAP-25 into a reaction buffer solution together, uniformly mixing to obtain an enzyme digestion reaction solution, and then carrying out enzyme digestion reaction on the enzyme digestion reaction solution at the temperature of 37 ℃ for 1-30 min to obtain a hydrolysis product of the SNAP-25.
- 6. The method according to claim 5, wherein in step three, the final concentration of PG1-LC protein in the cleavage reaction solution is 0.1. Mu.M, and the final concentration of SNAP-25 is 0.3mg/mL.
- 7. The method according to claim 5, wherein in step three, the pH of the reaction buffer is 7.5, and the reaction buffer comprises the following components: 50mM Tris,150mM NaCl and 0.1mM ZnCl 2 。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310545267.8A CN116693638B (en) | 2023-05-15 | 2023-05-15 | Application of PG1-LC protein as hydrolase of SNAP-25 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310545267.8A CN116693638B (en) | 2023-05-15 | 2023-05-15 | Application of PG1-LC protein as hydrolase of SNAP-25 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116693638A CN116693638A (en) | 2023-09-05 |
| CN116693638B true CN116693638B (en) | 2024-03-08 |
Family
ID=87824794
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310545267.8A Active CN116693638B (en) | 2023-05-15 | 2023-05-15 | Application of PG1-LC protein as hydrolase of SNAP-25 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116693638B (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7374896B2 (en) * | 2001-08-28 | 2008-05-20 | Allergan, Inc. | GFP-SNAP25 fluorescence release assay for botulinum neurotoxin protease activity |
| ATE483158T1 (en) * | 2005-04-05 | 2010-10-15 | Allergan Inc | CLOSTRIDIAL TOXIN ACTIVITY TESTS |
-
2023
- 2023-05-15 CN CN202310545267.8A patent/CN116693638B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| NCBI Reference Sequence: WP_250673407.1;无;Genbank;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116693638A (en) | 2023-09-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105601720B (en) | Signal peptide capable of effectively improving protein secretion expression efficiency and application thereof | |
| CN111575265B (en) | A keratinase mutant with improved thermal stability | |
| Roux et al. | Non-contiguous finished genome sequence and description of Oceanobacillus massiliensis sp. nov. | |
| Khelaifia et al. | Noncontiguous finished genome sequence and description of Virgibacillus massiliensis sp. nov., a moderately halophilic bacterium isolated from human gut | |
| CN116693638B (en) | Application of PG1-LC protein as hydrolase of SNAP-25 | |
| WO2025007626A1 (en) | Bacillus subtilis, selective breeding method therefor and use thereof | |
| CN116769756B (en) | Application of PG2-LC protein as hydrolase of SNAP-25 | |
| CN116121215B (en) | Mutant of glycerophosphate oxidase and application thereof | |
| Guilhot et al. | Genome sequence and description of Anaeromassilibacillus senegalensis gen. nov., sp. nov., isolated from the gut of patient with kwashiorkor | |
| CN116574710A (en) | DNA polymerase with strand displacement function and application thereof | |
| RU2447151C1 (en) | ALKALINE PHOSPHATASE CmAP SYNTHESIS-DETERMINING 40Ph PLASMID, E. coli rosetta(DE3)/40Ph STRAIN - PRODUCER OF CHIMERIC PROTEIN, CONTAINING AMINO ACID SEQUENCE OF RECOMBINANT ALKALINE PHOSPHATASE CmAP, AND PRODUCTION METHOD THEREOF | |
| CN111349585B (en) | A marine-derived collagen expansion protease VP9 and its encoding gene and application | |
| KR102152142B1 (en) | The preparation method of cyclic oligoadenylate using Cas10/Csm4 | |
| US11560582B2 (en) | Fusion moieties and microbial hosts for protein production | |
| CN114214351A (en) | A Shigella polysaccharide expression plasmid and use thereof | |
| CN105017399A (en) | Schistosoma japonicum SjPPase recombinant antigen protein and preparation method and application thereof | |
| Dubourg et al. | Risungbinella massiliensis sp. nov., a new member of Thermoactinomycetaceae isolated from human gut | |
| US20240360477A1 (en) | Systems and methods for transposing cargo nucleotide sequences | |
| US20250042953A1 (en) | Alkali-tolerant mutation method of protein a | |
| CN109022471A (en) | Produce the escherichia expression system of oxalate oxidase, the production method and its application of oxalate oxidase | |
| JP2025064490A (en) | Peptides or their salts, and nucleic acids | |
| KR100330688B1 (en) | Gene Coding for Heat-resistant Alanine Racemase of Aquifex pyrophilus, Heat-resistant Alanine Racemase Expressed therefrom, and Method for Preparing the Same | |
| CN109055419A (en) | Construction method and application with the active recombinant microorganism of Soluble phosphorus | |
| CN116694600A (en) | A Novel Polyurethane Plastic Degrading Enzyme and Its Application | |
| KR20090060124A (en) | Novel hydrogenase isolated from Ｔｈｅｒｍｏｃｏｃｃｕｓ ｓｐｐ. |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240401 Address after: Room 123, Building D, 288 Shenzhou Road, Huangpu District, Guangzhou City, Guangdong Province, 510663 Patentee after: Lemondi (Guangzhou) Biotechnology Co.,Ltd. Country or region after: China Address before: 712100 Shaanxi Province, Xi'an city Yangling District Tai Road No. 3 demonstration Patentee before: NORTHWEST A & F University Country or region before: China |