CN117761021A - Protein interaction analysis method based on fluorescence resonance energy transfer - Google Patents
Protein interaction analysis method based on fluorescence resonance energy transfer Download PDFInfo
- Publication number
- CN117761021A CN117761021A CN202311717390.XA CN202311717390A CN117761021A CN 117761021 A CN117761021 A CN 117761021A CN 202311717390 A CN202311717390 A CN 202311717390A CN 117761021 A CN117761021 A CN 117761021A
- Authority
- CN
- China
- Prior art keywords
- protein
- dye
- fluorescent
- energy transfer
- resonance energy
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 56
- 238000002866 fluorescence resonance energy transfer Methods 0.000 title claims abstract description 51
- 230000006916 protein interaction Effects 0.000 title claims abstract description 33
- 238000012482 interaction analysis Methods 0.000 title abstract description 11
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 97
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 80
- 108091006047 fluorescent proteins Proteins 0.000 claims abstract description 28
- 102000034287 fluorescent proteins Human genes 0.000 claims abstract description 27
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 20
- 230000014509 gene expression Effects 0.000 claims abstract description 20
- 230000004927 fusion Effects 0.000 claims abstract description 16
- 230000003993 interaction Effects 0.000 claims description 32
- 101710141454 Nucleoprotein Proteins 0.000 claims description 16
- 241000711573 Coronaviridae Species 0.000 claims description 9
- 239000007853 buffer solution Substances 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 7
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 7
- 238000002165 resonance energy transfer Methods 0.000 claims description 7
- 238000007865 diluting Methods 0.000 claims description 5
- 241001678559 COVID-19 virus Species 0.000 claims description 4
- 238000006467 substitution reaction Methods 0.000 claims description 4
- 239000012114 Alexa Fluor 647 Substances 0.000 claims description 3
- 108090001074 Nucleocapsid Proteins Proteins 0.000 claims description 3
- 238000012937 correction Methods 0.000 claims description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 2
- 239000007995 HEPES buffer Substances 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 20
- 239000000975 dye Substances 0.000 abstract description 15
- 238000002474 experimental method Methods 0.000 abstract description 9
- 238000012512 characterization method Methods 0.000 abstract description 5
- 239000000523 sample Substances 0.000 description 25
- 108700002099 Coronavirus Nucleocapsid Proteins Proteins 0.000 description 20
- 238000006243 chemical reaction Methods 0.000 description 17
- 238000010168 coupling process Methods 0.000 description 17
- 239000003446 ligand Substances 0.000 description 16
- 230000008878 coupling Effects 0.000 description 14
- 238000005859 coupling reaction Methods 0.000 description 14
- 108020004414 DNA Proteins 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 12
- 102000037865 fusion proteins Human genes 0.000 description 12
- 108020001507 fusion proteins Proteins 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 230000027455 binding Effects 0.000 description 11
- 239000003153 chemical reaction reagent Substances 0.000 description 11
- 238000005516 engineering process Methods 0.000 description 11
- 238000002372 labelling Methods 0.000 description 10
- 230000008569 process Effects 0.000 description 10
- 238000000746 purification Methods 0.000 description 10
- 239000011347 resin Substances 0.000 description 10
- 229920005989 resin Polymers 0.000 description 10
- 239000012634 fragment Substances 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 8
- 239000011248 coating agent Substances 0.000 description 8
- 238000000576 coating method Methods 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 238000012216 screening Methods 0.000 description 8
- 238000002156 mixing Methods 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 230000009466 transformation Effects 0.000 description 7
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 239000011521 glass Substances 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000004114 suspension culture Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000012575 bio-layer interferometry Methods 0.000 description 5
- 238000004364 calculation method Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 238000001502 gel electrophoresis Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000000985 reactive dye Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 239000003242 anti bacterial agent Substances 0.000 description 4
- 229940088710 antibiotic agent Drugs 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 229920002521 macromolecule Polymers 0.000 description 4
- 238000002823 phage display Methods 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 238000002818 protein evolution Methods 0.000 description 4
- 230000004850 protein–protein interaction Effects 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 238000002877 time resolved fluorescence resonance energy transfer Methods 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 101710132601 Capsid protein Proteins 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 240000000220 Panda oleosa Species 0.000 description 3
- 235000016496 Panda oleosa Nutrition 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 238000001742 protein purification Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 3
- 235000017557 sodium bicarbonate Nutrition 0.000 description 3
- 230000003595 spectral effect Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- 238000012815 AlphaLISA Methods 0.000 description 2
- 108020004638 Circular DNA Proteins 0.000 description 2
- 101710094648 Coat protein Proteins 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 2
- 101710125418 Major capsid protein Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 101710083689 Probable capsid protein Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 108010003723 Single-Domain Antibodies Proteins 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 238000003016 alphascreen Methods 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 239000012466 permeate Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- -1 small molecule compounds Chemical class 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 2
- 239000013603 viral vector Substances 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 101150003288 C2 gene Proteins 0.000 description 1
- 241000282836 Camelus dromedarius Species 0.000 description 1
- 108091036055 CccDNA Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 244000182067 Fraxinus ornus Species 0.000 description 1
- 235000002917 Fraxinus ornus Nutrition 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 102000044126 RNA-Binding Proteins Human genes 0.000 description 1
- 108700020471 RNA-Binding Proteins Proteins 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 102400000368 Surface protein Human genes 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 235000012511 Vaccinium Nutrition 0.000 description 1
- 241000736767 Vaccinium Species 0.000 description 1
- 241001416177 Vicugna pacos Species 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 238000004847 absorption spectroscopy Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009830 antibody antigen interaction Effects 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000005056 compaction Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 125000002228 disulfide group Chemical group 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000005281 excited state Effects 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000005283 ground state Effects 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000009878 intermolecular interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000012948 isocyanate Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 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 1
- 230000002934 lysing effect Effects 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000004001 molecular interaction Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 238000001050 pharmacotherapy Methods 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 108020001580 protein domains Proteins 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 238000002424 x-ray crystallography Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of protein interaction analysis, and particularly relates to a protein interaction analysis method based on fluorescence resonance energy transfer. The invention provides a protein interaction analysis method based on fluorescence resonance energy transfer, which adopts fluorescent protein FP and fluorescent Dye donor-acceptor to respectively carry out fusion expression and fluorescent marking on a protein to be detected and molecules participating in protein interaction, and realizes rapid qualitative characterization on protein interaction by utilizing the fluorescence resonance energy transfer phenomenon between fluorescent protein and Dye. The invention can express the three-dimensional structure of the whole protein; the device has low dependence on equipment, can complete experiments (constant temperature shaking table, water bath, PCR instrument, etc. are all necessary instruments in molecular laboratory) by only using conventional laboratory equipment, and has important application value.
Description
Technical Field
The invention belongs to the technical field of protein interaction analysis, and particularly relates to a protein interaction analysis method based on fluorescence resonance energy transfer.
Background
Proteins can interact with a variety of molecules in a variety of forms, which are important in maintaining cellular function, regulating biological processes, and in pharmacotherapy. Among the most common forms of interaction are mainly the following: (1) protein-protein interactions: interactions between proteins and other proteins occur. These interactions may include protein-protein binding, protein-protein recognition, and the like. For example, binding between enzyme and substrate, binding of receptor to ligand during signaling, and the like. (2) Protein-nucleic acid interactions: interactions between proteins and nucleic acids (e.g., DNA and RNA) occur. These interactions may include binding of transcription factors to DNA to regulate gene expression, participation of RNA-binding proteins in transcription, translation, or RNA degradation, among others. (3) Protein-small molecule interactions: interactions between proteins and small molecule compounds occur. These small molecules may be drugs, metabolites, hormones, etc. For example, binding of a drug to a protein may alter the function of the protein or inhibit its activity, thereby achieving a therapeutic effect. (4) Protein-polysaccharide interactions: interactions between proteins and polysaccharides (e.g., glycans, glycoproteins, etc.).
Display technology is a commonly used method for expressing and displaying a foreign protein or polypeptide in a host cell or organism. Among them, phage Display technology (Phage Display) uses Phage (bacteriophage) as a vector, and inserts the coding sequence of a target protein or polypeptide into the Phage genome to link it with Phage coat protein (coat protein) gene. Phage particles that are capable of binding to a particular ligand can be selected by infecting the host cell and selectively culturing. Yeast Display technology (Yeast Display) utilizes the protein domain on the surface of Yeast cells to link the coding sequence of a protein or polypeptide of interest to a Yeast surface protein, thereby achieving Display of the protein or polypeptide on the Yeast surface. However, these methods can only display domains of proteins, where phage display and yeast display technologies can generally only display surface domains of proteins or certain linear peptide fragments, and cannot display the three-dimensional structure of the entire protein. This may result in an inability to effectively screen for certain proteins or complex antibody-antigen interactions.
Surface-based techniques have found wide application in interaction studies, including surface plasmon resonance (Surface Plasmon Resonance, SPR) and biological chromatography (Biolayer Interferometry, BLI) techniques. These techniques utilize interactions between biomolecules on the sensor surface and target molecules for real-time monitoring and measurement. However, SPR and BLI techniques also suffer from several common disadvantages: (1) complicated experimental procedures: SPR and BLI techniques require precise instrument setup and operation, including sample preparation, sensor surface modification, data analysis, and the like, which may require specialized training and experience, which may be complex for a beginner. (2) high equipment cost: the instrumentation required for SPR and BLI techniques is relatively expensive, which may limit the scope of use and applications in some laboratories.
The technology based on no-clean has wider application, and the common technology mainly comprises the following steps: (1) AlphaLISA and AlphaScreen technologies. These techniques are based on the amplification of near infrared fluorescent signals and are commonly used in high throughput screening, protein-protein interactions, and cell signaling studies. The AlphaLISA and AlphaScreen kits can assess the activity of drug candidates by detecting interactions between ligands and targets. (2) LanthaScreen TR-FRET technique: the technique utilizes the principle of Time-resolved fluorescence resonance energy transfer (Time-Resolved Fluorescence Resonance Energy Transfer, TR-FRET) for measuring protein-protein interactions, protein kinase activity, inhibitor screening, and the like. However, there are also some drawbacks to the cleaning-free based technique that are notable: (1) high cost: perkinElmer's drug development reagents are typically expensive, which can burden a laboratory or project with a limited budget. (2) specialized equipment requirements: some PerkinElmer reagents require the use of specific equipment and instrumentation, which may require additional investment and training to accommodate the relevant technology and operational procedures. (3) specificity limitation: while PerkinElmer reagents perform well in particular applications, certain reagents may have limited specificity in other fields or on different types of samples. (4) limited application range: different PerkinElmer reagents are suitable for a particular experimental design and purpose and may therefore have limitations in terms of certain research problems or technical requirements.
Fluorescence Resonance Energy Transfer (FRET) is a phenomenon of non-radiative energy transfer, suitable for systems that meet spectral overlap and proximity requirements. Energy is transferred from the excited state of the donor fluorophore (D) to the ground state of the acceptor fluorophore (a) by dipole-dipole coupling. FRET has an inverse square distance dependence and can therefore be used for detection of nanoscale distances, typically ranging from 1 to 10 nanometers (up to 20 nanometers is possible for some atypical FRET pairs). Compared with other technologies (such as X-ray crystallography), FRET has the characteristics of being simpler and easier to implement, can be measured in living cells, and can be operated using standard experimental equipment. FRET is widely used in research fields matching with the length scale of biological macromolecules, and is widely focused on nanoscale distance measurement, called "molecular ruler" for detecting biological macromolecule structures. In addition to structural studies, FRET can also be used to monitor the dynamic process of intracellular protein-protein interactions and to determine signal transduction of organisms. FRET assays and sensors are very powerful in biological research because they can be detected in situ in real time without any intervening steps (e.g. washing). Some targeted processes suitable for FRET include mainly binding and dissociation of biological macromolecules, conformational changes, local environmental changes, etc., as they can lead to switching between high (on) and low (off) FRET states. FRET is an important technical tool, and has wide application in biological research. It aids in understanding the structure and interactions of biological macromolecules through nanoscale distance measurement and is useful for dynamic monitoring of signal transduction in many bioassays.
In summary, the current surface-based and cleaning-free protein interaction analysis method has the defects of higher research and development threshold, high equipment and consumable cost and the like; the technology based on phage and yeast display has a certain limitation in protein expression, and has the defects of complex flow, long period and the like. Therefore, there is a need to develop a simple and rapid assay for protein interactions based on the phenomenon of fluorescence resonance energy transfer.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a protein interaction analysis method based on fluorescence resonance energy transfer, which utilizes the phenomenon of fluorescence resonance energy transfer between fluorescent protein and dye to realize rapid qualitative characterization of protein interaction.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention provides a protein interaction analysis method based on fluorescence resonance energy transfer, which comprises the following steps:
s1, selecting a donor-acceptor pair D-A pair, wherein the D-A pair consists of a fluorescent protein FP and a fluorescent Dye;
s2, carrying out fusion expression on the protein alpha and FP to be detected, wherein the protein after fusion expression is named as FP-alpha (the molecular weight of FP-alpha is the sum of FP and alpha), combining a fluorescent Dye with molecules involved in protein interaction, and naming the fluorescent-labeled molecules as Dye-beta;
S3, diluting the FP-alpha and the Dye-beta by using a buffer solution to avoid that the FP-alpha and the Dye-beta are always within the distance of resonance energy transfer;
s4, incubating the diluted FP-alpha and Dye-beta, setting a control group by using a buffer solution, detecting FRET signals by using an enzyme-labeled instrument, and recording the fluorescence intensity I of the control group D And fluorescence intensity I of the experimental group DA ；
S5, the fluorescence intensity I of the control group is calculated according to the following formula D And fluorescence intensity I of the experimental group DA Substitution, calculate F RET Efficiency, when E FRET Above 0, an interaction between proteins is indicated:
calculation of fluorescence resonance energy transfer efficiency (FRET efficiency, E FRET ) The formula (formula source: medintz, I.L.&Hildebrandt,N.(eds)FRET-Resonance Energy Transfer from Theory to Applications (Wiley-VCH, 2013) are: />
Preferably, in S1, the donor-acceptor pair D-A pair includes, but is not limited to, donor mCherry-XL and acceptor Alexa Fluor 647.
D-A pair consists of a Fluorescent Protein (FP) and a fluorescent Dye (Dye), which has two forms: (1) FP as donor, dye as acceptor; (2) Dye as donor and FP as acceptor. The invention selects mCherry-XL as a donor and Alexa Fluor 647 as an acceptor to form the D-A pair of the invention, wherein the D-A pair has a Forst distance of 7.6nm distance,R 0 ). However, the present invention is not limited to the above two donor-acceptor pairs, in principle, only the donor and acceptor labeled molecules can generate fluorescence resonance energy transfer, and the donor-acceptor pair with larger R0 is preferably selected, so that the user can match the D-A pair according to the published data of fluorescent protein and dye, experimental environment and experimental existing conditions.
For R 0 The calculation of (a) can be referred to the following equations 1 and 2: (origin: medintz, I.L.&Hildebrandt,N.(eds)FRET–Resonance Energy Transfer from Theory to Applications(Wiley-VCH,2013).)：
Wherein,
the following are the meanings of the letters in the formula:
R 0 : the ferter distance represents the distance at which the energy transfer efficiency between the donor and acceptor drops to 50%.
κ 2 : the orientation factor, taking into account the relative orientation relationship between the donor and the acceptor.
n -4 : refractive index correction factor, taking into account the mediumEffect of refractive index on FRET efficiency.
Q D : the quantum yield of a donor indicates how many of the photons absorbed by the donor can be emitted as fluorescence.
J (λ): spectral overlap integration is used to describe the degree of overlap between the donor fluorescence spectrum and the acceptor absorbance spectrum.
F_d (λ): emission spectral intensity of the donor.
Epsilon_a (λ): the molar absorption coefficient of an acceptor indicates the intensity of light absorbed by the acceptor per unit concentration.
Lambda: wavelength means a specific wavelength of light.
Preferably, in S2, the protein α to be tested comprises (but is not limited to) a C2 nanobody of the N protein of the novel coronavirus, and the molecules involved in protein interactions comprise (but are not limited to) the N protein (nucleocapsid protein) of the novel coronavirus (SARS-CoV-2).
Preferably, in S3, the FP- α and Dye- β are diluted according to the following formula so that the distance between the FP- α and Dye- β is greater than 1.5 times R 0 ：
Wherein C is the concentration of FP-alpha or Dye-beta, d is R 0 。
The dilution of FP-alpha and Dye-beta is performed so that the final concentrations of both in the test system are such that the FP-alpha and Dye-beta are always within the distance (d) where resonance energy transfer occurs. Because the spatial distance of the donor-acceptor pair is 1.5 times R according to the FRET principle 0 Within this, the current instrument is able to sensitively detect the corresponding energy transfer signal (FRET as a biomolecular research tool-understanding its potential while avoiding pitfalls, https:// www.nature.com/statics/s 41592-019-0530-8). Thus, when two molecules are too large in concentration, they are all the time in close proximity, even if the two molecules do not interact themselves, there will be FRET signals, which are false interactions at this time, i.e. false positive results. For example, assuming that the distance (d) of resonance energy transfer is 10nm, the resonance energy transfer is calculated according to the following formula (public equation A source of formula (la): chandrasekhar, s.stochastic problems in physics and astronomy.rev.mod.Phys.15,1-89 (1943):
when the concentration (C) of Dye- β exceeds 200. Mu.M, the distance between FP- α and Dye- β is always less than 10nm, and therefore, in this case, it is necessary to make the concentration of Dye- β 200. Mu.M or less.
Preferably, in S4, the control group is a buffer solution used in place of Dye- β.
Preferably, in S3, the buffer solution includes (but is not limited to) PBST, HEPES.
Preferably, in S4, F is detected RET In signal processing, it is necessary to acquire a background signal from a non-fluorescent sample and subtract the background signal from the actual data to correct for background noise and perform signal correction.
The test sample is placed in a suitable assay device, which needs to contain PMT detectors to ensure that FRET signals can be detected. The gain or sensitivity of the PMT detector is set to accommodate the expected fluorescent signal intensity range. This can maximize the detection sensitivity of the signal and avoid oversaturation or noise interference. The FRET signal starts to be collected. FRET effects are detected by exciting a donor molecule and observing the emitted fluorescent signal of an acceptor molecule. During acquisition, stable operation of the PMT detector is ensured, and the resulting signal strength is recorded. According to experimental requirements, the donor or the acceptor can be excited and detected separately to obtain fluorescence intensity reference values of the donor and the acceptor.
Preferably, in S4, equal volumes of diluted FP-alpha and Dye-beta are incubated.
The diluted FP-alpha and Dye-beta are placed in the same reaction vessel or test tube in equal volumes, and the labeled molecular samples are gently mixed to ensure that they are uniformly mixed together. Mixing can be performed using a tube shaker, flip tube, or the like. After the completion of the mixing, the reaction vessel or test tube is sealed to prevent the volatilization of the solution and the contamination of the outside. The mixed labeled molecule sample is placed in a thermostat or incubator for incubation. The incubation conditions (e.g., temperature and humidity) are determined based on the experimental requirements and the nature of the labeling molecule. The incubation time was set to 5 minutes and ensured that a constant temperature was maintained during the incubation.
Preferably, in S5, the calculation method for determining whether the interaction between proteins occurs comprises calculation according to the Donor-accepter ratio or F-based calculation RET The efficiency formula is calculated. The method also comprises the following steps:
(1)I D and I DA The difference of (2) is greater than 0, i.e. interaction occurs;
(2)I DA and I D The difference of (2) is less than 0, i.e. interaction occurs;
(3)I D greater than I DA I.e. interaction occurs;
(4)I DA less than I D I.e. interaction takes place
Preferably, in S2, the specific steps of fusion expression of the protein α to be tested and FP together are as follows:
(1) Obtaining fusion genes: the fusion gene is obtained by connecting the target protein gene and the fluorescent protein gene and synthesizing or amplifying by PCR. Ensuring that the correct sequence and function of the protein of interest and fluorescent protein are preserved upon ligation.
(2) Inserting the fusion gene into an expression vector: the fusion gene is inserted into an appropriate expression vector for efficient expression in a host cell. The expression vector may be a plasmid, viral vector or other suitable expression system.
(3) Transfecting a host cell: the expression vector is transfected into the host cell of interest, such that it receives and expresses the fusion gene. Transfection may be performed using chemical transfection, electroporation, viral vector delivery, and the like.
(4) Culturing and expressing: culturing the transfected host cell under appropriate culture conditions to express the fusion protein of the target protein and the fluorescent protein. Culture conditions can be optimized as needed to ensure efficient protein expression and folding.
(5) Protein purification and analysis: the fusion protein of the protein of interest and the fluorescent protein is extracted and purified from the host cell by suitable protein purification techniques (e.g., affinity chromatography, gel filtration, centrifugation, etc.). The purified proteins are then analyzed, such as SDS-PAGE, western blot, etc., to verify the presence of the protein of interest and the fluorescent protein and the functionality of the fusion protein.
Preferably, in S2, the fluorescent Dye is bound to the molecule involved in the protein interaction in such a way that the fluorescent Dye is bound to the molecule involved in the protein interaction by a chemical reaction. This chemical reaction causes fluorescent dyes to become part of the molecule, and these fluorescently labeled molecules are designated Dye- β (where "Dye" represents a Dye and "β" represents a label).
The following are general steps and descriptions: (1) activating: first, a functional group of a fluorescent dye is activated with a target molecule. This can be achieved by introducing reactive groups (e.g., amine groups, hydroxyl groups, disulfide groups, etc.) on the fluorescent dye molecule; (2) and (3) connection: the activated fluorescent dye chemically reacts with a functional group having a corresponding reactive site in the target molecule to form a covalent linkage. Common reactions include amine-to-isocyanate reactions, hydroxyl-to-anhydride reactions, thiol-to-diene reactions, and the like; (3) purifying: after the fluorescent dye has been attached, a purification step is typically required to remove unreacted fluorescent dye and other impurities. This can be accomplished by column chromatography, gel electrophoresis, solution concentration, and the like. (4) And (3) verification: it was verified whether the labeled molecule was successfully attached to the fluorescent dye. Common methods include ultraviolet-visible absorption spectroscopy, fluorescence spectroscopy, mass spectrometry, and the like, to ensure that the labeled molecules have the desired optical properties and structure.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a protein interaction analysis method based on fluorescence resonance energy transfer, which adopts fluorescent protein FP and fluorescent Dye donor-acceptor to respectively carry out fusion expression and fluorescent marking on a protein to be detected and molecules participating in protein interaction, and realizes rapid qualitative characterization on protein interaction by utilizing the fluorescence resonance energy transfer phenomenon between fluorescent protein and Dye. The advantages are as follows:
(1) The invention can express the three-dimensional structure of the whole protein (the whole gene sequence of the protein is cloned into an expression vector in the fusion expression stage);
(2) The invention has low dependency on equipment, and can complete experiments (constant temperature shaking table, water bath, PCR instrument, etc. are all necessary instruments for molecular laboratory) only by using conventional laboratory equipment, while the existing commonly used protein interaction research method based on SPR adopts Biacore 8K+ equipment which needs about 500 ten thousand yuan;
(3) The donor-acceptor pair of the invention, R 0 Greater than 7.6nm, whereas conventional donor acceptor pairs have R of around 5nm only 0 The application range of FRET in protein interaction is expanded, so that molecules with larger interaction volume can be researched;
(4) The fluorescent dye has no influence on the combination of protein and molecules, and is suitable for protein screening items (antibody screening, enzyme screening and the like).
Drawings
FIG. 1 is an image of gel electrophoresis of a DNA fragment of interest; wherein lane 4 is the DNA fragment of interest, 369 bp).
FIG. 2 shows the result of gel electrophoresis of the enzyme-cleaved products; wherein, lane 2 is the cleavage product, and pET-28a (+) -mCherry-XL plasmid is subjected to single cleavage, so that a single band is about 5941bp during gel electrophoresis; if the cleavage fails, 3 bands (covalently closed circular DNA (cccDNA, SC configuration), open circular DNA (OC configuration) and linear molecules (L configuration)) appear around 5941 bp.
FIG. 3 is a comparison of the sequencing results and the plasmid sequences of interest, wherein both forward and reverse sequencing structures are aligned, demonstrating successful plasmid construction.
FIG. 4 shows the results of His tag purification, where SC is suspension culture; LC is the lysed supernatant of the suspension culture; e1, E2 and E3 are samples eluted first, second and third times, respectively.
FIG. 5 shows the results of AKTA purification, wherein E1 is His eluted sample; c1, C2, C3 and C4 are the first, second, third and fourth collections of AKTA purification, respectively.
FIG. 6 shows the results of the coupling of N proteins of a novel coronavirus, wherein MT COV-2N 5. Mu.g/mL in the row Ligand, pH5.5, refers to the N protein of the novel coronavirus, 5. Mu.g/mL refers to the coupling concentration, and pH5.5 refers to the pH of the buffer (10 nM sodium acetate). The Level (RU) column indicates the coupling amount, and the coupling amounts of Channel 3, channel 4 and Channel 5 were all about 220 RU.
FIG. 7 shows the functional verification results of the fusion proteins.
Detailed Description
The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.
The equipment, materials and reagent solutions involved in the invention are as follows:
apparatus (one)
(II) materials (sequence information including genes and primers)
pET-28a (+) -mCherry-XL was purchased from Rui Bo organism; pGEX-6P-1-C2 was purchased from Rui Bo organism; the forward primer (agatataccATGGCCGAAGTTCAG) and the reverse primer (CTTTACTCACCATggcGCCACTGCCTGAACTCACAGTTAC) delegate Rui Bo biosynthesis; the novel coronavirus N Protein is a Human N Protein (E.coil) Protein available from Michaelis, guangzhou. Coli BL21 (DE 3), E.coli DH 5. Alpha. Were purchased from Vaccinium.
Material description:
(1) pET-28a (+) -mCherry-XL: mCherry-XL is a fluorescent protein consisting of 236 amino acids and having a molecular weight of 26.7kD and a gene encoding mCherry-XL of 708bp in length (see, in particular, the literature "Mukherjee S, manna P, hung S T, et al directed evolution of a bright variant of mCherry: suppression of nonradiative decay by fluorescence lifetime selections [ J ]. The Journal of Physical Chemistry B,2022,126 (25): 4659-4668. Doi:10.26434/chemrxiv-2022-v3t03"). The gene sequence of mCherry-XL was constructed on this plasmid vector pET-28a, while pET-28a (+) -mCherry-XL was the vector for the subsequent expression of fusion proteins, the gene length being 5941bp.
(2) pGEX-6P-1-C2: the pGEX-6P-1-C2 plasmid has a gene length of 5338bp and has a sequence encoding the C2 protein. C2 is a nanobody of N protein of a novel coronavirus, consists of 123 amino acids, has a molecular weight of about 13.5kD, and has a gene length of 369bp (PDB ID:7N 0R) encoding C2. Nanobody is a small antibody molecule, typically a monoclonal antibody derived from a camel, alpaca or other similar animal. The C2 gene on pGEX-6P-1-C2 was cloned into the pET-28a (+) -mCherry-XL vector during the plasmid construction step.
(3) Coli BL21 (DE 3): BL21 (DE 3) is a commonly used Escherichia coli strain, and is widely applied to the fields of biotechnology and protein expression, and BL21 (DE 3) is adopted for expression of fusion proteins in the examples;
(4) Coli dh5α (competent): DH5 alpha (competent) is a commonly used laboratory strain, commonly used in molecular biology and genetic engineering research. In the examples, DH 5. Alpha. Was used for homologous recombination of genes.
(5) Forward primer: it is a DNA fragment having a specific sequence for selectively binding to one side of the target DNA and serving as a starting point of PCR reaction. In the examples, the forward primer of the target gene C2 was used.
(6) Reverse primer: it is a DNA fragment having a specific sequence for selectively binding to the other side of the target DNA and is used together with a forward primer as a starting point of PCR reaction. In the examples, the reverse primer of the target gene C2 was used.
(7) N protein (nucleocapsid protein) of novel coronavirus (SARS-CoV-2): is an important structural protein, also called core protein. The N protein plays a key role in the viral particle and is involved in the packaging, replication and transcription process of the viral genome.
N protein is a highly conserved protein, which is present in SARS-CoV-2 and its closely related viruses. Its main functions are to bind and protect the viral RNA genome and promote the formation of viral particles. The N protein also interacts with other viral proteins, regulating the viral replication process. Because of the importance of the N protein in viruses, antibodies have important biological and clinical implications for the recognition and binding of this protein. Anti-novel coronavirus N protein antibodies are specific antibodies against N protein produced by immunization of animals (e.g., mice, rabbits, etc.) or humans.
(III) consumable
(IV) reagents and solutions
Example FRET-based interaction studies of nanobodies and novel coronavirus N proteins
Construction of plasmid
1. PCR amplification of DNA fragment of interest (C2 nanobody)
PCR amplification was performed using Phanta Max Super-Fidelity DNA Polymerase (Northenzan, P505) reagent:
PCR reaction system:
1) Gently mixing, and centrifuging for 5s if necessary;
2) The reaction procedure: storing at 95 ℃ for 3 min- [95 ℃ for 15 s- & gt 65 ℃ for 15 s- & gt 72 ℃ for 42s ]. Times.35 cycles- & gt 72 ℃ for 5 min- & gt 4 ℃.
2. Purification recovery of amplified fragments
1) Taking 50 mu L of PCR product, adding the sample into a comb hole with the diameter of 6mm multiplied by 1.5mm, and carrying out constant-voltage electrophoresis for about 50min at 150V;
2) Cutting gel blocks containing the target DNA fragments from the gel cutting instrument (figure 1);
3) The PCR amplification product was recovered by electrophoretic purification using Gel Extraction Kit D2500 (omega brand, D2500), eluting with autoclaved pure water.
3. Restriction enzyme cutting, electrophoresis purifying and recovering carrier
1) Double enzyme cutting of the carrier: the reaction components are added sequentially to the sterile tube wall.
| Composition of the components | Volume (mu L) |
| ddH 2 O | 13 |
| rCutSmart 3.1 | 2 |
| pET-28a(+)-mCherry-XL(2μg/μL) | 4μL |
| NcoI-HF | 1 |
| Total volume of | 20 |
2) Enzyme digestion is carried out at 37 ℃ overnight;
3) The purified recovery of the cleaved product (FIG. 2) was performed using Gel Extraction Kit D2500 (omega brand, D2500), eluting with sterile water.
4. Homologous recombination of DNA
1) The temperature of the thermostatic water bath is adjusted to 42 ℃ in advance;
2) Taking out a tube (100 mu L) of competent bacterium DH5 alpha from a-70 ℃ ultralow temperature freezer, and thawing on ice;
3) 1. Mu.L of the cleavage product from step 3 (total 30 ng) was added, stirred with a pipette tip for 10s, and left on ice for 10min;
4) mu.L of the target DNA fragment (total 150 ng) recovered in step 2 was added, stirred with a pipette tip for 10s, and left on ice for 20min;
5) Slightly shaking, then inserting into a water bath at 42 ℃ for 90s for heat shock, then quickly putting back into ice, and standing for 3-5 min;
6) Adding 500 mu L of LB culture medium (without antibiotics) into the centrifuge tube standing in the step 5) in an ultra-clean workbench, gently mixing, and then fixing the mixture on a spring frame of a shaking table to oscillate for 1h at 37 ℃;
7) Taking out the transformation mixture in the step 6), dripping the transformation mixture into a solid LB plate culture dish containing Kana antibiotics in an ultra-clean bench, and uniformly coating the transformation mixture with a glass coating rod burnt by an alcohol lamp (note: the alcohol on the glass coating rod is extinguished for a little while, and the glass coating rod is coated after being cooled;
8) Marking the coated culture dish in the step 7), firstly placing the culture dish in a constant temperature incubator at 37 ℃ for 30-60min until the surface liquid permeates into the culture medium, and then placing the culture dish in the constant temperature incubator at 37 ℃ for overnight after inversion;
9) Picking single bacterial colony growing on the LB plate in the step 8) and sequencing;
10 The successfully constructed plasmid was designated pmC-C2 (FIG. 3).
Expression and isolation and purification of (II) fusion protein (FP-Nanobody)
1. Transformation of BL21 (DE 3)
1) The temperature of the thermostatic water bath is adjusted to 42 ℃ in advance;
2) Taking out a tube (100 mu L) BL21 (DE 3) from a-70 ℃ ultralow temperature refrigerator, and thawing on ice;
3) mu.L of pmC-C2 (total 100 ng) was added, stirred with a suction head for 10s and placed on ice for 30min;
4) Slightly shaking, then inserting into a water bath at 42 ℃ for 90s for heat shock, then quickly putting back into ice, and standing for 3-5 min;
5) Adding 500 mu L of LB culture medium (without antibiotics) into the centrifuge tube standing in the step 4) in an ultra-clean workbench, gently mixing, and then fixing the mixture on a spring frame of a shaking table to oscillate for 30min at 37 ℃;
6) Taking out the transformation mixture in the step 5), dripping the transformation mixture into a solid LB plate culture dish containing Kana antibiotics in an ultra-clean bench, and uniformly coating the transformation mixture with a glass coating rod burnt by an alcohol lamp (note: the alcohol on the glass coating rod is extinguished for a little while, and the glass coating rod is coated after being cooled;
7) Marking the coated culture dish in the step 6), firstly placing the culture dish in a constant temperature incubator at 37 ℃ for 30-60min until the surface liquid permeates into the culture medium, and then placing the culture dish in the constant temperature incubator at 37 ℃ for overnight after inversion;
8) After overnight, single colonies were grown on LB plates in step 7).
2. Induction suspension culture of fusion protein (mC-C2)
1) Picking single colony in the step 1, and carrying out suspension culture at 37 ℃ for overnight in 25mL LB+Kana 50 mug/mL;
2) Transfer to 1L LB to make OD 600 Suspension culture at 37℃to give OD 600 ＝0.4～0.6；
3) Adding IPTG to make its final concentration be 0.4mmol/L;
4) Suspension culture was carried out at 16℃for 16h.
3. Extraction of mC-C2 protein
1) The 1L culture solution in the step 2 was centrifuged at 6000g for 8 minutes at 25℃in two 500mL portions, and the cells were collected and E.coli was suspended in 40mL PBS (20 mM imidazole);
2) Lysing the thalli in the step 1) under an ultrasonic breaker;
3) Centrifuging at 8000rpm for 30min to precipitate thallus fragments in the bacterial liquid in the step 2), and collecting supernatant;
4) Mixing the supernatant with 4mL NTA resin, and incubating on a roller mixer at 4 ℃ overnight;
5) Adding the solution into a hollow column tube, and purifying and eluting by using a kit (His tag protein purification kit (reduction-resistant chelate type), P2226) (FIG. 4);
6) Performing a second purification on the protein sample obtained in step 5) using an AKTA instrument (fig. 5);
7) The proteins in step 6) were dialyzed using 1 XPBS, substituting buffer with PBS.
The fluorescent protein mCherry-XL used in the invention has a molecular weight of 26.7kD, the nano antibody C2 has a molecular weight of 13.5kD, and the theoretical molecular weight of the fluorescent protein mCherry-XL and the nano antibody C2 are about 40.2kD after fusion expression. Finally, a band was indeed formed around 43kD, so that the expression was successful (the difference between the molecular weight and the theoretical molecular weight shown by gel electrophoresis was normal). Compared with the His eluted sample E1, the bands of the AKTA collection liquid C1, C2, C3 and C4 are obviously less than E1, which indicates that part of the impurity protein is removed by multiple purification, so that the purity of the target protein is improved, and the subsequent experiment is facilitated.
(III) fluorescent dye labelling of novel coronavirus N protein (step reference kit Alexa)647Microscale Protein Labeling Kit specification
1. Protein preparation
1) The purified protein was prepared to a concentration of 1mg/mL in a buffer that did not contain major amine groups (e.g., ammonium ion, tris, glycine, ethanolamine, triethylamine, glutathione) or imidazole. The specific operation is as follows: adding 74 mu L of phosphate buffer (Phosphate Buffer Saline, PBS) into a 1.5mL centrifuge tube, and then adding 50 mu L of 2.48mg/mL of new coronavirus N protein into the 1.5mL centrifuge tube, namely diluting 2.48mg/mL of new coronavirus N protein to 1mg/mL;
2) Measurement of 1mg/mL New coronavirus N protein A 280 : after turning on the ultraviolet-visible spectrum function of the micro-spectrophotometer and using PBS as a blank control, 2 μL of 1mg/mL of novel coronavirus N protein is dripped on a probe of the micro-spectrophotometer, and the absorption light of the novel coronavirus N protein at 280nm is recorded and recorded as A 280 A is measured 280 ＝1.057。
2. Labelling reaction
1) The following formula was used to calculate the appropriate volume of reactive dye solution to be used, the appropriate volume specification being accompanied by instructions for the new coronavirus N protein, mr=12.
Wherein μg protein is the mass of the protein to be labeled, protein MW is the relative molecular mass of the protein to be labeled, MR is the dye: the molar amount of protein, and 7.94 is the concentration of the reactive dye. Substitution of 100 μg 55000Da of the new coronavirus N protein and mr=12 into the above formula calculated the need to add 2.75 μl of reactive dye.
2) 1M sodium bicarbonate solution was prepared by adding 1mL of deionized water to a sodium bicarbonate vial (component B), and extracting up and down with a shaker or pipette until the reagents were completely dissolved.
3) 100. Mu.L of 1mg/mL of the novel coronavirus N protein was transferred to a reaction tube (component C), and 10. Mu.L of 1M sodium bicarbonate was added thereto, followed by pipetting up and down multiple times and mixing well.
4) To a bottle Alexa555carboxylic acid,succinimidyl ester (component A) 10. Mu.L of deionized water was added and the contents of the flask were completely dissolved by pipetting up and down, the concentration of the reactive dye stock solution being 7.94 nmol/. Mu.L.
5) As calculated according to equation (18), 2.75. Mu.L of the reactive dye solution was added to the reaction tube containing the pH-adjusted protein and thoroughly mixed by pipetting up and down multiple times.
6) The reaction mixture was incubated at room temperature for 15 minutes.
3. Purification of markers
1) To separate the labeled proteins from the unreacted dye, the gel resin container (component E) and one spin filter (component D) were removed from the kit, and the gel resin was completely suspended by gently shaking the container without the use of vortex or magnetic stirring bars to agitate the material. The upper chamber of the spin-on filter was filled with suspended gel resin until the lip was reached, approximately 800 μl of resin was required. Spin filters were centrifuged in a microcentrifuge at 16,000Xg for 15 seconds (including start-up time). The use of a fixed angle rotor results in compaction of the resin on the low side and the high side, where the resin bed edge on the low side should be about 2-3mm above the green collar and the edge on the high side should not exceed the top lip of the rotary filter. If a pendulum rotor is used, the resin bed should fill half of the upper chamber, about 5mm.
2) After the spin filter was prepared, no more than 50. Mu.L of the conjugate reaction mixture was added dropwise to the center of the resin bed surface using a pipette. If the conjugation reaction has a volume of 51-100. Mu.L, it is split into two small fractions and purified on different spin filters. The spin filter was placed in a microcentrifuge with the high side of the resin bed facing outward and centrifuged at 16,000Xg for a total of 1 minute.
3) Finally, each collection tube yielded about 60-100. Mu.L of purified dye-labeled protein in buffer.
The procedure described in the labeling step for the novel coronavirus N protein can be completed in about 30 minutes.
4. Determining the degree of marking (Degree of Labeling, DOL)
To determine Alexa647 dye-labeled DOL of protein conjugate by spectrophotometry by absorption at 280nm (A 280 ) And650nm(A 650 ) Absorbance at to obtain protein concentration:
1) Opening a NANO-500 spectrophotometer, opening ultraviolet-visible spectrum function, using 2 mu L PBS as blank control, dripping 2 mu L labeled new coronavirus N protein, and recording A 650 And A 280 . Measurement A 650 ＝13.544，A 280 ＝1.159。
2) The concentration of protein in the sample was calculated according to the following formula:
in this equation, 0.03 is a factor correcting the contribution of the fluorophore to A280, A 650 Absorbance at 265nm of the labeled protein (A 650 ＝13.544)，A 280 Absorbance at 280nm of the protein after labeling (A 280 ＝1.159)，A 280 1mg/mL of protein is the absorbance of the pre-labeled protein at 280nm (A 280 =1.057), the Dilution factor is the Dilution ratio of the sample to be measured (the Dilution factor=1 in this example). In the implementation process, the concentration of the labeled novel coronavirus N protein is calculated to be 0.71mg/mL, and the labeled novel coronavirus N protein is named 647-N.
3) The molar concentration of protein was calculated according to the following formula:
substituting the result of the formula (19) and calculating the molecular weight 55000Da of 555-N to obtain 1.29E-05M;
the whole protein labeling process can be completed within one hour.
(IV) verification of antibody function Using fluorescence resonance energy transfer
1. FRET test sample preparation and microplate reader detection
1) Diluting mC-C2 in step (two) to 100nM;
2) Diluting 647-N of step (three) to 250nM;
3) The preparation of test samples (3 multiple wells) was performed as follows, and well sites as follows were added to 384 well plates. Wherein, the A behavior control group is named as I for the fluorescence intensity value D The method comprises the steps of carrying out a first treatment on the surface of the B behavioural experiment group, for which the fluorescence intensity value was named I DA ：
4) Excitation light is 558/8nm, and emission light is 588/8nm;
5) The following are the detection results of the enzyme-labeled instrument:
| 1 | 2 | 3 | |
| A | 441 | 426 | 427 |
| B | 222 | 230 | 230 |
2. data processing and analysis
1) I in the step 1 is as follows D And I DA Taking the average value, substituting the average value into a formula (6), and calculating to obtain E FRET ＝0.47；
2) According to E FRET Because of E in (1) FRET > 0, indicating that the fusion protein and 647-N interacted.
Comparative example SPR-based interaction study of nanobodies and novel coronaviruses
Steps (one) and (two) are the same as in example 1, and the subsequent steps are as follows:
(III) SPR verifies interactions of mC-C2 and novel coronavirus N proteins:
1. coupling of ligands
Coupling refers to the process of immobilizing a ligand on a chip. The coupling method varies from chip to chip and ligand to ligand. The most typical and most commonly used coupling method, the amino coupling method, will be used in this experiment. Ligand coupling was accomplished using the coupling (hybridization) procedure in Biacore 8K Control Software.
1) Click New method at upper right of Biacore 8K Control Software main interface, click Surface preparation, select the organization, click the lower right open button, and enter editing interface. In the Chip type drop-down menu, CM5 is selected. The default coupling mode of the program is amino coupling (Amine), and the experiment only uses channel3-5, so that the square frame before channel 1-2 and 6-8 is removed. The ligand was immobilized on flow cell 2 (Fc 2) of channel3-5, while flow cell 1 (Fc 1) was only activated and blocked, and ligand immobilization was not performed, so Fc2, activated/deactivated in 1 was selected. The Ligand (Ligand) sampling time was 105s and the Ligand name was modified.
2) Clicking on "Positioning and Plate layout" above, the system will jump to a new window, check if the volume of solution in the Bottles displayed on the screen is sufficient, then according to the sample location information displayed on the screen, prepare a sample of sufficient volume (7.5 μg/mL of new coronavirus N protein diluted with sodium acetate at pH 5.0), and place in the designated location on the Plate corresponding to Trays (placing the sample volume slightly greater than the display volume). The 96-well Plate is placed in the designated Plate position for the corresponding sample and locked, and returned to the sample compartment, closing the compartment door.
3) Clicking a file Save icon at the upper right, saving the method file in the user-defined folder, and clicking Save. Clicking the Send to queue again, the system will automatically jump to the new interface, confirm whether the screen display items are placed correctly, confirm Buffer, water, reagent if sufficient, and confirm whether the track and plate positions are consistent with the display, the whole process running for about 26 minutes. The lower Ready to start is clicked. And storing the result file into the corresponding folder in the jumped-out window. The system formally and automatically runs an immobization program.
4) After the coupling (organization) program is finished, click Runs above the Biacore 8K Control Software main interface to find the result file, double click or click the lower right Open button to Open. The Results (FIG. 6) show the final ligand protein coupling amount, which was about 220RU on flow cell 2 of channel 3-5 of this example. Clicking on the Sensorgram can look at the coupling overall process.
2. Multicycle kinetic detection affinity:
1) Clicking the New method on the right upper part of the main interface at the opened Biacore 8K Control Software, clicking the anti/general, selecting the kinetic/Affinity, and double clicking the Multi-cycle kinetic/Affinity on the right side.
2) In the Method definition interface, the sample compartment temperature was chosen to be set to fixed 25 ℃, concentration units were chosen to be nM, and others were unchanged. In the Analysis window below, the Analysis temperature filled in a default of 25 ℃, contact time of 600s at 120s,Dissociation time and flow rate of 30. Mu.L/min. Click Add command adds the Regeneration command, solution is Glycine 1,6, contact time is 30s, flow rate is 30 μL/min. Each item in Startup is unchanged according to the default value of the system.
3) Click "Variables and positioning" above, enter the new interface, click Analysis. Channel 3-5 (the Fc2 channels of these 3 channels were all coupled to the same ligand) was selected for affinity/kinetic detection, removing the. Clicking on Startup, clicking on Add cycle in the jumped-out window until 5 Startup.
4) Clicking Analysis, filling in sample name and concentration gradient (zero concentration for the first cycle) in Analysis 1Solution of the jumped-out form, and increasing the concentration of the second cycle by channels 1-6 in turn.
5) Clicking "Plate Layout" on the upper right, the system jumps to a new window, checks whether the volume of solution in the Bottles displayed on the screen is sufficient, prepares a sample of sufficient volume according to the sample position information displayed on the screen (preparation method of mC-C2 is shown in Table 1), and places the sample in the designated position on the Plate corresponding to Trays (placing the sample volume slightly larger than the display volume). The 96-well Plate is placed in the designated Plate position for the corresponding sample and locked, and returned to the sample compartment, closing the compartment door.
TABLE 1 preparation of mC-C2
| Sequence number | Concentration (nM) | The preparation method comprises the following steps: |
| 1 | 1000 | mC-C2 was diluted to 1000nM and the total volume was 400. Mu.L. |
| 2 | 500 | 200. Mu.L of 1 and 200. Mu.L of PBST were mixed to give 500nM mC-C2. |
| 3 | 250 | 200. Mu.L of 2 and 200. Mu.L of PBST were mixed to give 250nM mC-C2. |
| 4 | 125 | 200. Mu.L of 3 and 200. Mu.L of PBST were mixed to give 125nM mC-C2. |
| 5 | 62.5 | 200. Mu.L of 4 and 200. Mu.L of PBST were mixed to give 62.5nM mC-C2. |
| 6 | 31.25 | 200. Mu.L of 5 and 200. Mu.L of PBST were mixed to give 31.25nM mC-C2. |
6) Clicking a file Save icon at the upper right, saving the method file in the user-defined folder, and clicking Save. Clicking the Send to queue again, the system will jump automatically to the new interface, confirm whether the screen display items are placed correctly, confirm Buffer, water, reagent if sufficient, and confirm whether the track and plate positions are consistent with the display, the whole process running for about 2 hours. The lower Ready to start is clicked. And storing the result file into the corresponding folder in the jumped-out window. The system formally and automatically runs Multi-cycle graphics programs.
7) The results of the operation are shown in FIG. 7. The results in FIG. 7 show that the fusion protein has an affinity of 17.7nM for the novel coronavirus N protein, indicating that the fusion protein binds to the novel coronavirus N protein.
According to the invention, qualitative characterization of protein interaction can be realized by utilizing fluorescence resonance energy transfer phenomenon between fluorescent protein and small molecule dye, which shows that the invention can achieve analysis effect of molecular interaction of commercial instrument widely accepted in industry.
In summary, compared with the existing method for analyzing intermolecular interaction, the method has the following advantages:
(1) The donor-acceptor pair of the invention, R 0 About 7.6nm, whereas conventional donor-acceptor pairs have R of around 5nm only 0 The application range of FRET in protein interaction is expanded, so that molecules with larger interaction volume can be researched;
(2) The fluorescent protein and the protein to be detected are fused and expressed, so that the space three-dimensional structure of the whole protein can be reserved;
(3) The device dependence is low, and experiments (constant temperature shaking table, water bath, PCR instrument, etc. are all necessary instruments for molecular laboratory) can be completed only by conventional laboratory devices (compared with surface-based and cleaning-free technologies);
(4) The fluorescent dye has no influence on the combination of protein and molecule, and has outstanding advantages in protein screening work (antibody screening and enzyme screening). Because the structure of the protein in these screening works is constantly changing, the molecule interacting with the protein is unchanged, and the region where the protein and the molecule bind is determined, the construction of the fusion protein cannot affect the binding site of the protein, but the protein interacts with any one of the structures of the molecule to bind, and thus the labeling of the molecule cannot affect the interaction of the molecule and the protein, i.e., the labeling of the molecule cannot mask the interaction region and destroy the interacted structure. The use of fluorescent dyes to label molecules meets the above requirements, and if both the protein and the molecule are expressed in fusion with the fluorescent protein, fusion expression of the molecule and the fluorescent protein tends to block the interaction region.
Therefore, the invention utilizes the fluorescence resonance energy transfer phenomenon between fluorescent protein and dye to realize rapid qualitative characterization of protein interaction, and has important application value.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.
Claims (9)
1. A method for analyzing protein interactions based on fluorescence resonance energy transfer, comprising the steps of:
s1, selecting a donor-acceptor pair D-A pair, wherein the D-A pair consists of a fluorescent protein FP and a fluorescent Dye;
s2, carrying out fusion expression on the protein alpha and FP to be detected, wherein the protein after fusion expression is named as FP-alpha, combining a fluorescent Dye with molecules involved in protein interaction, and naming the fluorescent-labeled molecules as Dye-beta;
s3, diluting the FP-alpha and the Dye-beta by using a buffer solution to avoid that the FP-alpha and the Dye-beta are always within the distance of resonance energy transfer;
s4, incubating the diluted FP-alpha and Dye-beta, setting a control group by using a buffer solution, detecting FRET signals by using an enzyme-labeled instrument, and recording the fluorescence intensity I of the control group D And fluorescence intensity I of the experimental group DA ；
S5, the fluorescence intensity I of the control group is calculated according to the following formula D And fluorescence intensity I of the experimental group DA Substitution, calculate F RET Efficiency, when E FRET Above 0, an interaction between proteins is indicated:
2. the method of claim 1, wherein in S1, the donor-acceptor pair D-a pair comprises donor mCherry-XL and acceptor Alexa Fluor 647.
3. The method according to claim 1, wherein in S2, the protein α comprises C2 nanobody of N protein of new coronavirus, and the molecule involved in protein interaction comprises N protein (nucleocapsid protein) of new coronavirus (SARS-CoV-2).
4. The method for analyzing protein interactions based on fluorescence resonance energy transfer according to claim 1, wherein in S3, the FP- α and Dye- β are diluted to a distance of greater than 1.5 times R according to the following formula 0 ：
Wherein C is the concentration of FP-alpha or Dye-beta, d is R 0 。
5. The method of claim 1, wherein in S4, the control group is buffer solution used in place of Dye- β.
6. The method of claim 1, wherein in S3, the buffer solution comprises PBST, HEPES.
7. The method according to claim 1, wherein in S4, when detecting FRET signals, it is necessary to obtain background signals from non-fluorescent samples and subtract the background signals from actual data to correct for background noise and perform signal correction.
8. The method of claim 1, wherein in S4, the diluted FP- α and Dye- β equal volumes are incubated.
9. The method of claim 1, wherein in S5, determining whether an interaction occurs between proteins further comprises:
(1)I D and I DA The difference of (2) is greater than 0, i.e. interaction occurs;
(2)I DA and I D The difference of (2) is less than 0, i.e. interaction occurs;
(3)I D greater than I DA I.e. interaction occurs;
(4)I DA less than I D I.e. interaction occurs.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311717390.XA CN117761021A (en) | 2023-12-14 | 2023-12-14 | Protein interaction analysis method based on fluorescence resonance energy transfer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311717390.XA CN117761021A (en) | 2023-12-14 | 2023-12-14 | Protein interaction analysis method based on fluorescence resonance energy transfer |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117761021A true CN117761021A (en) | 2024-03-26 |
Family
ID=90315605
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311717390.XA Pending CN117761021A (en) | 2023-12-14 | 2023-12-14 | Protein interaction analysis method based on fluorescence resonance energy transfer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117761021A (en) |
-
2023
- 2023-12-14 CN CN202311717390.XA patent/CN117761021A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101335560B1 (en) | Fluoroimmunoassay method | |
| JP4571499B2 (en) | Biosensor | |
| US20080064025A1 (en) | Cooperative reporter systems, components, and methods for analyte detection | |
| US20240168032A1 (en) | Glucose biosensors and uses thereof | |
| US7601510B2 (en) | Development and use of fluorescent probes of unbound analytes | |
| Traxlmayr et al. | Directed evolution of protein thermal stability using yeast surface display | |
| Van Deventer et al. | A platform for constructing, evaluating, and screening bioconjugates on the yeast surface | |
| CN107698683B (en) | CK-MB fusion protein, preparation method thereof and detection kit | |
| De Picciotto et al. | Design principles for SuCESsFul biosensors: Specific fluorophore/analyte binding and minimization of fluorophore/scaffold interactions | |
| JP2004502130A (en) | Methods for identifying compounds that bind to a target species under isothermal denaturation | |
| JP7307158B2 (en) | Anti-human cardiac troponin I antibody and application thereof | |
| Schröter et al. | Isolation of pH-sensitive antibody fragments by fluorescence-activated cell sorting and yeast surface display | |
| EP1731908B1 (en) | Method of biosubstance trapping, structural analysis or/and identification with use of a labeling substance | |
| Kim et al. | Improving single-molecule antibody detection selectivity through optimization of peptide epitope presentation in OmpG nanopore | |
| CN117761021A (en) | Protein interaction analysis method based on fluorescence resonance energy transfer | |
| JP6983419B2 (en) | Antigen detection or measurement kit | |
| Yu et al. | Fc-specific and covalent conjugation of a fluorescent protein to a native antibody through a photoconjugation strategy for fabrication of a novel photostable fluorescent antibody | |
| KR101551925B1 (en) | Target-specific probe comprsing t7 bacteriophage and detecting for biomarker using the same | |
| US9023998B2 (en) | Near infrared fluorogen and fluorescent activating proteins for in vivo imaging and live-cell biosensing | |
| CN110615844A (en) | Multi-species universal detection protein with green fluorescence activity and application thereof | |
| Cater et al. | Development of a generalisable tryptophan-optimised quenchbody biosensor based on a synthetic nanobody library | |
| CN114058623B (en) | Aptamer for recognizing and combining integrin alpha 3 subunit and related functions thereof | |
| Schofield et al. | Sequencing and affinity determination of antigen-specific B lymphocytes from peripheral blood | |
| CN110437341B (en) | Detection protein with red fluorescence activity and application thereof | |
| TWI779550B (en) | Method and biosensor for lead ions detection |
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 |