CN117587042A - Recombinant lactic acid oxidase and preparation method and application thereof - Google Patents
Recombinant lactic acid oxidase and preparation method and application thereof Download PDFInfo
- Publication number
- CN117587042A CN117587042A CN202210982263.1A CN202210982263A CN117587042A CN 117587042 A CN117587042 A CN 117587042A CN 202210982263 A CN202210982263 A CN 202210982263A CN 117587042 A CN117587042 A CN 117587042A
- Authority
- CN
- China
- Prior art keywords
- polynucleotide
- host cell
- protein
- vector
- seq
- 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
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N Lactic Acid Natural products CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title abstract description 36
- 235000014655 lactic acid Nutrition 0.000 title abstract description 32
- 239000004310 lactic acid Substances 0.000 title abstract description 32
- 108090000854 Oxidoreductases Proteins 0.000 title abstract description 29
- 102000004316 Oxidoreductases Human genes 0.000 title abstract description 29
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 108091033319 polynucleotide Proteins 0.000 claims abstract description 63
- 102000040430 polynucleotide Human genes 0.000 claims abstract description 63
- 239000002157 polynucleotide Substances 0.000 claims abstract description 63
- 239000013598 vector Substances 0.000 claims abstract description 33
- 241000588724 Escherichia coli Species 0.000 claims abstract description 21
- 108090000623 proteins and genes Proteins 0.000 claims description 74
- 102000004169 proteins and genes Human genes 0.000 claims description 49
- 238000000034 method Methods 0.000 claims description 48
- 108010073450 Lactate 2-monooxygenase Proteins 0.000 claims description 28
- 239000013604 expression vector Substances 0.000 claims description 21
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 238000000502 dialysis Methods 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 229930027917 kanamycin Natural products 0.000 claims description 10
- 229960000318 kanamycin Drugs 0.000 claims description 10
- 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 claims description 10
- 229930182823 kanamycin A Natural products 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 9
- 239000012137 tryptone Substances 0.000 claims description 9
- 230000000295 complement effect Effects 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 8
- 108020004705 Codon Proteins 0.000 claims description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 7
- 230000001131 transforming effect Effects 0.000 claims description 4
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 229960005150 glycerol Drugs 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 2
- 239000006228 supernatant Substances 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 17
- 108700010070 Codon Usage Proteins 0.000 abstract description 11
- 210000004027 cell Anatomy 0.000 description 51
- 229920001184 polypeptide Polymers 0.000 description 17
- 102000004196 processed proteins & peptides Human genes 0.000 description 17
- 108090000765 processed proteins & peptides Proteins 0.000 description 17
- 238000010828 elution Methods 0.000 description 16
- 239000002609 medium Substances 0.000 description 16
- 238000005457 optimization Methods 0.000 description 14
- 239000012634 fragment Substances 0.000 description 13
- 239000002773 nucleotide Substances 0.000 description 13
- 125000003729 nucleotide group Chemical group 0.000 description 13
- 102000004190 Enzymes Human genes 0.000 description 12
- 108090000790 Enzymes Proteins 0.000 description 12
- 108020004414 DNA Proteins 0.000 description 10
- 125000003275 alpha amino acid group Chemical group 0.000 description 10
- 238000010829 isocratic elution Methods 0.000 description 10
- 239000013612 plasmid Substances 0.000 description 10
- 239000000243 solution Substances 0.000 description 9
- 150000007523 nucleic acids Chemical group 0.000 description 8
- 238000000746 purification Methods 0.000 description 7
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 description 6
- 125000000539 amino acid group Chemical group 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000004587 chromatography analysis Methods 0.000 description 6
- 238000007796 conventional method Methods 0.000 description 6
- 238000012217 deletion Methods 0.000 description 6
- 230000037430 deletion Effects 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000006467 substitution reaction Methods 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 238000001042 affinity chromatography Methods 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 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 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- PXHVJJICTQNCMI-UHFFFAOYSA-N nickel Substances [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 3
- 238000010188 recombinant method Methods 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 108091033380 Coding strand Proteins 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 2
- 235000019797 dipotassium phosphate Nutrition 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- FVTCRASFADXXNN-SCRDCRAPSA-N flavin mononucleotide Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O FVTCRASFADXXNN-SCRDCRAPSA-N 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 239000008057 potassium phosphate buffer Substances 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000027756 respiratory electron transport chain Effects 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 208000010444 Acidosis Diseases 0.000 description 1
- 108020004638 Circular DNA Proteins 0.000 description 1
- 241001478240 Coccus Species 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000010911 Enzyme Precursors Human genes 0.000 description 1
- 108010062466 Enzyme Precursors Proteins 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 108010003272 Hyaluronate lyase Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 208000026545 acidosis disease Diseases 0.000 description 1
- 238000005377 adsorption 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
- 238000004458 analytical method Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000000385 dialysis solution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000014726 immortalization of host cell Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 208000006443 lactic acidosis Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
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.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- 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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The application discloses a recombinant lactic acid oxidase and a preparation method and application thereof. The application provides a polynucleotide sequence for coding the lactic acid oxidase, which is optimized by synonymous codon preference, and has high expression efficiency in escherichia coli; the invention also provides a vector adapting to the polynucleotide sequence, and the high-activity lactic acid oxidase is prepared and obtained through the cooperative coordination of the vector and the polynucleotide sequence.
Description
Technical Field
The invention relates to the field of biochemical detection, in particular to recombinant lactic acid oxidase and a preparation method and application thereof.
Background
The lactic acid oxidase is taken as a flavin protein, FMN or FAD is taken as a spreading factor, the electron transfer form is different from a classical electron transfer chain, and oxygen is directly taken as a substrate, so that the FMN or FAD is very firmly combined with enzyme protein, free exogenous coenzyme is not needed in the whole reaction process, the regeneration problem of the spreading enzyme is thoroughly solved, and the lactic acid oxidase is an ideal enzyme for preparing pyruvic acid. The lactate oxidase can also be immobilized on a carrier by covalent bonding to prepare a lactate oxidase enzyme membrane, and can be applied to enzyme electrodes and biosensors. In addition, the lactic acid oxidase can be used as a medical diagnostic reagent for measuring the concentration of lactic acid in blood, and has important clinical value for early diagnosis and treatment of hyperlactic acid and lactic acidosis.
In the prior art, researches related to the production of the lactic acid oxidase are mainly on upstream technologies such as screening of lactic acid oxidase strains and optimization of culture conditions. Patent EP0376163B1 discloses a method for preparing lactic acid oxidase, wherein DNA is extracted from green coccus (IFO 12219) and spliced in pACYC184 plasmid, the plasmid is introduced into escherichia coli for culture, and the lactic acid oxidase with activity of 75U/mg is obtained after separation and purification, and the activity of the product obtained by the method is not high. Thus, there is still a need in the art to develop more efficient methods for preparing lactate oxidase.
Disclosure of Invention
The invention aims to provide a method for efficiently preparing lactate oxidase based on genetic engineering.
Another object of the present invention is to provide a polynucleotide sequence encoding a lactate oxidase.
It is another object of the present invention to provide a vector adapted to a polynucleotide sequence encoding a lactate oxidase.
It is another object of the present invention to provide a kit comprising a polynucleotide sequence encoding a lactate oxidase.
To solve the above technical problem, according to a first aspect of the present invention, there is provided a polynucleotide encoding a lactate oxidase, the polynucleotide being codon-optimized, and the polynucleotide being selected from any one of the following:
(i) A polynucleotide having a sequence as shown in SEQ ID NO.1 or SEQ ID NO. 4;
(ii) Polynucleotides having greater than 95% homology to the sequences as set forth in SEQ ID No.1 or SEQ ID No. 4; and
(iii) Having a polynucleotide complementary to the polynucleotide sequence set forth in (i) or (ii).
In a second aspect of the invention there is provided an expression vector comprising a polynucleotide provided in the first aspect of the invention.
In some preferred embodiments, the expression vector is an E.coli expression vector, more preferably pS28a, pBAD-hisA or pET28a, more preferably pET28a.
In some preferred embodiments, the expression vector includes a pro-lytic tag, such as sumo.
In a third aspect of the invention there is provided a host cell comprising an expression vector provided in the second aspect of the invention; or alternatively
The host cell has integrated into its genome a polynucleotide as provided in the first aspect of the invention.
In some preferred embodiments, the host cell is E.coli (Escherichia coli).
In some preferred embodiments, the host cell is an E.coli BL21 (DE 3) strain.
In a fourth aspect, the present invention provides a method for preparing a lactate oxidase, the method comprising the steps of: culturing the host cell of the third aspect of the invention to express the protein of interest; and
separating the target protein to obtain the lactic acid oxidase.
In some preferred embodiments, the host cell is obtained by transforming E.coli with a plasmid comprising a polynucleotide according to the first aspect of the invention.
In some preferred embodiments, the medium in which the host cells are cultured includes water, tryptone, yeast extract, glycerol, and kanamycin.
In some preferred embodiments, the medium in which the host cells are cultured comprises water, sodium chloride, tryptone, yeast powder, and kanamycin, and more preferably the medium is formulated as follows:
NaCl 10g/L，
10g/L of tryptone,
5g/L of yeast powder,
kanamycin 50ug/ml.
In some preferred embodiments, the medium includes water, sodium chloride, tryptone, yeast powder, and ampicillin.
In some preferred embodiments, the temperature of the culturing is 17 to 19 ℃ when the host cell is cultured.
In some preferred embodiments, the host cell is cultured for a period of time greater than 10 hours, more preferably greater than 14 hours, and even more preferably from 14 to 20 hours.
In some preferred embodiments, the host cell is cultured in a shaking environment.
In some preferred embodiments, the medium used in culturing the host cell comprises a kanamycin resistance gene.
In some preferred embodiments, the host cell is cultured using IPTG to induce expression of the protein of interest.
In some preferred embodiments, the host cell is cultured until an OD600 of 0.6 to 0.8 is reached, and is then induced with IPTG to express the protein of interest.
In some preferred embodiments, the step of isolating the protein of interest comprises:
and (3) passing the crushed target protein supernatant through a chromatographic column, eluting, and collecting eluent for dialysis to obtain the target protein.
In some preferred embodiments, the chromatography column is a Ni-column affinity chromatography column, such as Ni-NTA.
In some preferred embodiments, the elution procedure includes a first isocratic elution phase, a gradient elution phase, and a second isocratic elution phase performed sequentially; the mobile phase used in the first isocratic elution stage is bufferA, the mobile phase used in the gradient elution stage is a mixed solution of bufferA and bufferB, the content of the bufferA is gradually reduced while the content of the bufferB is gradually increased, and the mobile phase used in the second isocratic elution stage is bufferB.
In some preferred embodiments, the mobile phase flow rate in the first isocratic elution stage is 12-18ml/min; for example 15ml/min.
In some preferred embodiments, the mobile phase flow rate in the gradient elution phase is 12-18ml/min; for example 15ml/min.
In some preferred embodiments, the mobile phase in the second isocratic elution stage has a flow rate of 0.8 to 1.5ml/min; for example 1ml/min.
In some preferred embodiments, the elution procedure includes a gradient elution phase using a mixture of BufferA and BufferB as the mobile phase, with the BufferA content gradually decreasing and the BufferB content gradually increasing.
In some preferred embodiments, the volume percentage of the BufferA in the mobile phase used in the gradient elution phase is gradually reduced from 100% to 40% while the volume percentage of the BufferB is gradually increased from 0% to 60%.
In some preferred embodiments, the step of dialyzing comprises: the eluate was placed in a 3.5KD dialysis bag containing a dialysate for dialysis, and the target protein was collected.
Wherein the composition of BufferA and BufferB is shown in Table 1 below.
TABLE 1
| Reagent(s) | BufferA | BufferB |
| Tris | 50mM | 50mM |
| NaCl | 50mM | 50mM |
| Glycerol | 5％ | 5％ |
| Imidazole | - | 500mM |
| pH | 8.0 | 8.0 |
In a fifth aspect, the invention provides a kit comprising: a polynucleotide as provided in the first aspect of the invention; or alternatively
An expression vector as provided in the second aspect of the invention; or alternatively
A host cell according to the third aspect of the invention; or alternatively
Or a lactate oxidase prepared according to the method of the fourth aspect of the present invention.
Compared with the prior art, the invention has at least the following advantages:
(1) The invention provides a polynucleotide sequence for coding lactate oxidase, which is optimized by synonymous codon preference, and has high expression efficiency in escherichia coli;
(2) The invention also provides a vector adapting to the polynucleotide sequence, and the high-activity lactic acid oxidase is prepared and obtained through the cooperative coordination of the vector and the polynucleotide sequence;
(3) The invention provides a method for preparing recombinant lactic acid oxidase, which optimizes the culture condition and improves the yield of the lactic acid oxidase.
(4) In a preferred embodiment of the invention, a polynucleotide obtained by codon optimization of a gene sequence corresponding to the mutated lactate oxidase is provided, and the application of the polynucleotide to the expression system of the invention greatly improves the activity of the product.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings.
FIG. 1 is a diagram showing SDS-PAGE identification of a TB medium cultured lactate oxidase according to an embodiment of the present invention;
FIG. 2 is a diagram showing SDS-PAGE identification result of a lactic oxidase cultured in LB medium according to an embodiment of the present invention;
FIG. 3 is an electrophoretogram of unmutated lactate oxidase after chromatography according to an embodiment of the invention;
FIG. 4 is an electrophoretogram of mutant lactate oxidase after chromatography according to an embodiment of the present invention;
FIG. 5 is a graph of absorbance versus lactate oxidase concentration according to an example of the invention.
Detailed Description
The inventor provides an expression system for preparing the lactic acid oxidase by efficiently expressing heterologous genes in microorganisms through extensive and intensive research, and the polynucleotide sequence matched with a target escherichia coli vector is obtained through codon preference optimization in the expression system, so that the yield of the lactic acid oxidase is greatly improved, the subsequent purification steps are simplified, the economic cost is saved, and the activity of the recombinant lactic acid oxidase obtained by a lactic acid oxidase preparation method based on the expression system is improved, thereby having good application prospect.
Furthermore, in the preferred embodiment of the present invention, the use of the polynucleotide obtained by codon optimization of the gene sequence corresponding to the mutated lactate oxidase greatly improves the activity of the resulting recombinant lactate oxidase.
The embodiment of the invention constructs a method for efficiently preparing high-activity lactic acid oxidase, which comprises the following steps: obtaining a target gene sequence; carrying out synonymous codon preference optimization on a target gene sequence; obtaining a vector of a target gene, and connecting the target gene to the vector; transforming a host cell with a vector comprising a gene of interest; culturing the host cell to express the target protein lactate oxidase; and separating the target protein.
Acquisition of a Gene of interest/acquisition of a nucleic acid sequence related to a protein of interest
The full-length nucleotide sequence of the target protein or its element or a fragment thereof of the present invention can be usually obtained by PCR amplification, recombinant methods or artificial synthesis. For the PCR amplification method, primers can be designed based on the disclosed nucleotide sequences, particularly open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.
Methods of amplifying DNA/RNA using PCR techniques are preferred for obtaining the genes of the present invention. Primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.
In one embodiment of the present invention, the amino acid sequence of the target protein (SEQ ID NO: 2) is analyzed by NCBI database to obtain the sequence information of the target gene. The sequence of the gene of interest encoding the enzyme is obtained, for example, by analysis of the amino acid sequence of the green balloon lactate oxidase by NCBI database.
Synonymous codon bias optimization
To overcome the potential problem of reduced yield when expressing heterologous proteins in E.coli, the present invention relates to synonymous codon-biased optimized polynucleotide sequences. The obtained target gene sequence is subjected to synonymous codon preference optimization, the target gene sequence (SEQ ID NO: 1) subjected to synonymous codon preference optimization can express the same amino acid sequence as the target protein, but the stability and efficiency of the expression process are improved, and finally the obtained target protein maintains higher activity.
The invention also relates to polynucleotides having a homology of more than 95% with the sequence shown in SEQ ID NO. 1; and a polynucleotide complementary to the sequence shown in SEQ ID No. 1.
In order to further increase the activity of the product, the polynucleotide sequence corresponding to the mutant target protein obtained unexpectedly by the inventor has higher enzyme activity after synonymous codon preference optimization (SEQ ID NO: 4).
The invention also relates to polynucleotides having a homology of more than 95% with the sequence shown in SEQ ID NO. 4; and a polynucleotide complementary to the sequence shown in SEQ ID No. 1.
Vector of target gene
The invention also relates to vectors comprising the polynucleotides of the invention. "vector" in the present invention means a linear or circular DNA molecule comprising a fragment encoding a protein of interest operably linked to other fragments providing for its transcription. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, a vector, and the like. The vector fragment may be derived from the host organism, another organism, plasmid or viral DNA, or may be synthetic. The vector may be any expression vector, synthetic or conveniently subjected to recombinant DNA procedures, the choice of vector generally being dependent on the host cell into which the vector is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one that, when introduced into the host cell, integrates into the host cell genome and replicates with the chromosome with which it is integrated. In one embodiment, the vector of the invention is an expression vector. In one embodiment of the invention pS28a, pBAD-hisA or pET28a is selected as vector to obtain more efficient expression, most preferably pET28a is selected as expression vector.
Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequences of the proteins of the invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Illustratively, the insertion of the exogenous DNA fragment is accomplished by cleaving the vector DNA molecule with a DNA endonuclease into a linear molecule that can be linked to the exogenous gene, and then ligating the codon optimized fragment of the gene of interest to the vector, optionally with a single restriction site cohesive end ligation, double restriction site directional cloning, cohesive end ligation of different restriction sites, blunt end ligation, artificial linker ligation, or end ligation with an oligonucleotide.
Functional tags, e.g., dissolution enhancing tags, etc., may be included in the expression vectors of the present invention, which in preferred embodiments of the present invention include the dissolution enhancing tag sumo.
Transformation of host cells with vectors containing genes of interest
The invention also relates to host cells genetically engineered with the vector or fusion protein coding sequences of the invention. The vector containing the codon-optimized gene of interest may be inserted, transfected or otherwise transformed into a host cell by known methods to obtain a transformant containing the codon-optimized gene of interest of the present invention and capable of expressing the protein of interest. A "host cell" in the present invention is a cell into which an exogenous polynucleotide and/or vector has been introduced. The host cell may be a eukaryotic host cell or a prokaryotic host cell, the host cell is preferably a bacterium, and is preferably E.coli, more preferably E.coli BL21 (DE 3) strain (Escherichia coli BL (DE 3) strain).
Method for producing target protein
The invention also relates to a method for preparing the target protein, and the polynucleotide sequence can be used for expressing or producing recombinant protein. Generally, there are the following steps:
(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a protein of the invention, or with a recombinant expression vector comprising the polynucleotide;
(2) A host cell cultured in a suitable medium;
(3) Separating and purifying the protein from the culture medium or the cells.
Wherein, the transformation or transduction of the recombinant expression vector containing the polynucleotide of the step (1) into a suitable host cell can be performed by conventional techniques well known to those skilled in the art, and when the host is E.coli, a heat shock method, an electrotransformation method, etc. can be selected.
The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.
In order to enhance the expression efficiency of the soluble protein, it is preferable to culture the host cells in LB or TB medium, and in a preferred embodiment of the present invention, the medium in which the host cells are cultured includes water, tryptone, yeast extract, glycerol and kanamycin. In another more preferred embodiment of the present invention, the medium in which the host cells are cultured comprises water, sodium chloride, tryptone, yeast powder and kanamycin, and more preferably the medium is formulated as follows:
NaCl 10g/L，
10g/L of tryptone,
5g/L of yeast powder,
kanamycin 50ug/ml.
The temperature of culturing the host cells can be conventionally carried out with reference to the art, but in order to enhance the expression efficiency of soluble proteins, it is preferable to use a low-temperature culture, for example, in a preferred embodiment of the present invention, the temperature of the culture is 17 to 19 ℃.
In a preferred embodiment of the present invention, the medium used contains a kanamycin resistance gene, is cultured until the OD600 is 0.6 to 0.8, and is then induced with IPTG to express the target protein.
The protein in the above method may be expressed in the cell, or on the cell membrane, or secreted outside the cell. If desired, the proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods. In one embodiment of the invention, the protein of interest is isolated using affinity chromatography.
It should be noted that, although the manner of isolating the target protein is known to those skilled in the art, the purity and activity of the target protein obtained by various isolation methods are far from each other depending on the specific properties of the target protein. In the invention, the inventor has verified that compared with other common modes, the method of separating target protein by using an affinity chromatography combined with a dialysis method has very high activity and extremely high purity of the obtained product through a large amount of experiments.
In affinity chromatography, the effect of the relative separation of the column and flow used is significant. In one embodiment of the present invention, chromatography is performed using a Ni-NTA column, and the elution procedure is set to a first isocratic elution phase, a gradient elution phase, and a second isocratic elution phase, which are sequentially performed; the mobile phase used in the first isocratic elution stage is bufferA, the mobile phase used in the gradient elution stage is a mixed solution of bufferA and bufferB, the content of the bufferA is gradually reduced while the content of the bufferB is gradually increased, and the mobile phase used in the second isocratic elution stage is bufferB. In some preferred embodiments of the present invention, the volume percentage of the BufferA in the mobile phase used in the gradient elution phase is gradually reduced from 100% to 40% while the volume percentage of the BufferB is gradually increased from 0% to 60%.
In another embodiment of the present invention, on the premise that the composition of the BufferA and the BufferB is unchanged, the single-stage gradient elution is used for chromatography, and the elution procedure comprises a gradient elution stage, wherein the mobile phase used in the gradient elution stage is a mixed solution of the BufferA and the BufferB, and the content of the BufferA is gradually reduced while the content of the BufferB is gradually increased. The inventors found that the purity and activity of the target protein obtained in this embodiment were inferior to those of the third stage method.
In the dialysis method, the collected chromatographic eluate is placed in a 3.5KD dialysis bag containing a dialysis solution for dialysis, and the dialysis time is preferably not less than 8 hours, for example, 10 hours or 12 hours.
In the present disclosure, any exemplary or exemplary language (e.g., ") provided for certain embodiments herein is used merely to better present the disclosure and does not limit the scope of the disclosure as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
If the definition or use of a term in a reference is inconsistent or inconsistent with the definition of that term described herein, the definition of the term described herein applies and the definition of the term in the reference does not apply.
Various terms used herein are shown below. If a term used in a claim is not defined below, the broadest definition persons in the pertinent art have given that term are given as reflected in publications or issued patents that are printed at the time of application.
As used herein, the term "isolated" refers to a nucleic acid or polypeptide that is separated from at least one other component (e.g., a nucleic acid or polypeptide) that the nucleic acid or polypeptide is found in its natural source. In one embodiment, the nucleic acid or polypeptide is found to be present only (if any) in solvents, buffers, ions or other components that are normally present in its solution. The terms "isolated" and "purified" do not include nucleic acids or polypeptides that are present in their natural source.
As used herein, the terms "polynucleotide" and "polynucleotide sequence" may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.
The invention also relates to variants of the above polynucleotides which encode protein fragments, analogs and derivatives having the same amino acid sequence as the invention. Variants of the polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. Such nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded polypeptide.
As used herein, the term "codon optimization" refers to a manner of improving the efficiency of gene synthesis by avoiding the use of low-availability or rare codons according to codon usage differences exhibited by organisms (including e.coli, yeast, mammalian blood cells, plant cells, insect cells, etc.) that actually do protein expression or production.
As used herein, the terms "homology" and "identity" are used interchangeably to refer to the percentage of identical (i.e., identical) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be measured by the following methods. The nucleotide or amino acid sequence of a polynucleotide or polypeptide is aligned, the number of positions in the aligned polynucleotide or polypeptide that contain the same nucleotide or amino acid residue is scored and compared to the number of positions in the aligned polynucleotide or polypeptide that contain a different nucleotide or amino acid residue. Polynucleotides may differ at one position, for example, according to the inclusion of different nucleotides (i.e., substitutions or variations) or deletions of nucleotides (i.e., insertions or deletions of one or two nucleotides in the polynucleotide). The polypeptides may differ at one position, for example, by containing an amino acid (i.e., substitution or variation) or a deletion of an amino acid (i.e., an amino acid or deletion of an amino acid inserted into one or both polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide, and then multiplying by 100.
As used herein, the terms "sequence complementary" and "reverse sequence complementary" are used interchangeably to refer to a sequence that is opposite in direction to and complementary to the original polynucleotide sequence. For example, if the original polynucleotide sequence is actaac, then its reverse complement is GTTCAT.
As used herein, the term "expression" includes any step involving the production of a polypeptide in a host cell, including, but not limited to, transcription, translation, post-translational modification, and secretion. After expression, the host cells or expression products can be harvested, i.e.recovered.
The present invention will be further described with reference to specific embodiments in order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, it is to be noted that the terms used herein are used merely to describe specific embodiments and are not intended to limit the exemplary embodiments of this application.
Example 1 construction of lactate oxidase plasmid
(1) The gene sequence (SEQ ID NO: 1) of the green balloon lactic acid oxidase provided by NCBI was used as a reference, and was subjected to optimization of E.coli synonymous codon preference, and then ligated to different vectors, as shown in Table 1.
(2) Recombinant plasmid transformed E.coli BL21 (DE 3)
Taking 1 mu L of plasmid, adding the plasmid into 30 mu L of escherichia coli competent BL21 (DE 3) under ice bath condition, standing for 20min in ice bath, carrying out heat shock for 45s in water bath at 42 ℃, standing for 2min on ice immediately, adding 400 mu L of SOC culture medium without antibiotics, and carrying out shaking culture at 220rpm for 45min at 37 ℃. mu.L of the bacterial liquid was uniformly spread on LB plates containing 100. Mu.g/mL of kana resistance, and incubated overnight at 37 ℃.
The codon optimized lactic acid oxidase nucleic acid sequence SEQ ID NO. 1:
ATGAACAACAACGATATCGAATACAACGCTCCGAGCGAGATCAAGTATATTGATGTTGTAAACACCTATGATCTGGAAGAAGAAGCTTCCAAGGTTGTTCCGCACGGTGGCTTCAACTACATCGCGGGTGCATCCGGCGACGAATGGACGAAACGTGCCAATGATCGCGCATGGAAGCATAAACTGCTGTACCCACGTCTGGCTCAGGATGTTGAAGCTCCGGACACTAGCACTGAAATCCTGGGTCATAAAATCAAAGCACCGTTCATCATGGCCCCGATTGCTGCCCATGGCCTGGCGCATACTACTAAAGAAGCAGGTACTGCTCGTGCAGTCAGCGAATTTGGCACCATTATGTCTATCTCTGCGTACTCTGGTGCTACTTTTGAAGAAATCTCTGAGGGTCTGAACGGCGGTCCGCGTTGGTTCCAGATCTATATGGCGAAAGATGACCAGCAGAACCGTGATATCCTGGATGAAGCAAAATCCGATGGTGCTACTGCTATCATCCTGACTGCGGACTCCACTGTATCCGGCAACCGCGATCGTGATGTTAAAAACAAATTTGTGTACCCGTTCGGTATGCCGATTGTTCAGCGTTACCTGCGTGGTACTGCCGAAGGCATGTCCCTGAACAACATCTATGGCGCGTCTAAACAGAAAATTAGCCCGCGTGATATTGAGGAGATCGCGGGTCACTCTGGCCTGCCGGTGTTCGTGAAGGGTATCCAGCATCCGGAGGATGCCGACATGGCAATCAAACGCGGTGCCTCTGGTATCTGGGTCTCCAACCACGGTGCACGCCAGCTGTATGAGGCTCCGGGCTCCTTCGATACGCTGCCGGCTATTGCCGAACGTGTGAACAAACGCGTTCCGATTGTCTTCGATTCCGGCGTGCGTCGTGGCGAACACGTTGCTAAGGCGCTGGCGAGCGGCGCGGACGTTGTAGCTCTGGGTCGCCCGGTTCTGTTTGGTCTGGCACTGGGCGGTTGGCAGGGCGCGTACAGCGTGCTGGACTACTTTCAGAAAGACCTGACCCGTGTGATGCAGCTGACCGGTTCCCAGAACGTTGAAGATCTGAAAGGCCTGGATCTGTTCGATAACCCGTACGGTTACGAG
lactic acid oxidase amino acid sequence SEQ ID NO 2:
MNNNDIEYNAPSEIKYIDVVNTYDLEEEASKVVPHGGFNYIAGASGDEWTKRANDRAWKHKLLYPRLAQDVEAPDTSTEILGHKIKAPFIMAPIAAHGLAHTTKEAGTARAVSEFGTIMSISAYSGATFEEISEGLNGGPRWFQIYMAKDDQQNRDILDEAKSDGATAIILTADSTVSGNRDRDVKNKFVYPFGMPIVQRYLRGTAEGMSLNNIYGASKQKISPRDIEEIAGHSGLPVFVKGIQHPEDADMAIKRGASGIWVSNHGARQLYEAPGSFDTLPAIAERVNKRVPIVFDSGVRRGEHVAKALASGADVVALGRPVLFGLALGGWQGAYSVLDYFQKDLTRVMQLTGSQNVEDLKGLDLFDNPYGYE
TABLE 1
| Group of | The carrier used |
| 1 | pET28a |
| 2 | pS28a |
| 3 | pBAD-hisA |
Example 2 mutation optimization
Mutation optimization is carried out on the basis of the amino acid sequence (SEQ ID NO: 2) of lactic acid oxidase to obtain an amino acid sequence SEQ ID NO:3, synonymous codon preference optimization is carried out on the amino acid sequence SEQ ID NO:3 (SEQ ID NO: 4), the connecting carrier is pET-28a, and a dissolution promoting tag sumo is arranged in front of a multiple cloning site of the carrier pET-28 a. The recombinant plasmid was transformed into E.coli BL21 (DE 3) in the same manner as in example 1.
After mutation, the amino acid sequence of the lactic acid oxidase is SEQ ID NO:3:
MNNNDIEYNAPSEIKYIDVVNTYDLEEEASKVVPHGGFNYIAGASGDEWTKRANDRAWKHKLLYPRLAQDVEAPDTSTEILGHKIKAPFIMAPIAAHGLAHTTKEAGTARAVSEFGTIMSISAYSGATFEEISEGLNGGPRWFQIYMAKDDQQNRDILDEAKSDGATAIILTADSTVSGNRDRDVKNKFVYPFGMPIVQRYLRGTAEGMSLNNIYGASKQKISPRDIEEIAAHSGLPVFVKGIQHPEDADMAIKAGASGIWVSNHGARQLYEAPGSFDTLPAIAERVNKRVPIVFDSGVRRGEHVAKALASGADVVALGRPVLFGLALGGWQGAYSVLDYFQKDLTRVMQLTGSQNVEDLKGLDLFDNPYGYEY
corresponding nucleic acid sequence SEQ ID NO. 4:
ATGAACAACAACGACATCGAATACAACGCCCCGTCCGAAATCAAATACATTGATGTAGTTAACACCTACGACCTGGAAGAAGAAGCAAGCAAAGTGGTCCCGCACGGTGGTTTCAACTATATCGCAGGCGCGAGCGGTGACGAATGGACCAAGCGTGCAAACGACCGTGCATGGAAACATAAACTGCTGTACCCGCGTCTGGCGCAGGATGTTGAAGCTCCTGACACCTCCACCGAAATCCTGGGCCATAAAATTAAAGCACCGTTTATCATGGCACCGATCGCTGCACACGGTCTGGCTCACACCACCAAGGAGGCAGGCACCGCGCGTGCAGTGAGCGAATTCGGTACCATCATGTCTATCTCTGCTTACAGCGGTGCGACTTTCGAGGAGATTTCTGAAGGTCTGAATGGTGGTCCTCGTTGGTTCCAGATCTATATGGCTAAAGATGATCAGCAGAACCGCGATATCCTGGATGAGGCTAAATCTGATGGTGCGACTGCTATTATCCTGACCGCGGACTCCACTGTATCCGGCAACCGTGATCGTGACGTTAAAAACAAATTCGTGTACCCGTTCGGTATGCCGATCGTGCAGCGTTATCTGCGTGGCACCGCCGAAGGTATGTCTCTGAACAACATCTATGGCGCATCTAAGCAGAAAATTTCTCCACGTGACATCGAAGAAATTGCTGCTCACAGCGGTCTGCCGGTTTTCGTGAAAGGTATCCAACACCCGGAAGATGCGGATATGGCCATTAAAGCAGGCGCATCTGGCATCTGGGTTTCTAATCACGGTGCCCGCCAGCTGTACGAAGCCCCGGGTTCCTTCGATACCCTGCCGGCCATCGCGGAACGTGTTAACAAACGTGTGCCGATCGTTTTCGATTCTGGTGTGCGTCGTGGCGAGCATGTTGCAAAAGCCCTGGCATCCGGTGCAGATGTTGTGGCGCTGGGTCGTCCGGTACTGTTTGGTCTGGCTCTGGGCGGTTGGCAGGGTGCTTACAGCGTGCTGGATTACTTCCAGAAAGATCTGACGCGTGTGATGCAGCTGACCGGTTCTCAAAACGTTGAGGATCTGAAGGGCCTGGATCTGTTTGACAACCCGTACGGTTACGAATAC
example 2 expression of target protein of lactate oxidase
In this example, the inventors optimized the induction conditions of lactate oxidase. The monoclonal in examples 1 and 2 were inoculated aseptically into different formulations of culture media and cultured at different temperatures, and the specific procedures were as follows: sterile procedures were inoculated into 100. Mu.g/mL of the medium of Table 2, each medium was replicated in 2 tubes, each of which was designated as TB (1), TB (2), LB (1), LB (2) was cultured with shaking at 37℃and 220rpm until OD600 was between 0.6 and 0.8, induction was performed with IPTG, and the culture was performed with shaking at 37℃and 18℃overnight, respectively.
SDS-PAGE identification was performed by sampling and ultrasonication, and the identification results are shown in FIGS. 1-2 and Table 2.
TABLE 2
Example 3 purification of lactate oxidase
pET28a is selected as a vector, and after sequences shown in SEQ ID NO.1 and SEQ ID NO.4 are respectively connected, escherichia coli is introduced for culture, and the culture conditions are the same as those of group 3 in Table 2.
After completion of the culture, 150g of the cells were homogenized and crushed, and then were once passed through a 0.45 μm membrane and once passed through a 0.22 μm membrane, followed by purification with 30ml of Ni-NTA. The flow rate was 15ml/min, and the loading was done using 90ml Lysis Buffer rinse UV and conductance to baseline. The elution procedure included: step 1:100% A+0% B,3CV,15ml/min;
Step 2:100-40％A+0-60％B，12CV，15ml/min；
Step 3:0％A+100％B,6CV，1ml/min。
the electrophoresis results after sample collection are shown in FIG. 3 and FIG. 4. The reagents used are shown in Table 3.
TABLE 3 Table 3
| Reagent(s) | BufferA | BufferB | Lysis Buffer |
| Tris | 50mM | 50mM | 50mM |
| NaCl | 50mM | 50mM | 300mM |
| Glycerol | 5％ | 5％ | 5％ |
| Imidazole | - | 500mM | - |
| pH | 8.0 | 8.0 | 8.0 |
FIG. 3 is an unmutated set, according to FIG. 3,0% B, with the target protein eluted, but with more bands, and a distinct band of the target protein was visible at about 70kd, possibly because the amount of this time cell was used too much, and some of the target protein was spiked out with the target protein; 100% B: the target protein is eluted, and the impurity bands are less. Collecting the sample from the No. 4-7 tube and the No. 35-40 tube respectively, wherein the sample collection volume of the No. 4-7 tube is 56ml; the sample collection volume of the 35-40 # tube is 84ml.
The samples after Ni column purification are respectively put into dialysis bags with 3.5KD and are put into 2000ml of dialysate for overnight dialysis, the samples after dialysis are collected in the morning the next day, the final concentration of the samples is 5mM DTT, the samples are uniformly mixed, the sample collection volume after 4-7 tube dialysis is 16.5ml, and the sample collection volume after 35-40 tube dialysis is 26ml. The concentrations were measured by BCA method and found to be 4.64mg/ml and 6.1mg/ml, respectively; yields were 120.64mg and 100.65mg, R 2 =0.996; the yield was 1.84mg/g of bacteria.
FIG. 4 shows the mutant group, the purification step is the same as the non-mutant group, 5g of bacteria are weighed, 1ml of nickel column is used for purification, samples are collected and dialyzed into PBS, and the concentration is measured by BCA, and the result is: standard curve R 2 =0.996, at a concentration of 2.8mg/ml, a volume of 11ml and a yield of 30.8mg; the total yield was 7.7mg/g bacteria.
Example 4 determination of recombinant lactate oxidase Activity
1) Solution preparation
(1) 100mM potassium phosphate buffer: 0.435g of dipotassium phosphate powder and 0.34g of potassium dihydrogen phosphate powder were weighed separately, dissolved in 50mL of deionized water, and 3.81mL of dipotassium phosphate solution and 6.19mL of potassium dihydrogen phosphate were mixed, and the pH was 6.6.
(2) 0.1% 4-aminoantipyrine: 0.05g of 4-aminoantipyrine powder was weighed and dissolved in 50mL of deionized water and stored in the dark.
(3) DMA:0.965g/mL, stored in the dark.
(4) 1M lactic acid: commercial lactic acid (13.4M) diluted 13.4 times
5mL of the working solution was prepared in accordance with Table 4 below.
TABLE 4 Table 4
| Reagent(s) | Volume added | Final concentration |
| 100mM potassium phosphate buffer | 2mL | 40mM |
| 0.1% 4-aminoantipyrine | 1.5mL | 1.5mM |
| Peroxidase (5U/ul) | 5μL | 25U |
| 1M lactic acid | 0.25mL | 50mM |
| DMA | 10μL | 0.2％(W/V) |
| ddH 2 O | 1.24mL |
2) Test procedure
(1) Configuration of positive enzymes: 100U positive enzyme was dissolved in 0.1mL PBS pH 7.4 buffer (containing 50% glycerol) to make 1U/. Mu.L enzyme solution, which was gradually diluted according to the gradient, and the dilution was PBS pH 7.4 buffer.
(2) The enzyme label instrument is preheated for 30min.
(3) Program setting of the enzyme label instrument: 100. Mu.L of the reaction solution was added, and the absorbance A1 was measured at 565nm, and then 1. Mu.L of each concentration of the enzyme solution was added, followed by reaction for 1 minute, and the absorbance A2 was measured at 565 nm.
(4) The OD difference A2-A1 before and after the reaction was calculated and a standard curve of OD value as a function of concentration was made as shown in fig. 3.
The absorbance of the lactic acid oxidase purified in example 3 was measured as described above, and the results are shown in Table 5 below.
TABLE 5
The calculated mutant zymogen solution concentration was 0.711mg/mL, the average viability was 0.133U/μl, and the specific viability was (0.1333 x 1000)/0.711=187.5U/mg. Compared with the wild type, the specific activity is obviously improved.
It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples of carrying out the invention and that various changes in form and details may be made therein without departing from the spirit and scope of the invention.
Claims (10)
1. A polynucleotide encoding a lactate oxidase, wherein the polynucleotide is codon optimized and the polynucleotide is selected from any one of the following:
(i) A polynucleotide having a sequence as shown in SEQ ID NO.1 or SEQ ID NO. 4;
(ii) Polynucleotides having greater than 95% homology to the sequences as set forth in SEQ ID No.1 or SEQ ID No. 4; and
(iii) Having a polynucleotide complementary to the polynucleotide sequence set forth in (i) or (ii).
2. An expression vector comprising the polynucleotide of claim 1.
3. The expression vector of claim 2, wherein the expression vector is an escherichia coli expression vector, pS28a, pBAD-hisA or pET28a, more preferably pET28a.
4. A host cell comprising the expression vector of any one of claims 2 to 4; or alternatively
The polynucleotide of claim 1 integrated into the genome of the host cell.
5. A method of preparing a lactate oxidase, the method comprising the steps of:
transforming a host cell with a vector comprising the polynucleotide of claim 1;
culturing the host cell to express the protein of interest; and
isolating the protein of interest.
6. The method of claim 5, wherein the medium in which the host cells are cultured comprises water, tryptone, yeast extract, glycerol, and kanamycin.
7. The method of claim 5, wherein the medium in which the host cells are cultured comprises water, sodium chloride, tryptone, yeast powder, and kanamycin.
8. The method of claim 5, wherein the temperature of the culturing is from 17 to 19 ℃ when the host cell is cultured;
and/or, the time of the culturing is greater than 10 hours.
9. The method of claim 6, wherein the step of isolating the protein of interest comprises:
and (3) passing the crushed target protein supernatant through a chromatographic column, eluting, and collecting eluent for dialysis to obtain the target protein.
10. A kit, comprising: the polynucleotide of claim 1; or alternatively
The expression vector of any one of claims 2 to 3; or alternatively
The host cell of claim 4; or alternatively
A recombinant lactate oxidase prepared according to the method of any one of claims 5-9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210982263.1A CN117587042A (en) | 2022-08-16 | 2022-08-16 | Recombinant lactic acid oxidase and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210982263.1A CN117587042A (en) | 2022-08-16 | 2022-08-16 | Recombinant lactic acid oxidase and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117587042A true CN117587042A (en) | 2024-02-23 |
Family
ID=89913877
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210982263.1A Pending CN117587042A (en) | 2022-08-16 | 2022-08-16 | Recombinant lactic acid oxidase and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117587042A (en) |
-
2022
- 2022-08-16 CN CN202210982263.1A patent/CN117587042A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107828755B (en) | Hot start TaqDNA polymerase and preparation method and application thereof | |
| CN108865962B (en) | Escherichia coli engineering bacterium capable of efficiently and soluble expressing 4-alpha-glycosyltransferase | |
| JP4486009B2 (en) | DNA ligase mutant | |
| CN114262697B (en) | Bsu DNA polymerase and Bsu DNA polymerase mutant as well as gene, plasmid and genetic engineering bacteria thereof | |
| CN113801240B (en) | D-psicose-3-epimerase activity aggregate and preparation method and application thereof | |
| CN116426546A (en) | Recombinant cholesterol oxidase, and preparation method and application thereof | |
| CN117587042A (en) | Recombinant lactic acid oxidase and preparation method and application thereof | |
| CN114875002B (en) | Arginine mutated DNA/RNA ligase | |
| CN110184289B (en) | Recombinant glycerol phosphate oxidase expression vector and establishment method thereof | |
| CN117187210B (en) | Mutant Bst DNA polymerase large fragment and preparation method thereof | |
| CN112662642B (en) | Reverse transcriptase of skin sarcoma virus of mutant type large-eyed zander and application thereof | |
| CN116515781A (en) | Phosphoglycerate oxidase, mutant thereof, preparation method and application | |
| CN117587041A (en) | Recombinant uricase, and preparation method and application thereof | |
| CN116200408A (en) | Preparation method and application of 3 alpha-steroid dehydrogenase | |
| CN117587049A (en) | Recombinant D-3-hydroxybutyrate dehydrogenase and preparation method and application thereof | |
| CN116042664A (en) | Preparation method and application of acyl-CoA oxidase | |
| CN116042662A (en) | Preparation method and application of acyl-coa synthetase | |
| CN116042588A (en) | Preparation method and application of recombinant creatine enzyme | |
| CN116121280A (en) | Preparation method and application of recombinant cystathionine-beta-lyase | |
| CN116606874A (en) | Preparation method and application of cystathionine-beta-synthetase | |
| CN117587046A (en) | Recombinant hexokinase and preparation method and application thereof | |
| CN116622744A (en) | Preparation method and application of recombinant terminal deoxynucleotidyl transferase | |
| CN112680431B (en) | DNA library preparation method based on fragmenting enzyme | |
| WO2023115517A1 (en) | Dna polymerase mutant and use thereof | |
| CN116426545A (en) | Recombinant pyranose oxidase, and preparation method and application thereof |
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 |