CN118324860A - Construction of phage display cyclopeptide library based on natural cyclopeptide skeleton - Google Patents
Construction of phage display cyclopeptide library based on natural cyclopeptide skeleton Download PDFInfo
- Publication number
- CN118324860A CN118324860A CN202311346656.4A CN202311346656A CN118324860A CN 118324860 A CN118324860 A CN 118324860A CN 202311346656 A CN202311346656 A CN 202311346656A CN 118324860 A CN118324860 A CN 118324860A
- Authority
- CN
- China
- Prior art keywords
- phage
- cyclic peptide
- display system
- cysteine
- peptide library
- 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
- 108010069514 Cyclic Peptides Proteins 0.000 title claims abstract description 200
- 102000001189 Cyclic Peptides Human genes 0.000 title claims abstract description 200
- 238000002823 phage display Methods 0.000 title claims abstract description 65
- 238000010276 construction Methods 0.000 title description 8
- 235000018417 cysteine Nutrition 0.000 claims abstract description 101
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 78
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 75
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims abstract description 64
- 235000001014 amino acid Nutrition 0.000 claims abstract description 63
- 150000001413 amino acids Chemical class 0.000 claims abstract description 60
- 238000000034 method Methods 0.000 claims abstract description 51
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 41
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 claims abstract description 7
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 184
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 142
- 229920001184 polypeptide Polymers 0.000 claims description 129
- 229960002433 cysteine Drugs 0.000 claims description 63
- 239000013598 vector Substances 0.000 claims description 59
- 229940024606 amino acid Drugs 0.000 claims description 58
- 230000015572 biosynthetic process Effects 0.000 claims description 56
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 44
- 239000006228 supernatant Substances 0.000 claims description 40
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 38
- 150000001945 cysteines Chemical class 0.000 claims description 37
- 238000012216 screening Methods 0.000 claims description 36
- 235000018102 proteins Nutrition 0.000 claims description 33
- 238000005406 washing Methods 0.000 claims description 29
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 27
- 239000000047 product Substances 0.000 claims description 25
- 239000013612 plasmid Substances 0.000 claims description 23
- 238000012163 sequencing technique Methods 0.000 claims description 20
- 125000000539 amino acid group Chemical group 0.000 claims description 19
- 239000011780 sodium chloride Substances 0.000 claims description 19
- 230000035772 mutation Effects 0.000 claims description 15
- 239000004471 Glycine Substances 0.000 claims description 14
- 238000012258 culturing Methods 0.000 claims description 14
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- 241000588724 Escherichia coli Species 0.000 claims description 12
- 241000701959 Escherichia virus Lambda Species 0.000 claims description 12
- 241000724791 Filamentous phage Species 0.000 claims description 12
- 239000006166 lysate Substances 0.000 claims description 12
- 238000011534 incubation Methods 0.000 claims description 11
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 claims description 10
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 claims description 10
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 10
- 229960001153 serine Drugs 0.000 claims description 10
- 235000004400 serine Nutrition 0.000 claims description 10
- 230000008685 targeting Effects 0.000 claims description 10
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 9
- 229960003767 alanine Drugs 0.000 claims description 9
- 235000004279 alanine Nutrition 0.000 claims description 9
- 229910052757 nitrogen Inorganic materials 0.000 claims description 9
- 238000006467 substitution reaction Methods 0.000 claims description 9
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 8
- 102000047725 alpha7 Nicotinic Acetylcholine Receptor Human genes 0.000 claims description 8
- 108700006085 alpha7 Nicotinic Acetylcholine Receptor Proteins 0.000 claims description 8
- 230000003698 anagen phase Effects 0.000 claims description 8
- 108091033319 polynucleotide Proteins 0.000 claims description 8
- 102000040430 polynucleotide Human genes 0.000 claims description 8
- 239000002157 polynucleotide Substances 0.000 claims description 8
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 7
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 7
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 7
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 7
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 7
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 7
- 239000004472 Lysine Substances 0.000 claims description 7
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 7
- 229960001230 asparagine Drugs 0.000 claims description 7
- 235000009582 asparagine Nutrition 0.000 claims description 7
- 235000004554 glutamine Nutrition 0.000 claims description 7
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 7
- 239000003112 inhibitor Substances 0.000 claims description 7
- 235000018977 lysine Nutrition 0.000 claims description 7
- 229960002429 proline Drugs 0.000 claims description 7
- 235000013930 proline Nutrition 0.000 claims description 7
- 239000004475 Arginine Substances 0.000 claims description 6
- 102100037840 Dehydrogenase/reductase SDR family member 2, mitochondrial Human genes 0.000 claims description 6
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 6
- 101100450591 Human adenovirus B serotype 3 PVIII gene Proteins 0.000 claims description 6
- 102000004310 Ion Channels Human genes 0.000 claims description 6
- 108090000862 Ion Channels Proteins 0.000 claims description 6
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 6
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 6
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 6
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 6
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 6
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 6
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 6
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 6
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims description 6
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 6
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 6
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 claims description 6
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 claims description 6
- 101710188053 Protein D Proteins 0.000 claims description 6
- 101710132893 Resolvase Proteins 0.000 claims description 6
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 6
- 239000004473 Threonine Substances 0.000 claims description 6
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 6
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 claims description 6
- 238000007792 addition Methods 0.000 claims description 6
- 229960003121 arginine Drugs 0.000 claims description 6
- 235000009697 arginine Nutrition 0.000 claims description 6
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 6
- 229960005261 aspartic acid Drugs 0.000 claims description 6
- 235000003704 aspartic acid Nutrition 0.000 claims description 6
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 6
- 235000013922 glutamic acid Nutrition 0.000 claims description 6
- 239000004220 glutamic acid Substances 0.000 claims description 6
- 235000014304 histidine Nutrition 0.000 claims description 6
- 229960002885 histidine Drugs 0.000 claims description 6
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 6
- 229960000310 isoleucine Drugs 0.000 claims description 6
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims description 6
- 229960003136 leucine Drugs 0.000 claims description 6
- 229930182817 methionine Natural products 0.000 claims description 6
- 235000008729 phenylalanine Nutrition 0.000 claims description 6
- 229960005190 phenylalanine Drugs 0.000 claims description 6
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 6
- 229910052717 sulfur Inorganic materials 0.000 claims description 6
- 235000008521 threonine Nutrition 0.000 claims description 6
- 229960002898 threonine Drugs 0.000 claims description 6
- 229960004441 tyrosine Drugs 0.000 claims description 6
- 235000002374 tyrosine Nutrition 0.000 claims description 6
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 6
- 229960004295 valine Drugs 0.000 claims description 6
- 239000004474 valine Substances 0.000 claims description 6
- 108010078791 Carrier Proteins Proteins 0.000 claims description 5
- 102000014914 Carrier Proteins Human genes 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- 239000002244 precipitate Substances 0.000 claims description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 claims description 3
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 3
- 102000015296 acetylcholine-gated cation-selective channel activity proteins Human genes 0.000 claims description 3
- 108040006409 acetylcholine-gated cation-selective channel activity proteins Proteins 0.000 claims description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 3
- 238000006664 bond formation reaction Methods 0.000 claims description 3
- 238000012217 deletion Methods 0.000 claims description 3
- 230000037430 deletion Effects 0.000 claims description 3
- 238000003780 insertion Methods 0.000 claims description 3
- 230000037431 insertion Effects 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- 239000001301 oxygen Substances 0.000 claims description 3
- 229910052711 selenium Inorganic materials 0.000 claims description 3
- 239000011669 selenium Substances 0.000 claims description 3
- 239000011593 sulfur Substances 0.000 claims description 3
- 239000003446 ligand Substances 0.000 abstract description 11
- 108050003126 conotoxin Proteins 0.000 abstract description 7
- 239000007788 liquid Substances 0.000 description 62
- 210000004027 cell Anatomy 0.000 description 60
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 57
- 238000003786 synthesis reaction Methods 0.000 description 54
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 51
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 45
- 239000003153 chemical reaction reagent Substances 0.000 description 34
- 239000002609 medium Substances 0.000 description 32
- 108020004414 DNA Proteins 0.000 description 27
- 230000001580 bacterial effect Effects 0.000 description 26
- 239000000243 solution Substances 0.000 description 26
- 239000002904 solvent Substances 0.000 description 26
- 239000000203 mixture Substances 0.000 description 23
- 239000011347 resin Substances 0.000 description 23
- 229920005989 resin Polymers 0.000 description 23
- 239000012148 binding buffer Substances 0.000 description 19
- 102000009660 Cholinergic Receptors Human genes 0.000 description 18
- 108010009685 Cholinergic Receptors Proteins 0.000 description 18
- 239000007787 solid Substances 0.000 description 18
- 125000005647 linker group Chemical group 0.000 description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 17
- 108010067902 Peptide Library Proteins 0.000 description 16
- 241000894006 Bacteria Species 0.000 description 13
- 125000002619 bicyclic group Chemical group 0.000 description 13
- 230000000694 effects Effects 0.000 description 13
- 238000002474 experimental method Methods 0.000 description 13
- 229960002449 glycine Drugs 0.000 description 13
- 229920001223 polyethylene glycol Polymers 0.000 description 13
- 238000002360 preparation method Methods 0.000 description 13
- 238000005119 centrifugation Methods 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 12
- 238000003776 cleavage reaction Methods 0.000 description 12
- 230000007017 scission Effects 0.000 description 12
- 238000004140 cleaning Methods 0.000 description 11
- 230000002401 inhibitory effect Effects 0.000 description 11
- 239000000499 gel Substances 0.000 description 10
- 238000004128 high performance liquid chromatography Methods 0.000 description 9
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 8
- 230000000903 blocking effect Effects 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
- 238000010790 dilution Methods 0.000 description 8
- 239000012895 dilution Substances 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 7
- 238000001976 enzyme digestion Methods 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 238000004949 mass spectrometry Methods 0.000 description 7
- 239000008188 pellet Substances 0.000 description 7
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 6
- 238000005481 NMR spectroscopy Methods 0.000 description 6
- 108010019160 Pancreatin Proteins 0.000 description 6
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 6
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 6
- 229960000723 ampicillin Drugs 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 230000018109 developmental process Effects 0.000 description 6
- 238000001962 electrophoresis Methods 0.000 description 6
- 229960002743 glutamine Drugs 0.000 description 6
- 230000005764 inhibitory process Effects 0.000 description 6
- 229960003646 lysine Drugs 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 239000011259 mixed solution Substances 0.000 description 6
- 229940055695 pancreatin Drugs 0.000 description 6
- 229910001868 water Inorganic materials 0.000 description 6
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 238000010511 deprotection reaction Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 238000007865 diluting Methods 0.000 description 5
- 231100000673 dose–response relationship Toxicity 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 229960002989 glutamic acid Drugs 0.000 description 5
- 229960004452 methionine Drugs 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 238000005086 pumping Methods 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 231100000765 toxin Toxicity 0.000 description 5
- 229960004799 tryptophan Drugs 0.000 description 5
- 238000012795 verification Methods 0.000 description 5
- 101710132601 Capsid protein Proteins 0.000 description 4
- 101710094648 Coat protein Proteins 0.000 description 4
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 4
- XLYOFNOQVPJJNP-ZSJDYOACSA-N Heavy water Chemical compound [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 4
- 101710125418 Major capsid protein Proteins 0.000 description 4
- 101710141454 Nucleoprotein Proteins 0.000 description 4
- 101710083689 Probable capsid protein Proteins 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 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 4
- 230000004071 biological effect Effects 0.000 description 4
- 238000000861 blow drying Methods 0.000 description 4
- 239000011575 calcium Substances 0.000 description 4
- 238000005520 cutting process Methods 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 229960002715 nicotine Drugs 0.000 description 4
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 description 4
- 150000007523 nucleic acids Chemical group 0.000 description 4
- 229910052700 potassium Inorganic materials 0.000 description 4
- 238000004153 renaturation Methods 0.000 description 4
- 239000007790 solid phase Substances 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- NDKDFTQNXLHCGO-UHFFFAOYSA-N 2-(9h-fluoren-9-ylmethoxycarbonylamino)acetic acid Chemical compound C1=CC=C2C(COC(=O)NCC(=O)O)C3=CC=CC=C3C2=C1 NDKDFTQNXLHCGO-UHFFFAOYSA-N 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 description 3
- 108091006146 Channels Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 3
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- 239000004793 Polystyrene Substances 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000011230 binding agent Substances 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 239000012149 elution buffer Substances 0.000 description 3
- 238000011010 flushing procedure Methods 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000003306 harvesting Methods 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 238000005360 mashing Methods 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 229920002223 polystyrene Polymers 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- -1 small molecule compounds Chemical class 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 230000003595 spectral effect Effects 0.000 description 3
- 230000008961 swelling Effects 0.000 description 3
- 230000002194 synthesizing effect Effects 0.000 description 3
- 239000003053 toxin Substances 0.000 description 3
- 108700012359 toxins Proteins 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- KLBPUVPNPAJWHZ-UMSFTDKQSA-N (2r)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-tritylsulfanylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)SC(C=1C=CC=CC=1)(C=1C=CC=CC=1)C1=CC=CC=C1 KLBPUVPNPAJWHZ-UMSFTDKQSA-N 0.000 description 2
- LHEJDBBHZGISGW-UHFFFAOYSA-N 5-fluoro-3-(3-oxo-1h-2-benzofuran-1-yl)-1h-pyrimidine-2,4-dione Chemical compound O=C1C(F)=CNC(=O)N1C1C2=CC=CC=C2C(=O)O1 LHEJDBBHZGISGW-UHFFFAOYSA-N 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 2
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- OUVXYXNWSVIOSJ-UHFFFAOYSA-N Fluo-4 Chemical compound CC1=CC=C(N(CC(O)=O)CC(O)=O)C(OCCOC=2C(=CC=C(C=2)C2=C3C=C(F)C(=O)C=C3OC3=CC(O)=C(F)C=C32)N(CC(O)=O)CC(O)=O)=C1 OUVXYXNWSVIOSJ-UHFFFAOYSA-N 0.000 description 2
- 239000012981 Hank's balanced salt solution Substances 0.000 description 2
- 239000012880 LB liquid culture medium Substances 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 239000008049 TAE buffer Substances 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Substances CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- HGEVZDLYZYVYHD-UHFFFAOYSA-N acetic acid;2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid Chemical compound CC(O)=O.OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O HGEVZDLYZYVYHD-UHFFFAOYSA-N 0.000 description 2
- 125000001931 aliphatic group Chemical group 0.000 description 2
- 239000005557 antagonist Substances 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 238000010170 biological method Methods 0.000 description 2
- 230000009460 calcium influx Effects 0.000 description 2
- 229910001424 calcium ion Inorganic materials 0.000 description 2
- 230000003185 calcium uptake Effects 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 229960003067 cystine Drugs 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 235000021186 dishes Nutrition 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 238000009509 drug development Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- JGBUYEVOKHLFID-UHFFFAOYSA-N gelred Chemical compound [I-].[I-].C=1C(N)=CC=C(C2=CC=C(N)C=C2[N+]=2CCCCCC(=O)NCCCOCCOCCOCCCNC(=O)CCCCC[N+]=3C4=CC(N)=CC=C4C4=CC=C(N)C=C4C=3C=3C=CC=CC=3)C=1C=2C1=CC=CC=C1 JGBUYEVOKHLFID-UHFFFAOYSA-N 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 230000004941 influx Effects 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000007480 spreading Effects 0.000 description 2
- 238000003892 spreading Methods 0.000 description 2
- 239000012128 staining reagent Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000001551 total correlation spectroscopy Methods 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- QWXZOFZKSQXPDC-NSHDSACASA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C)C(O)=O)C3=CC=CC=C3C2=C1 QWXZOFZKSQXPDC-NSHDSACASA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- CEIIEALEIHQDBX-UHFFFAOYSA-N 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methyl-3-isoxazolyl)urea Chemical compound C1=C(Cl)C(OC)=CC(OC)=C1NC(=O)NC1=NOC(C)=C1 CEIIEALEIHQDBX-UHFFFAOYSA-N 0.000 description 1
- 238000001026 1H--1H correlation spectroscopy Methods 0.000 description 1
- 238000000362 1H--1H nuclear Overhauser enhancement spectroscopy Methods 0.000 description 1
- 238000012593 1H–1H TOCSY Methods 0.000 description 1
- 241000242759 Actiniaria Species 0.000 description 1
- 241000239290 Araneae Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 101710176384 Peptide 1 Proteins 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 108010038988 Peptide Hormones Proteins 0.000 description 1
- 102000015731 Peptide Hormones Human genes 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 241000239226 Scorpiones Species 0.000 description 1
- 241000270295 Serpentes Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 125000003158 alcohol group Chemical group 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 231100000659 animal toxin Toxicity 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 108010055460 bivalirudin Proteins 0.000 description 1
- OIRCOABEOLEUMC-GEJPAHFPSA-N bivalirudin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)CNC(=O)CNC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 OIRCOABEOLEUMC-GEJPAHFPSA-N 0.000 description 1
- 229960001500 bivalirudin Drugs 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 239000008139 complexing agent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 125000000392 cycloalkenyl group Chemical group 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 125000002228 disulfide group Chemical group 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000007709 intracellular calcium signaling Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000004001 molecular interaction Effects 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000000813 peptide hormone Substances 0.000 description 1
- 108700010839 phage proteins Proteins 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000003123 plant toxin Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 108010087782 poly(glycyl-alanyl) Proteins 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 108010054442 polyalanine Proteins 0.000 description 1
- 108010094020 polyglycine Proteins 0.000 description 1
- 229920000232 polyglycine polymer Polymers 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 238000012483 real time interaction analysis Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 210000002265 sensory receptor cell Anatomy 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000003696 structure analysis method Methods 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 238000002076 thermal analysis method Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- BPKIMPVREBSLAJ-QTBYCLKRSA-N ziconotide Chemical compound C([C@H]1C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]2C(=O)N[C@@H]3C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CSSC2)C(N)=O)=O)CSSC[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CSSC3)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(N1)=O)CCSC)[C@@H](C)O)C1=CC=C(O)C=C1 BPKIMPVREBSLAJ-QTBYCLKRSA-N 0.000 description 1
- 229960002811 ziconotide Drugs 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
A phage display cyclopeptide library constructed based on a natural cyclopeptide backbone is provided. The present invention relates to a phage displayed cyclic peptide library, wherein the cyclic peptide comprises an amino acid sequence shown by X pCC-Xn-C-Xm-CXq, wherein p = 0-10, q = 0-10, n = 4 and m = 4-16, X is any of 20 natural amino acids, said cyclic peptide library comprising a cyclic peptide having two disulfide bonds. According to the method, a strategy of fixing the position of cysteine and embedding random amino acids is adopted according to a conserved conotoxin skeleton, so that phage capable of displaying cyclic peptides is generated, more target proteins are targeted, and the cyclic peptide ligand with high affinity and high selectivity is screened out.
Description
Technical Field
The application relates to the field of polypeptide libraries, in particular to constructing phage display cyclopeptides libraries based on natural cyclopeptides backbones.
Background
Polypeptides are an important class of bioactive molecules, and are widely available in nature, including polypeptide components derived from animal and plant venom, polypeptide hormones secreted by human bodies, and more artificially designed and synthesized polypeptides. These polypeptides act primarily by modulating effector proteins of the organism. Because polypeptides do not penetrate cell membranes well, proteins located on the surface of cell membranes become the primary targets for polypeptides, including ion channels, G-protein coupled receptors (GPCRs), transport proteins, and the like.
Compared with small molecule compounds, the sequence and structure of the polypeptide are more complex, and the functions of the polypeptide are more various. More complex chemical compositions and larger molecular weights, polypeptides also exhibit more excellent target selectivity and affinity than small molecules. Based on these advantages, polypeptides are widely used as pharmacological tools to study the structure and function of target proteins; also as molecular probes, it is important to study the key node Σ of intracellular signal pathways, and polypeptides are drug candidate molecules that are now of increasing interest.
The polypeptide is taken as an important candidate molecule for drug development, a plurality of drugs based on the polypeptide development are approved to be marketed at present, including artificial designed enfuwei peptide, bivalirudin and ziconotide derived from animal toxin, rope Ma Lutai based on human hormone development and the like, and the polypeptide has important application and commercial value in the treatment of different diseases.
Cyclic peptides have three major advantages over linear peptides: the cyclopeptide has higher affinity with a macromolecular target; the targeting selectivity of the cyclic peptide is better, and similar proteins with the same tertiary structure and high sequence homology can be distinguished; cyclic peptides are more stable than linear peptides.
However, the source of cyclic peptides generated in nature is very limited, so that the source of cyclic peptides is widened, and the diversity of cyclic peptide ligands is enriched.
Thus, there is a need in the art to develop more polypeptide libraries, such as phage-displayed polypeptide libraries, for de novo screening for desired cyclopeptide ligands.
Disclosure of Invention
The inventors have found that biotoxins are very valuable drugs or drug-directed compounds, with conotoxins being a new area of rapid development in biotoxins. However, the source of toxins produced in nature is very limited, and it is necessary to widen the source of cyclopeptide toxins and enrich the diversity of cyclopeptide ligands. The inventor hopes to generate phage capable of displaying cyclic peptide according to a strategy of fixing the position of cysteine and embedding random amino acid according to a conserved conotoxin skeleton, thereby targeting more target proteins and screening out the cyclic peptide ligand with affinity and selectivity. According to the conservation of conotoxin polypeptide skeleton, the screened cyclopeptide ligand has a predictable three-dimensional structure.
Phage display technology, i.e., the display of polypeptides or proteins on the surface of phage, is an in vitro screening technology that is capable of extracting desired polypeptides from a large library of polypeptides, and has become an extremely powerful tool in drug discovery and development as a basic research method. For example, the Chinese patent application publication number is CN111945231A, the application date is 2020 and 25 is 08, the application name is "method for constructing phage display multi-element cyclopeptide library based on disulfide bond accurate pairing", and discloses a method for constructing phage display multi-element cyclopeptide library based on disulfide bond accurate pairing, which relates to cyclopeptide compounds. The application discloses a brand-new cyclopeptide skeleton different from the prior patent, which is one of a few strategies for phage display cyclopeptide screening at present.
In one aspect, the invention provides a cyclic peptide library, wherein the cyclic peptide comprises an amino acid sequence shown by X pC1C2-Xn-C3-Xm-C4Xq, wherein p = 0-10, q = 0-10, n = 4 and m = 4-16, X is any one of 20 natural amino acids, the cyclic peptide library comprises a cyclic peptide having two disulfide bonds comprising a disulfide bond formed by cysteine C 1 with cysteine C 3 and a disulfide bond formed by cysteine C 2 with cysteine C 4, and/or the disulfide bond comprises a disulfide bond formed by cysteine C 1 with cysteine C 4 and a disulfide bond formed by cysteine C 2 with cysteine C 3. The cyclic peptide library may comprise disulfide bonds formed by any two cysteines (C1-C2, C1-C3, C1-C4, C2-C3, C2-C4, or C3-C4). The number of disulfide bonds may be 1 or 2. Linear peptides may also be included in the cyclic peptide library. The cyclic peptide library may be a phage-displayed cyclic peptide library. Preferably, X is any amino acid other than cysteine.
In one embodiment, p=0-10 or 2-6, e.g. p may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
In one embodiment, q=0-10 or 2-6, e.g. q may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
In one embodiment, m=4-16 or 6-8, e.g. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.
In one embodiment, X is any one of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, cysteine, tyrosine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.
In one embodiment, the phage-displayed cyclic peptide library is a single-chain filamentous phage display system, lambda phage display system, T4 phage display system, or T7 phage display system-displayed cyclic peptide library.
In one embodiment, the single-chain filamentous phage display system is a PIII display system or PVIII display system.
In one embodiment, the lambda phage display system is a PV display system or a protein D display system.
In one embodiment, a cyclic peptide library, preferably a phage-displayed cyclic peptide library, is provided, wherein the cyclic peptide comprises an amino acid sequence shown at X1X2C1C2X3X4X5X6C3X7X8X9X10X11X12X13C4, X 1-X13 is any of the 20 natural amino acids. in one embodiment, the phage-displayed cyclic peptide library comprises cyclic peptides, Wherein two disulfide bonds (C1-C3/C2-C4) are formed between cysteines C 1 and C 3 and between cysteines C 2 and C 4, Or two disulfide bonds (C1-C4/C2-C3) are formed between cysteines C 1 and C 4 and between cysteines C 2 and C 3. To facilitate ligation of the cyclic peptide backbone to the phage vector, a linker sequence (such as G or a) comprising one or more (e.g., 2-10,3, 4, 5, 6, 7, 8, 9) amino acid residues may be added to the C-terminus of the cyclic peptide backbone. The amino acid sequence may have a linker at the C-terminus. In one embodiment, the joint is a flexible joint or a rigid joint. In one embodiment, the linker comprises 1-15, 1-12, 1-10, or 1-5 amino acid residues, e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues. Preferably, the linker comprises (G) n1 or (A) n2, wherein n 1 and n 2 are 1-15, 1-12, 1-10 or 1-5, for example each independently is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
For example, the cyclic peptide may comprise X1X2C1C2X3X4X5X6C3X7X8X9X10X11X12X13C4(G/A), wherein (G/a) represents an optional linker residue G or a. In one embodiment, the phage-displayed cyclic peptide library is produced in a host cell, such as an E.coli cell. There may be cyclic peptides in which X1X2C1C2X3X4X5X6C3X7X8X9X10X11X12X13C4 amino acids may form disulfide bonds (e.g., C1-C2,C1-C3、C1-C4、C2-C3、C2-C4、C3-C4 linkages) two by two between cysteines C 1-C4(C1、C2、C3 and C 4. In one embodiment, a phage-displayed cyclopeptide library according to the present invention is provided comprising a cyclopeptide having an amino acid sequence of X1X2C1C2X3X4X5X6C3X7X8X9X10X11X12X13C4 or X1X2C1C2X3X4X5X6C3X7X8X9X10X11X12X1 3C4(G/A).
In one embodiment, X 1-X13 is any one of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, cysteine, serine, tyrosine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.
In one embodiment, the phage-displayed cyclic peptide library has one or more of the following properties: each of the plurality of cyclic peptides is present in the cyclic peptide library in an equal or substantially equal percentage; the cyclic peptide library capacity is at least 1X 10 9.
In one embodiment, the cyclic peptide library is prepared by a method comprising plasmid PCR and a self-ligation step; wherein plasmid PCR is performed on a phage vector using primers, and the PCR product is digested and ligated into the phage vector, wherein the phage vector is a pCANTAB 5E phage vector. In one embodiment, the pCANTAB 5E phage vector is a pCANTAB 5E phage vector comprising a S240 site base mutation (TCG). The pcatab 5E phage vector may contain an S240L mutation. In one embodiment, the primer comprises SEQ ID NO 9 and SEQ ID NO 10.
In one embodiment, the method comprises introducing a phage vector into a host cell, and culturing the host cell comprising the phage vector to log phase with the addition of helper phage, separating after culturing to obtain a supernatant, separating phage precipitate from the supernatant, preferably by adding a solution of PEG and NaCl to the supernatant, preferably such that 1/5 of the volume of PEG and NaCl is added. Preferably, the PEG is PEG-8000. Preferably, 1-5M, for example 2.5M NaCl is added. Preferably, the host cell is an E.coli cell.
The method may include treating the phage under conditions suitable for formation of cysteines to form disulfide bonds (e.g., oxidizing conditions) to promote disulfide bond formation.
In another aspect, the invention provides a method of preparing a phage-displayed cyclic peptide library comprising displaying the cyclic peptide library by a phage display system.
In one embodiment, the phage-displayed cyclic peptide library is a single-chain filamentous phage display system, lambda phage display system, T4 phage display system, or T7 phage display system-displayed cyclic peptide library.
In one embodiment, the single-chain filamentous phage display system is a PIII display system or PVIII display system.
In one embodiment, the lambda phage display system is a PV display system or a protein D display system.
In one embodiment, the PIII display system is the pcatab 5E display system.
In one embodiment, the phage display system uses a pCANTAB 5E phage vector, preferably a pCANTAB 5E phage vector comprising an S240 site base mutation TCG.
In one embodiment, the method comprises plasmid PCR and a self-ligation step; wherein plasmid PCR is performed on a phage vector using primers, and the PCR product is digested and ligated into the phage vector, wherein the phage vector is a pCANTAB 5E phage vector. In one embodiment, the pCANTAB 5E phage vector is a pCANTAB 5E phage vector comprising a S240 site base mutation (TCG). In one embodiment, the primer comprises SEQ ID NO 9 and SEQ ID NO 10.
In one embodiment, the method further comprises introducing a phage vector into the host cell, and adding a helper phage when the host cell comprising the phage vector is cultured to log phase, separating after culturing to obtain a supernatant, separating phage precipitate from the supernatant, preferably by adding a solution of PEG and NaCl to the supernatant, preferably such that 1/5 volume of PEG and NaCl is added. Preferably, the PEG is PEG-8000. Preferably, 1-5M, for example 2.5M NaCl is added.
In another aspect, the invention provides a method of screening for a target protein comprising
(1) Adding the phage-displayed cyclic peptide library of claim 1 to a target protein coated well plate, washing the plate after incubation, and eluting phage bound to the target protein;
(2) Optionally amplifying the eluted phage and contacting the amplified phage with a target protein coated well plate, washing the plate, and eluting phage that bind to the target protein;
(3) Optionally repeating steps (1) - (2) one or more times;
(4) Optionally sequencing the eluted phage surface displayed polypeptides, determining abundance from sequencing results and ordering according to abundance, and selecting phage with high abundance.
In one embodiment, the target protein is selected from the group consisting of ion channels, G Protein Coupled Receptors (GPCRs), transport proteins, α7 nicotinic acetylcholine receptor (α7 nAChR) proteins.
In one embodiment, the well plate is a multi-well plate, such as a 96-well plate.
Herein, the target protein may be any kind of protein, such as a cell surface protein or a membrane integrating protein, such as a receptor, etc. In one embodiment, the target protein may be an ion channel, a G Protein Coupled Receptor (GPCR), a transporter, an α7nAChR protein.
In yet another aspect, a cyclic peptide is provided comprising one or more of the following sequences: SEQ ID NOS 1-8 or 32-39 or variants thereof.
In one embodiment, the variant comprises an amino acid sequence that is mutated by 1-4 amino acid residues compared to any of SEQ ID NOs 1-8 or 32-39 or an amino acid sequence that has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of SEQ ID NOs 1-8 or 32-39.
In one embodiment, the amino acids at positions corresponding to positions 3 and 9 and the amino acids at positions 4 and 17 of any one of SEQ ID NOS 1-8 or 32-39 are linked by a bond, or the amino acids at positions 3 and 17 and the amino acids at positions 4 and 9 of any one of SEQ ID NOS 1-8 or 32-39 are linked by a bond. In one embodiment, the amino acids at positions corresponding to positions 3 and 9 and the amino acids at positions 4 and 17 of any one of SEQ ID NOS 1-8 or 32-39 are covalently linked in a side chain to side chain. In one embodiment, the amino acids at positions corresponding to positions 3 and 17 and the amino acids at positions 4 and 9 of any one of SEQ ID NOS 1-8 or 32-39 are covalently linked in a side chain to side chain.
In one embodiment, the bond comprises one or more atoms of carbon, sulfur, oxygen, nitrogen, or selenium.
In one embodiment, the bond is -S-S-、-S-O-、-S-Se-、-O-Se-、-O-(CH2)n3-、-S-(CH2)n3-、-Se-(CH2)n3-、-(CH2)n3-、-O-(NH)n3-、-S-(NH)n3-、-Se-(NH)n3-、 or- (NH) n3 -, where n 3 = 1-6, e.g. 1,2, 3, 4 or 5.
In one embodiment, the variant retains the original cysteine of any of SEQ ID NOs 1-8 or 32-39 and the mutated amino acid is not a cysteine residue. In one embodiment, two disulfide bonds are formed between the cysteines at positions corresponding to positions 3 and 9 and between the cysteines at positions 4 and 17 of any one of SEQ ID NOS 1-8 or 32-39, or two disulfide bonds are formed between the cysteines at positions 3 and 17 and between the cysteines at positions 4 and 9 of any one of SEQ ID NOS 1-8 or 32-39.
In one embodiment, the mutation is a substitution, addition, deletion or insertion. In one embodiment, the mutation is a conservative amino acid substitution.
In one embodiment, the amino acid sequence of the cyclic peptide is SEQ ID NO. 1 or 32, wherein two disulfide bonds are formed between the cysteines at positions 3 and 17 and between the cysteines at positions 4 and 9.
In yet another aspect, a method of preparing a cyclic peptide is provided, comprising preparing a linear peptide from an amino acid sequence comprising SEQ ID NOs 1-8 or 32-39 or variants thereof and chemically cyclizing the linear peptide to a cyclic peptide; alternatively, the polynucleotide encoding the amino acid sequence is introduced into a host cell and expressed, and then the cell supernatant or lysate comprising the cyclic peptide is harvested, optionally purifying the cyclic peptide.
The advantages of the invention include:
(1) The cyclic peptide library of the present invention is a novel phage display cyclic peptide library comprising cyclic peptides (e.g., the amino acid sequence shown by X pC1C2-Xn-C3-Xm-C4Xq, wherein p=0-10, q=0-10, n=4 and m=4-16) having two disulfide bonds linked at specific linkage positions (disulfide bond formed by cysteine C1 and cysteine C3 and disulfide bond formed by cysteine C2 and cysteine C4, and/or disulfide bond comprising disulfide bond formed by cysteine C1 and cysteine C4). The inventor proves that the cyclic peptide library can be used for screening the bicyclic peptide for inhibiting the activity of the alpha 7 type acetylcholine receptor;
(2) The present invention provides a cyclic peptide library having the characteristic sequence that the 1 st and 2 nd (C1 and C2) cysteines are directly adjacent, the 2 nd cysteine is separated from the 3 rd cysteine (C2 and C3) by 4 amino acid residues, and the 3 rd cysteine is separated from the 4 th cysteine (C3 and C4) by 4-16, preferably 4-9 amino acid residues.
(3) The invention also provides a cyclic peptide with a simple polypeptide sequence and only containing 17 or 18 (with a single amino acid residue joint) amino acids, and the preparation is convenient.
(4) The library preparation of the invention is efficient and convenient, and can construct phage display library by means of commercial DNA synthesis and plasmid construction methods.
(5) The invention also provides a cyclic peptide library which comprises double-ring peptides constructed based on conotoxin skeletons, and two pairing modes of C3-C9/C4-C17 and C3-C17/C4-C9 exist. These cyclic peptides are well-defined in conformation.
(6) The invention proves that the screening success rate of the cyclopeptide library of the invention against target proteins is high. The application of the double-ring peptide library can successfully screen and obtain a plurality of double-ring peptides with the activity of inhibiting the alpha 7-type acetylcholine receptor.
(7) The present invention provides a plurality of bicyclic peptides having inhibitory activity against the alpha 7 acetylcholine receptor.
Drawings
FIG. 1 shows the DNA gel identification of PCR products.
FIG. 2 shows the results of DNA purification after cleavage.
Figure 3 shows the chromatographic and mass spectral characterization of polypeptides after renaturation following 17AA cyclic peptide library screening.
Fig. 4 shows a concentration response curve of cyclic peptide KP2002-KP2009 inhibiting the activity of the α7-type acetylcholine receptor.
Figure 5 shows nuclear magnetic resonance structure of cyclic peptide KP2007 with highest inhibitory activity.
Figure 6 shows HPLC purification and mass spectrometry profiles of KP1794, KP1795 and KP1796 polypeptides.
FIG. 7 shows concentration response curves for inhibition of alpha 7 type acetylcholine receptor activity by KP1794, KP1795 and KP1796 polypeptides.
Figure 8 shows HPLC purification and mass spectrometry detection patterns of KP1877 polypeptides.
FIG. 9 shows concentration response curves for KP1877 polypeptide inhibiting the activity of the α7-type acetylcholine receptor.
FIG. 10 shows nuclear magnetic resonance structural diagrams of KP1877 polypeptide.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Cyclic peptides: cyclic peptides of cyclic structure have a conformational limitation compared to linear peptides, and have less entropy loss when bound to the target and higher binding affinity. Reduced structural flexibility of the polypeptide also stabilizes the specific structure of the cyclic peptide, increasing the specificity of binding to the target. Cyclic peptides have three major advantages over linear peptides: the cyclopeptide has higher affinity with a macromolecular target; the targeting selectivity of the cyclic peptide is better, and similar proteins with the same tertiary structure and high sequence homology can be distinguished; cyclic peptides are more stable than linear peptides. Cyclic peptides bind to protein targets with high affinity and Gao Babiao specificity, an attractive molecular species for the development of therapeutic drugs.
Natural amino acids: the basic amino acids constituting the protein are 20, including glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.
Phage display technology: the encoding gene or target gene fragment of polypeptide or protein is cloned into proper position of phage coat protein structure gene, and under the condition of correct reading frame and no influence on normal functions of other coat proteins, the exogenous polypeptide or protein and coat protein are fused and expressed, and the fusion protein is displayed on phage surface along with the recombination of progeny phage. The displayed polypeptides or proteins may maintain relatively independent spatial structures and biological activities to facilitate recognition and binding of target molecules. After the peptide library and the target protein-loaded molecules on the solid-phase medium are incubated for a certain time, unbound free phage is washed away, then the phage which is bound and adsorbed with the target molecules is eluted by competing receptors or acid, the eluted phage infects host cells, is propagated and amplified, and is eluted in the next round, and after 3-5 rounds of adsorption-elution-amplification, the phage which is specifically bound with the target molecules is highly enriched. The resulting phage preparation can be used to further enrich target phage with desired binding properties. The pCANTAB 5E phage display system is used herein, and is composed of a phagemid vector (pCANTAB 5E), a host bacterium (E.coli TG1, HB2151, etc.), helper phage M13K07, etc.
Host bacteria: the host bacteria in the pCANTAB 5E system include two species of Escherichia coli TG1 and Escherichia coli HB 2151. Infection of the phagemid with two host bacteria will result in two different products, and infection with E.coli TG1 will ultimately result in polypeptide-pIII fusion proteins.
Helper phage: the M13K07 helper phage is an important component of the pCANTAB 5E phage display system. The phage must be helper phage involved in smooth passage after transfer into the host cell.
Efficient way of directly screening proteins or polypeptides by phage display technology. Whether or not a target protein screen is successful depends on the size and diversity of the library screened, but generating large phage-encoded polypeptide libraries remains challenging. In recent years, newly developed polypeptide phage display libraries have complicated steps, complex procedures, low product efficiency and low library diversity. The present study established a library generation strategy based on whole plasmid PCR and self-ligation, with diversity of greater than 5 x 10 9. The peptide library sequence is introduced into the vector through the PCR primer, and the efficient DNA self-ligation is realized through terminal ligation, so that the difficulty and the complicated steps of inserting the DNA fragments into the vector are simplified, and a huge library is obtained. The high affinity ligand was obtained against the target screening peptide library, validating the quality of the library and thus validating the new library generation strategy. This simple and efficient strategy allows researchers to obtain larger libraries, thereby increasing the probability of ligands being screened.
Conotoxin: a large class of natural polypeptides in the conotoxin liquid of marine animals has the outstanding characteristics of high abundance, chemical diversity, strong specificity and the like, and has become a treasury for the development of innovative medicaments. Conotoxins act on many ion channels and neuroreceptors and are widely used in life sciences research. Conotoxins are generally composed of 10-30 amino acid residues, and currently known are the major superfamilies of α -, μ -, ω -, etc., which are cysteine-rich and have a highly conserved disulfide backbone. Compared with toxins of a plurality of animals such as spiders, scorpions, snakes, sea anemones and the like, the conotoxins have short peptide chains, rich disulfide bonds, more compact molecular structures and high biological activity. The invention is based on a strategy of conserved conotoxin skeleton, fixing the position of cysteine and embedding random amino acid, and generates phage capable of displaying cyclic peptide, thereby targeting more target proteins and screening out the cyclic peptide ligand with affinity and selectivity.
Target protein: refers to the protein against which the phage-displayed library is screened. The target protein may be an extracellular or intracellular component, a soluble factor (e.g., an enzyme, hormone, cytokine, growth factor, antibody, etc.), or a transmembrane protein (e.g., a cell surface receptor), etc.
And (3) joint: refers to a peptide or other chemical linkage that functions to link a polypeptide or cyclic peptide to a phage protein (e.g., coat protein pIII). Suitable linkers include, but are not limited to, polypeptide linkers, such as glycine linkers, serine linkers, mixed glycine/serine linkers, glycine and serine rich linkers, or linkers composed primarily of polar polypeptide fragments. The linker comprises an amino acid selected from the group consisting of: glycine, alanine, proline, asparagine, glutamine and lysine. For example, the linker comprises an amino acid selected from glycine, serine and/or alanine. In some embodiments, the peptide linker is selected from the group consisting of poly glycine (such as (Gly) 1-8, poly (Gly-Ala), and poly alanine (Ala) 1-8.
A cyclic peptide or polypeptide that binds to a target protein: the characteristics of binding to a target protein may be determined using assays, bioassays, and/or animal models known in the art for assessing the activity of a cyclic peptide or polypeptide for binding to a target protein. For example, the identity of a ligand can be measured directly by determination of the affinity constant (e.g., the amount of ligand that associates and dissociates at a given target protein concentration). There are several methods available for characterizing such molecular interactions, for example, competition analysis, equilibrium analysis and micro-thermal analysis, as well as real-time interaction analysis based on surface plasmon resonance interactions (e.g., using a BIACORE instrument). These methods are well known to those skilled in the art. In this context, the cyclic peptide or polypeptide that binds to the target protein is selected by determining the proportion of clones in the screening hits to the total number of clones. The hit high abundance cyclic peptides or polypeptides can be further assayed for activity at the cellular level (e.g., inhibitory activity against α7 nachrs).
Sequence identity: the degree of relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity". For the purposes of the present invention, sequence identity between two amino acid sequences is determined using the Nidlmann-Wen application algorithm (Needleman and Wunsch,1970, J.mol. Biol. [ J. Mol. J. Mol. 48:443-453) as implemented in the Nidel program of the EMBOSS software package (EMBOSS: european molecular biology open software suite, rice et al 2000,Trends Genet, [ genetics trend ]16:276-277, preferably version 5.0 or more). The parameters used are gap opening penalty of 10, gap extension penalty of 0.5, and EBLOSUM62 (the emoss version of BLOSUM 62) substitution matrix. The output of the nitel labeled "longest identity" (obtained using the non-simplified option) was used as the percent identity and calculated as follows:
(identical residues×100)/(alignment length-total number of gaps in the alignment).
Amino acid conservative substitutions: may be defined by substitutions within the amino acid categories reflected in one or more of the following tables:
Amino acid residues of conserved class:
Acidic residues D and E
Basic residues K, R and H
Hydrophilic uncharged residues S, T, N and Q
Aliphatic uncharged residues G, A, V, L and I
Nonpolar uncharged residues C, M and P
Aromatic residues F, Y and W.
Physical and functional classification of alternative amino acid residues:
residues S and T containing alcohol groups
Aliphatic residues I, L, V and M
Cycloalkenyl related residues F, H, W and Y
Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W and Y
Negatively charged residues D and E
Polar residue C, D, E, H, K, N, Q, R, S and T
Positively charged residues H, K and R
Small residues A, C, D, G, N, P, S, T and V
Very small residues A, G and S
Residues A, C, D, E, G, H, K, N, Q, R, S, P and T involved in corner formation
Flexible residues Q, T, K, S, G, P, D, E and R.
Phage display cyclic peptide library
The inventors have found a novel library of phage-displayed cyclic peptides in long-term work. The cyclic peptide may comprise a primary structure of an amino acid sequence shown by X pC1C2-Xn-C3-Xm-C4Xq, where p=0-10, q=0-10, n=4 and m=4-16, and X is any of 20 natural amino acids. The cyclic peptide library may comprise disulfide bonds formed by any two cysteines (C1-C2, C1-C3, C1-C4, C2-C3, C2-C4, or C3-C4). The number of disulfide bonds may be1 or 2. Linear peptides may also be included in the cyclic peptide library. In particular, the cyclic peptide library should contain cyclic peptides which may have two disulfide bonds comprising a disulfide bond formed by cysteine C1 with cysteine C3 and a disulfide bond formed by cysteine C2 with cysteine C4, and/or which comprises a disulfide bond formed by cysteine C1 with cysteine C4 and a disulfide bond formed by cysteine C2 with cysteine C3. There may be a4 amino acid residue interval between cysteine C2 and cysteine C4 in the above amino acid sequences.
The amino acid sequence of the primary structure of the cyclic peptide is not particularly limited in length, but it is preferable that the amino acid sequence is 12 to 50, 13 to 30, 14 to 20, or 15 to 18 amino acid sequences in length. Thus, there may be some variation in the p and q amino acid sequences, provided that the overall length of the sequence meets the requirements set forth above. For example, p and q in the amino acid sequence may independently be 1-8 (e.g., 1,2,3,4,5,6, 7, or 8), and m may be 5-9 (5, 6, 7,8, or 9).
The cyclic peptide libraries herein may be displayed by any suitable phage display system. For example, the phage display system may comprise a single-chain filamentous phage display system, a lambda phage display system, a T4 phage display system, or a cyclic peptide library displayed by a T7 phage display system. The cyclic peptide libraries herein may also be obtained by combining cyclic peptide libraries displayed by two or more display systems. For example, the single-stranded filamentous phage display system is a PIII display system or PVIII display system; the lambda phage display system is a PV display system or a protein D display system.
The amino acid sequence of the cyclic peptide has a linker at the N-terminus or C-terminus, preferably at the C-terminus, to link the displayed cyclic peptide to phage. The type of joint may be not particularly limited, and may be a flexible joint or a rigid joint. For example, a linker may comprise 1-15, 1-12, 1-10, or 1-5 amino acid residues. Preferably, the linker may comprise (G) n1 or (A) n2, where n 1 and n 2 are 1-15, 1-12, 1-10, or 1-5. In this context, the cyclic peptide may be linked to the protein of the phage by a single amino acid G/A to display the cyclic peptide on the phage surface.
The type of amino acid residue used in the cyclic peptide is not particularly limited, and may be any amino acid that can be incorporated into the cyclic peptide, and may be, for example, a naturally occurring amino acid such as any one of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, cysteine, tyrosine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.
Provided herein are phage-displayed cyclic peptide libraries comprising a plurality of cyclic peptides. The cyclic peptide may comprise the amino acid sequence shown at X1X2C1C2X3X4X5X6C3X7X8X9X10X11X12X13C4, X 1-X13 is any one of the amino acids other than cysteine and forms two disulfide bonds (i.e., C1-C3/C2-C4) between cysteines C1-C3 and between cysteines C2-C4, and/or forms two disulfide bonds (C1-C4/C2-C3) between cysteines C1-C4 and between cysteines C2-C3. X 1-X13 may be random amino acid residues, so that each of the plurality of cyclic peptides may be present in the cyclic peptide library in approximately equal percentages or fractions. For example, each of X 1-X13 can independently be any of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, cysteine, tyrosine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine. The phage-displayed cyclic peptide library described herein has one or more of the following properties: each of the plurality of cyclic peptides is present in the cyclic peptide library in an equal or substantially equal percentage; the cyclic peptide library capacity is at least 1X 10 9. The cyclopeptide pool capacity may represent the diversity of the cyclopeptide pool, referring to the number of different cyclopeptides in the cyclopeptide pool. A linker sequence (such as G or a) comprising one or more amino acid residues may be added at the C-terminus of the cyclic peptide backbone.
The cyclopeptide pool capacity is at least 1×10 9, for example 2×109、3×109、4×109、5×109、6×109、7×109、8×109、9×109、1×1010、2×1010、3×1010、4×1010、5×1010、6×1010、7×1010、8×1010、9×1010、1×1011、2×1011、3×1011、4×1011、5×1011、6×1011、7×1011、8×1011、9×1011、1×1012、2×1012、3×1012、4×1012、5×1012、6×1012、7×1012、8×1012 or 9×10 12.
The phage-displayed cyclic peptide library herein can be prepared by any conventional technique. In particular, phage-displayed cyclic peptide libraries herein can be generated by the preparation methods described below.
Preparation method of phage display cyclic peptide library
The cyclic peptide libraries described herein can be displayed by any suitable phage display system. For example, the phage-displayed cyclic peptide library may be a single-chain filamentous phage display system, a lambda phage display system, a T4 phage display system, and/or a T7 phage display system-displayed cyclic peptide library. The single-chain filamentous phage display system may be a PIII display system or PVIII display system. The lambda phage display system may be a PV display system or a protein D display system. Particularly preferred display systems herein are PIII display systems, such as the pcatab 5E display system. For example, phage display systems may use a pcatab 5E phage vector, preferably a pcatab 5E phage vector comprising an S240 site base mutation TCG;
More specifically, provided herein are methods of preparing phage-displayed cyclic peptide libraries, which may include plasmid PCR and self-ligation steps. Plasmid PCR can be performed on a phage vector using the primers of SEQ ID NO. 9 and SEQ ID NO. 10, and the PCR product is digested and ligated into the phage vector, wherein the phage vector is a pCANTAB 5E phage vector. In one embodiment, the pCANTAB 5E phage vector is a pCANTAB 5E phage vector comprising a S240 site base mutation (TCG).
SEQ ID NO:9：
TTTGGTCTCGGTGCGCCGGTGCCGTATCCGGATCCGCTG；
SEQ ID NO:10：
TTTGGTCTCAGCACCACCACCGCAMNNMNNMNNMNNMNNMNN MNNGCAMNNMNNMNNMNNGCAGCAMNNMNNACCGGCCATGGCCG GCTGGGCCGCATAG, Wherein N represents any one base in T/A/G/C, and M represents any one base in T/G.
In one embodiment, the method further comprises introducing a phage vector into the host cell, and adding a helper phage when the host cell comprising the phage vector is cultured to the logarithmic growth phase. After the addition of helper phage, cells can be separated from the supernatant after culturing. Phage pellet can be isolated from the supernatant. Preferably, PEG and NaCl may be added to the supernatant. Preferably, 1/5 volume of PEG and NaCl are added. Preferably, the PEG is PEG-8000. Preferably, 1-5M, for example 2.5M NaCl is added.
The present invention also encompasses phage populations produced by the above methods.
Screening method
The present invention provides a method of screening for cyclic peptides targeting a protein of interest comprising:
(1) Adding a phage-displayed cyclic peptide library according to the description to a target protein coated well plate, incubating the plate, and eluting phage bound to the target protein;
(2) Optionally amplifying the eluted phage and contacting the amplified phage with a target protein coated well plate, washing the plate, and eluting phage that bind to the target protein;
(3) Optionally repeating steps (1) - (2) one or more times;
(4) Optionally sequencing the eluted phage surface displayed polypeptides, determining abundance from sequencing results and ordering according to abundance, and selecting high abundance phages. Abundance refers to the proportion of polypeptides displayed on the surface of a selected phage to the total number of polypeptides selected.
The methods herein can use phage-displayed cyclic peptides to screen for binding agents (e.g., inhibitors or antagonists) to a target protein. The type of target protein described herein may be without particular limitation. The target protein may be selected from ion channels, G Protein Coupled Receptors (GPCRs), transport proteins, α7 nicotinic acetylcholine receptor (α7 nAChR) proteins.
In particular, the method may include the steps of adding a library of phage-displayed cyclic peptides as described herein to a plate (e.g., 96-well plate) coated with a protein of interest, incubating the plate, washing the plate after incubation, and eluting phage that bind to the protein of interest, the step being performed one or more times. The protein of interest may be any suitable protein, such as an α7nAChR protein. The method may further comprise amplifying and sequencing the eluted phage. The method may further comprise determining abundance from sequencing results and ordering according to abundance. The method may further comprise selecting a phage (clone) in high abundance. The above steps can be repeated for the selected phage (clone).
The screening methods of the invention can obtain a variety of cyclic peptides of interest, such as those directed against the α7 nachrs. The cyclic peptide may be a cyclic peptide as described herein.
Cyclic peptides
The inventors can obtain multiple cyclic peptides against a target (e.g., α7 nachrs) through phage-displayed cyclic peptide screening of the present invention. Such cyclic peptides may be binding agents, e.g., inhibitors or antagonists, of the target protein.
The cyclic peptide may comprise one or more of the following sequences: 1-8 or 32-39 or a variant thereof comprising an amino acid sequence that is mutated by 1-4 amino acid residues compared to any one of SEQ ID NOs 1-8 or 32-39 or an amino acid sequence that has 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs 1-8 or 32-39. The amino acids corresponding to positions 3 and 9 and the amino acids corresponding to positions 4 and 17 of any one of SEQ ID NOS.1 to 8 or 32 to 39 may be linked by any suitable bond, or the amino acids corresponding to positions 3 and 17 and the amino acids corresponding to positions 4 and 9 may be linked by any suitable bond. For example, the amino acids at positions 3 and 9 and the amino acids at positions 4 and 17 of any one of SEQ ID NO 1-8 or 32-39 are covalently linked in a side chain to a side chain, or the amino acids at positions 3 and 17 and the amino acids at positions 4 and 9 of any one of SEQ ID NO 1-8 or 32-39 are covalently linked in a side chain to a side chain. The type of linkage is not particularly limited as long as two amino acid residues can be covalently linked. The bond may comprise one or more atoms of carbon, sulfur, oxygen, nitrogen or selenium. For example, the bond is -S-S-、-S-O-、-S-Se-、-O-Se-、-O-(CH2)n3-、-S-(CH2)n3-、-Se-(CH2)n3-、-(CH2)n3-、-O-(NH)n3-、-S-(NH)n3-、-Se-(NH)n3-、 or- (NH) n3 -, where n 3 =1-6, e.g. 1, 2,3, 4 or 5. Preferably, the variant retains the original cysteine in any of SEQ ID NOs 1-8 or 32-39 and the mutated amino acid is not a cysteine residue. More preferably, two disulfide bonds are formed between the cysteines at positions corresponding to positions 3 and 9 and between the cysteines at positions 4 and 17 of any one of SEQ ID NOS 1-8 or 32-39, or two disulfide bonds are formed between the cysteines at positions 3 and 17 and between the cysteines at positions 4 and 9.
It is also preferred that the variant retains the original cysteine of any of SEQ ID NOS 1-8 or 32-39, and that the mutated amino acid is not a cysteine residue, and that two disulfide bonds are formed between the 1 st and 3 rd cysteines and between the 2 nd and 4 th cysteines from the N-terminus, or two disulfide bonds are formed between the 1 st and 4 th cysteines and between the 2 nd and 3 rd cysteines. The type of mutation is not particularly limited, and may be substitution, addition, deletion and/or insertion.
Herein, the expression "a position corresponding to position 3,4, 9 or 17 of any one of SEQ ID NOS: 1 to 8 or 32 to 39" refers to a position in the other sequences aligned with position 3,4, 9 or 17 of any one of SEQ ID NOS: 1 to 8 or 32 to 39 according to the sequence numbering of any one of SEQ ID NOS: 1 to 8 or 32 to 39. By alignment with the sequences of any of SEQ ID NOS 1-8 or 32-39, the corresponding amino acid positions in other variants can be found. For example, standard sequence alignment programs such as ALIGN, clustalW or similar programs can be used to align, typically under default settings, and with any of the sequences of SEQ ID NOS 1-8 or 32-39 with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. The amino acid sequence of the cyclic peptide may be SEQ ID NO 1 or 32, wherein two disulfide bonds are formed between C1-C4 and C2-C3 (i.e., two disulfide bonds are formed between cysteines at positions 3 and 17 and between cysteines at positions 4 and 9).
The cyclic peptides described herein can be used as binders (e.g., inhibitors) of the α7 nachrs. Multiple cyclic peptides may also be combined into a composition. The invention also provides compositions comprising a plurality of cyclic peptides as described herein. The cyclic peptides or compositions described herein can be used to treat or prevent diseases associated with a target protein (e.g., α7 nachrs).
Method for preparing cyclic peptides
The cyclic peptides screened in the present invention may be prepared by any suitable method. The method may comprise a chemical and/or biological method. The chemical method may comprise preparing a linear peptide according to an amino acid sequence comprising SEQ ID NOs 1-8 or 32-39 or variants thereof and chemically cyclizing the linear peptide to a cyclic peptide herein. Alternatively, the method may comprise introducing a polynucleotide encoding an amino acid sequence into a host cell and expressing, and then harvesting the cell supernatant or lysate comprising the cyclic peptide, optionally purifying the cyclic peptide.
In the case of biological methods, polynucleotide sequences encoding polypeptides may be provided. Thus, the present invention encompasses all forms of nucleic acid sequences, such as RNA and DNA encoding polypeptides. Vectors, such as plasmids, may also be provided that contain, in addition to the coding sequence, the signal sequences required for expression of the polypeptides according to the present disclosure. Such polynucleotides optionally further comprise one or more expression control elements. For example, a polynucleotide may comprise one or more promoters or transcription enhancers, ribosome binding sites, transcription termination signals and polyadenylation signals as expression control elements. The polynucleotide may be inserted into any suitable vector, which may be contained within any suitable host cell for expression. Expression of a nucleic acid encoding a polypeptide is typically achieved by operably linking the nucleic acid to a promoter in an expression vector. Typical expression vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence. Exemplary promoters useful for expression in E.coli include, for example, the T7 promoter. Expression of the polynucleotide may be carried out in any suitable expression host known in the art, including but not limited to bacterial cells, yeast cells, insect cells, plant cells, or mammalian cells. In particular, the intracellular environment or culture conditions of the host cell may favor disulfide bond formation by cysteines. The host cell may be, for example, an E.coli cell.
Various embodiments of the present invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
The present application will be explained in further detail with reference to examples. However, those skilled in the art will appreciate that these examples are provided for illustrative purposes only and are not intended to limit the present application.
Examples
Embodiments of the present application will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present application and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. All amounts listed are described in weight percent based on total weight unless otherwise indicated. The application should not be construed as being limited to the particular embodiments described.
Example 1: construction of phage display libraries
Reagents, vectors, strains and instruments
A. Reagent:
① Enzyme: dpn1 (NEB: R0176S); bsa1 (NEB: R0535S); t4 DNA ligase (NEB: M0202T)
②PrimeSTAR GXL(Takara:R050A)
③ The kit comprises: plasmid miniprep kit; DNA gel recovery kit; DNA recovery kit
B. and (3) a carrier: PCantab 5E' (mutation of S240 (TCT) to S240 (TCG))
Remarks: the S240 site base sequence of the carrier is mutated from TCT to TCG to avoid the BSA1 cleavage site.
C. Strains: TG1 electrotransport competence
D. Instrument: a PCR instrument; a DNA electrophoresis apparatus; electric turn-over instrument
Wherein plasmid PCR (template plasmid is commercial PCantab E 'vector) was performed using primers of SEQ ID NO:9 (TTTGGTCTCGGTGC GCCGGTGCCGTATCCGGATCCGCTG) and SEQ ID NO:10(TTTGGTCTC AGCACCACCACCGCAMNNMNNMNNMNNMNNMNNMNNGCAMNNMN NMNNMNNGCAGCAMNNMNNACCGGCCATGGCCGGCTGGGCCGCATA G, wherein N represents any one of the bases T/A/G/C and M represents any one of the bases T/G), and the PCR product was digested and ligated into phage vector (phage vector is commercial PCantab E' vector, see method section hereinafter for details).
(2) Primer design
17AA double-ring peptide library primer design:
F-ETAG:TTTGGTCTCGGTGCGCCGGTGCCGTATCCGGATCCGCTG
R-17AA:TTTGGTCTCAGCACCACCACCGCAMNNMNNMNNMNNM NNMNNMNNGCAMNNMNNMNNMNNGCAGCAMNNMNNACCGGCCAT GGCCGGCTGGGCCGCATAG
remarks: n is any one of T/A/G/C base, and M is any one of T/G base. This pattern is a universal codon pattern for library construction, which ensures that all amino acids are covered at each position.
(3) PCR reaction
PCR reaction System (50. Mu.L)
And (3) setting PCR reaction conditions:
| Denaturation temperature | 98℃ | 10s |
| Annealing temperature | 60℃ | 15s |
| Extension temperature | 68℃ | 6min(1min/kb) |
| Number of cycles | 30 Times |
DNA gel identification of PCR products
① Agarose gel (TAE buffer: 40mM Tris-acetic acid, 2mM EDTA, pH 8.0) was prepared at 1% (w/v), and 0.01% (v/v) Gelred dye was added.
② PCR products were mixed with loading buffer at a ratio of 5:1, the mixture was added to the sample well by a micropipette, and 10. Mu.L of the mixture was added to each well.
③ The horizontal electrophoresis tank was subjected to constant pressure electrophoresis at 15V/cm for 30 minutes, and when the bromophenol blue indicator was moved to 1-2cm from the front of the gel, the electrophoresis was stopped.
④ The gel was taken out, and the position with orange-red fluorescent band, i.e. DNA band, was observed under a 254nm ultraviolet lamp, and the electropherogram was recorded under a UV lamp (see FIG. 1).
PCR product purification
The kit comprises:SV Gel and PCR Clean-Up System(Promega,Lot No.A9281)
① An equal volume of membrane bound solution was added to the PCR amplification reaction solution.
② The prepared PCR product was transferred into SV adsorption column and incubated at room temperature for 1 minute.
③ The column was centrifuged at 16000 Xg (14,000 rpm) for 1 minute in a tabletop centrifuge and the collection tube was decanted.
④ To the adsorption column, 700. Mu.L of a membrane washing solution diluted with 95% ethanol in advance was added for washing. The column was centrifuged at 16,000Xg for 1 min. The collection tube was emptied and the adsorption column was returned to the collection tube. The washing step was repeated with 500. Mu.L of the membrane washing liquid, and then the column was centrifuged at 16,000Xg for 5 minutes.
⑤ The column was carefully transferred into a clean 1.5ml centrifuge tube. 50 μl of nuclease-free water was added directly to the center of the column. After 1min incubation at room temperature, centrifugation was performed at 16,000Xg for 1 min. And collecting liquid in the centrifuge tube, namely the purified DNA sample.
(4) PCR product enzyme digestion (DPN1+BSA1)
A. Enzyme cutting system
| BSA1 | 1μL |
| Dpn1 | 1μL |
| PCR products or PCantab E' vectors | 1μg |
| 10X Cutsmart buffer | 5μL |
| Total | 50μL |
37 ℃ For 2h in water bath
And (3) reserving a sample, and identifying an enzyme digestion product by DNA gel electrophoresis, wherein the experimental step is the same as (3) b.
B. and (3) purifying the DNA after enzyme digestion, wherein the experimental step is the same as (3) c, and the purification result of the DNA after enzyme digestion is shown in figure 2.
(5) T4 DNase ligation
A. Connection System (20 μL)
The ligation was carried out at 16℃overnight.
(6) Preparation of electrotransformation competent cells and electrotransformation
200Ml TG1 bacteria liquid electrocompetence preparation
① E.coli TG1 was selected as competent cell for this experiment. TG1 was selected and cultured in 10mL of LB medium at 37℃with shaking at 220rpm for 6h.
② 2ML of the bacterial liquid is added into 200mL of LB culture solution, and after shaking culture is carried out for 2 hours at 37 ℃ and 220rpm, OD 600 is measured. When the OD 600 reaches 0.6-0.7, pouring the mixture into a bacterial harvesting bottle pre-cooled in advance, and carrying out ice bath for 15min. Centrifugation was performed at 5,000Xg for 10min at 4℃and the supernatant was discarded to collect the cells.
③ The bacterial pellet was resuspended in 45mL of pre-chilled double distilled water. Centrifugation was performed at 5,000Xg for 10min at 4℃and the supernatant was discarded to collect the cells.
④ The cells were resuspended in 40mL of pre-chilled 10% (v/v) glycerol. Centrifugation was performed at 5,000Xg for 10min at 4℃and the supernatant was discarded to collect the cells.
⑤ The cells were resuspended in 10mL of pre-chilled 10% (v/v) glycerol. Centrifugation was performed at 5,000Xg for 10min at 4℃and the supernatant was discarded to collect the cells.
⑥ The cells were resuspended in 2mL of pre-chilled 10% (2 v/v) glycerol. The resuspended bacterial liquid is divided into EP tubes according to 100 mu L of each tube, and immediately transformed by an electrotransformation instrument, or frozen and stored at-80 ℃ for a long time.
⑦ Electrotransport DNA into competence
EC2 program with burle instrument (2.5 kv,0.2cm electric rotor)
A. Ice bath electric rotating cup
B. taking 1 mug of DNA+ PCantab 5E' connection product, adding 100 mug of competent, mixing uniformly, adding an electric rotating cup, placing on ice, and covering an electric rotating cup cover
C. put into a clamping groove of an electrotransport device, immediately taken out after electric shock, added with 1mL of SOC culture medium, poured into an EP tube after being uniformly mixed, and shake-cultured for 1h at 37 ℃ and 220 rpm.
D. Pouring the bacterial liquid after electrotransformation into 1L of LB liquid culture medium, uniformly mixing, taking out 1ml of bacterial liquid for later use, firstly taking out 100 mu L of coated plates, sequentially diluting the rest bacterial liquid with LB culture medium according to 10 times of gradients, and taking out 100 mu L of solid culture substrates coated with ampicillin antibiotics for each gradient. After incubation at 37℃for 12h, colonies on LB plates were counted.
Peptide pool capacity=10×gradient×10 3 pfu/mL, final constructed peptide pool capacity determination was 8×10 9
E. after the rest bacterial liquid is cultured for 12 hours at 37 ℃ and 220rpm in a shaking way, glycerol is added to a final concentration of 40% (v/v) and the bacterial liquid is frozen at-80 ℃.
(7) Phage preparation
① Taking out the phage glycerol bacteria library frozen at-80 ℃.
② 1ML of glycerol bacteria are inoculated into 1L of fresh sterilized LB liquid medium and evenly mixed. Ampicillin (100. Mu.g/mL) was added and shaking culture at 220rpm at 37℃was performed to achieve the logarithmic growth phase (OD 600 = 0.6-0.8). Adding helper phage (M13 KO7 helper phage), standing at 37deg.C in incubator for 60min, shake culturing for 60min.
③ Kanamycin (50. Mu.g/mL) was added to each flask. The culture was continued overnight at 30℃with shaking at 220 rpm.
④ 10,000Xg, 4 ℃ centrifugation for 15min, the supernatant was collected in a clean and sterilized large beaker, 1/5 volume of PEG/NaCl (20% (w/v) PEG-8000,2.5M NaCl) was added, and the ice bath was settled for 2h.
⑤ The above liquid was centrifuged in batches (10 min) at 10,000Xg at 4℃with 50mL of sterilized centrifuge tube, and the supernatant was decanted to leave a pellet. After all centrifugation, phage pellet at the bottom of the centrifuge tube was resuspended in 10ml PBS and pooled in one tube after complete lysis. Centrifuge at 4 ℃,10,000Xg for 10min, and recycle supernatant to new centrifuge tubes. 1/5 volume of PEG/NaCl was added to the tube and the mixture was allowed to settle in an ice bath for 2h.
⑥ 10,000Xg, 4 ℃ centrifugation for 10min, removing the supernatant, bottom off white solid is the phage.
⑦ Resuspension was performed with 10ml PBS, after complete dissolution, 40% (v/v) glycerol was added to the same volume, mixed well, sterilized by passing through a membrane, sub-packaged and frozen in a refrigerator at-80 ℃.
(8) Titer determination experiment
1. TG1 monoclonal (obtained after phage infection of TG1 e.coli) was picked and added to 10mL of LB liquid medium and cultured with shaking at 37 ℃ at 220rpm to logarithmic growth phase (OD 600 = 0.6-0.8).
2. A1.5 mL centrifuge tube was prepared, and 90. Mu.L of bacterial liquid was added to each tube.
3. 10 Mu L of phage to be tested is added into a first centrifuge tube, after being uniformly mixed by a gun head, 10 mu L is sucked and added into a lower tube, gradient dilution is carried out by the method, and in the process, the gun head needs to be replaced cleanly in each step to prevent errors.
4. Incubate at 37℃for 40 min in a stationary incubator.
5. Mu.L of the liquid in each tube was spread on LB-ampicillin plates and incubated overnight (10-12 hours) at 37 ℃.
6. Titer, titer = colony count on solid medium x fold of dilution x 100 = titer (pfu/ml) example 2: screening by taking acetylcholine receptor alpha 7-nAChR as target
Cyclic peptide screening targeting α7 nachrs
1. Solid phase screening procedure
1) Overnight coating of target proteins
Purified 10. Mu.g of α7nAChR protein was coated in polystyrene 96-well plates (coated protein from 10. Mu.g in the first round to 5. Mu.g in the second round to 2. Mu.g in the third round as the number of screening increases) using binding buffer (PBS+0.025% (w/v) GDN (Glyco-diosgenin)) to 100. Mu.l volume per well, gently shaking at 4 ℃,60rpm, and overnight incubation
2) Closure
The liquid in the plate was drained and unbound protein was removed. 200 μl of blocking buffer (1% BSA+0.025% (w/v) GDN, PBS) was added to the wells and incubated for 2h with shaking at 60rpm at 4deg.C. Mu.l of phage (titer: 1X 10 12 pfu/mL) and 100. Mu.l of blocking buffer were added to a 1.5mL EP tube and incubated for 2h with shaking at 60rpm at 4 ℃.
3) Co-incubation
The plate was removed from the liquid, the blocked phage were added to wells of a protein coated 96-well plate and incubated for 2h at 4℃with shaking at 60 rpm.
4) Washing plate
Unbound phage were removed by pouring and the plate was inverted to remove residual solution on a clean paper towel. 200 μl of wash buffer (PBS+0.025% (w/v) GDN++0.1% (v/v) Tween-20) was added, the 96-well plate was gently shaken, and the pipette was blotted off and repeated 10 times. 200. Mu.l of binding buffer (PBS+0.025% (w/v) GDN) was added. The 96-well plate was gently shaken to drain the liquid, and repeated 3 times.
5) Elution
The remaining liquid in the wells was removed, 200. Mu.l of elution buffer (0.2M Glycine-HCl, pH 2.2) was added, and the mixture was gently shaken at room temperature for 10min, and after the liquid was aspirated, 20. Mu.l of 1M Tris-HCl (pH=9) was added to neutralize the above-mentioned eluate.
6) Measuring titre
A. Selecting a colony of the monoclonal TG1, adding 15ml of LB liquid medium into a 50ml test tube, and culturing at 4 ℃ for 6 hours by shaking at 220rpm until the colony reaches the logarithmic growth phase (OD 600 = 0.6-0.8);
b. Taking 10 mu l of the screened phage, adding 90 mu l of TG1 bacterial liquid, and diluting the phage in sequence by 5-6 orders of magnitude according to the proportion;
c. Coating the diluted bacterial liquid on a solid culture medium, and culturing for 12 hours at 37 ℃;
d. Counting: colony count on solid medium x fold of dilution x 10 = titer (pfu/ml)
7) Amplifying the remaining phage
A. Phage were added to 20ml of TG1 bacteria solution (OD 600 = 0.6-0.8), cultured at 37℃with shaking at 220rpm for 1h. Centrifuging and removing the supernatant. LB liquid medium was added to the bacterial pellet, resuspended, spread on solid medium and cultured at 37℃for 12 hours. An appropriate amount of LB liquid medium was added, colonies were blown off, 40% (v/v) glycerol was added in an equal volume, and the mixture was stored at-80 ℃.
B. Phage were amplified, for specific procedures see "phage preparation" above. And then second and third rounds of screening were performed.
| Titer of | |
| First wheel | 3×106 |
| Second wheel | 5×108 |
| Third wheel | 2×109 |
8) Sequencing
The colonies on the solid medium in step 6 were picked up and sequenced (Shanghai Biotechnology Co., ltd.). And selecting high-abundance sequence synthesis according to a sequencing result.
| Sequence number | Name of the name | Polypeptide sequence | Abundance ratio |
| 1 | KP2007 | PACCEGNWCRWLRLDTCG | 6/30 |
| 2 | KP2002 | MWCCTGPFCLPSLEHRCG | 2/30 |
| 3 | KP2003 | EFCCNPFFCKMSQSGGCG | 2/30 |
| 4 | KP2004 | RSCCLGHECPIPMFWLCG | 2/30 |
| 5 | KP2005 | YECCMHPACAWLRSRECG | 2/30 |
| 6 | KP2006 | HICCTHPACASIREDLCG | 2/30 |
| 7 | KP2008 | AHCCKESWCLPAAAFLCG | 2/30 |
| 8 | KP2009 | GRCCWGHGCLPAELWVCG | 2/30 |
Example 3: chemical synthesis of polypeptide sequences targeting the acetylcholine receptor alpha 7-nachrs
(1) Swelling of the resin:
a) 0.2mmol (512 mg) of the chlorinated resin was weighed and put into a polypeptide synthesis tube. 5ml of DMF (N, N-dimethylformamide) and 5ml of DCM (dichloromethane) were added to the polypeptide synthesis tube, the mixture was left at room temperature for 30min, the solvent was pumped down with air pump, and the solvent was pumped down after washing with 10ml of DMF
Note that: i) DMF and DCM referred to in this item are both common reagents with 99.7% purity.
Ii) pumping the solvent to a suction bottle by using an air pump.
(2) Synthesis to the left (N-terminus) starting from the first amino acid to the right (C-terminus) of the peptide fragment sequence:
a) 0.8mmol Fmoc-Gly-OH (glycine) was weighed into a 10mL EP tube, 6mL DMF was added to the EP tube and dissolved, shaking thoroughly was performed, and 1.6mmol (264. Mu.L) DIEA was added to the EP tube
B) Transferring the above mixed solution into polypeptide synthesis tube, transferring polypeptide synthesis tube into 33 deg.C constant temperature shaking table, shaking overnight, and taking out polypeptide synthesis tube
C) Cleaning: the resin was washed three times (10 ml each) with DMF and the solvent was drained; the resin was rinsed three times (10 ml each) with DCM and the solvent was drained; finally, the solvent is drained after being washed three times (10 ml each time) by DMF
(3) Deprotection:
a) Adding 10ml of 20% piperidine solution to the polypeptide synthesis tube to submerge the resin, and transferring to a 33 ℃ constant temperature shaking table to oscillate for 5min;
b) Taking the polypeptide synthesis tube out of the shaker;
c) Cleaning: repeating the step (2) of cleaning.
(4) With a second amino acid
A) 0.8mmol (468.5 mg) of Fmoc-Cys (Trt) -OH (cysteine) and 0.76mmol (314 mg) of condensing agent (HCTU) were weighed into a 10ml EP tube, 6ml of DMF was added to the EP tube and dissolved, thoroughly shaken and 1.6mmol (264. Mu.L) of DIEA was added to the EP tube.
B) Transferring the mixed solution into a polypeptide synthesis tube, transferring the polypeptide synthesis tube into a shaking table at a constant temperature of 33 ℃ for 1h, and taking out the polypeptide synthesis tube.
C) Cleaning: repeating the step (2) of cleaning.
(5) Peptide chain extension: and (3) continuing to repeat the deprotection in the step (3) and the amino acid synthesis in the step (4) according to the polypeptide sequence until the synthesis of the last amino acid is finished.
(6) Cleavage of crude peptide
The polypeptide synthesis tube was removed and the resin was washed three times with DMF (10 ml each time), the solvent was drained after each washing, the resin was washed three times with DCM (10 ml each time), and the solvent was drained after each washing (drained to dry powder form). After draining, the TFA/H2O/phenol/Tips (10 ml/500. Mu.L/500 mg/500. Mu.L) volume ratio of cleavage reagent was formulated in a 50ml EP tube. Transferring the cutting reagent into the polypeptide synthesis tube, placing the polypeptide synthesis tube into a shaking table at a constant temperature of 26 ℃ for oscillating reaction for 2.5 hours, and taking out the polypeptide synthesis tube, wherein the solution in the tube is the peptide chain lysate.
(7) Blow-drying and flushing
A) Transferring 10ml peptide chain lysate with ear washing ball into 50ml EP tube, and blow drying the lysate with nitrogen at room temperature to below 5ml
B) Adding 40ml of glacial diethyl ether into 50ml of EP tube, properly shaking the EP tube, putting the EP tube into a centrifuge, and centrifuging for 3min at the rotating speed of 3,500 rpm; after centrifugation, the supernatant was discarded
C) Repeating
D) Air-drying at room temperature, mashing
(8) Crude peptide chromatography and separation: the crude peptide was analyzed for correctness by HPLC and mass spectrometry of shimadzu. After verification of correctness, the correct product is isolated and lyophilized.
(9) Polypeptide renaturation
Tris (1.6 g), urea (12 g), cystine (24 mg) and cysteine (10 mg) are weighed and added into a 500ml volumetric flask, 100ml purified water is weighed and added into the volumetric flask, the pH is adjusted to 7.5-8.5, 10mg polypeptide is added, the reaction is carried out at room temperature, and the correctness analysis of the crude peptide is carried out by utilizing HPLC and mass spectrum of Shimadzu. After verification of correctness, the correct product is isolated and lyophilized.
The polypeptide nuclear magnetic resonance structure analysis method comprises the following steps:
(1) The lyophilized polypeptide powder was dissolved in 400. Mu.L deionized water and 20. Mu.L heavy water (deuterium oxide, D 2 O) at a final concentration of about 2mM, and subjected to nuclear magnetic resonance experimental measurement.
(2) The 1H-1H COSY,1H-1H TOCSY and 1H-1H NOESY spectra of the samples were obtained by acquisition and measurement at 298K on a Bruker ADVANCE III HD MHz spectrometer equipped with a TCI H & F/C/N-D probe, the mixing times of the TOCSY and NOESY spectra being 150ms and 500ms, respectively.
(3) The 1H-1H distance constraint is extracted from the NOESY spectrum, and the dihedral angle constraint is extracted from TOCSY.
(4) The disulfide pairing mode was confirmed directly by short-range contacts specified on the NOESY spectrum.
(5) The measured data were subjected to spectral peak assignment by spark software and calculation by Xplor software to obtain the three-dimensional structure of the polypeptide in solution.
Figure 3 shows the chromatographic and mass spectral characterization of polypeptides after renaturation following 17AA cyclic peptide library screening. Fig. 4 shows a concentration response curve of cyclic peptide KP2002-KP2009 inhibiting the activity of the α7-type acetylcholine receptor. Figure 5 shows nuclear magnetic resonance structure of cyclic peptide KP2007 with highest inhibitory activity.
Example 4: cell level detection of the inhibitory Effect of chemically synthesized Polypeptides on alpha 7-nAChR
① Culture cells (HEK-293T)
A. the reagent used is as follows: PBS; trypsin; DMEM (Medium)
B. 10 6 HEK-293T cells were added to a petri dish, 4ml DMEM was added, and after gentle shaking, the cells were incubated at 37℃for 48 hours with 5% (v/v) CO 2.
C. the medium was removed, 1ml of PBS was added along the dish wall, and after gentle shaking, the PBS was removed.
D. add 500. Mu.l of pancreatin, shake the dish gently, cover the bottom of the dish with pancreatin, digest for 30s, remove pancreatin.
E. 1ml of DMEM was added and the cells resuspended.
F. culture dishes were taken, 4ml of DMEM medium was added to each dish, 250. Mu.l of the cell heavy suspension was added, and after gentle shaking, the cells were dispersed uniformly.
② Transfection plasmid
A. The reagent used is as follows: OMEM (Gibco: LOT# 2447664); lipo3000/p3000 (Invitrogen: LOT# 2476277); a master plasmid: alpha 7-PCDNA 3.1.1 (Uniprot: P36544); helper plasmid: NACHO-PCDNA 3.1.1 (Uniprot: Q53FP 2)
B. Preparing solution A: OMEM 100. Mu.l+lipo3000. Mu.l;
and (2) liquid B: OMEM 100. Mu.l+p3000. Mu.l+alpha.7-PCDNA 3.1.1. Mu.g+RIC-PCDNA 3.1.1. Mu.g+ NACHO-PCDNA 3.1.1. Mu.g, and standing for 5min.
C. adding the solution A into the solution B, and standing for 20min.
D. the mixture was added to a petri dish and incubated at 37℃for 24 hours with 5% (v/v) CO 2.
③ Cell plating
Transfected cells were plated in 96-well plates with 6X 10 4 cells per well.
④ Calcium ion influx experiments Using FLIPR Instrument
A. Preparation of calcium stream reagent binding buffer (Hanks solution+0.1% (w/v) BSA+2.5 mu M Probeacid (MCE CAS# 57-66-9), pH=7.4)
Staining reagent: 10ml binding buffer+10. Mu.l Fluo-4 (Invitrogen LOT# 2342741) +5. Mu.l 20% Pluronicplu (Sigma CAS# 9003-11-6)
Nicotine agent: 1 mu M Nicotine (CHEMFACES: CAS#54-11-5, binding buffer formulation)
PNU reagent: 40. Mu.M PNU (MCE: PNU-120596, formulation of reagents with buffer)
B. polypeptide-complexing agent for gradient dilution of polypeptides with binding buffer
C. The 96-well plate was removed, 100 μl of binding buffer was added to each well, and the cells were washed once to remove the liquid.
D. 50 μl of the prepared dye reagent was added to each well, and the mixture was placed in an incubator after light-shielding operation, and allowed to stand for 45min.
E. The 96-well plate was removed, the dye reagent was removed, 100. Mu.l of binding buffer was added, the cells were washed once, the liquid was removed, and the procedure was repeated twice.
F. The diluted polypeptide reagents are added one by one in sequence, and the diluted polypeptide reagents are slowly added by adherence.
G. UsingThe calcium flux detection kit measures the inhibition effect of the polypeptide on the target protein alpha 7 type acetylcholine receptor.
The α7 acetylcholine receptor is a Ca 2+ channel whose level of functional activation or inhibition can be characterized by detecting changes in intracellular Ca 2+ signaling. The detection principle is that after lipophilic Acetoxymethyl (AM) carries an indicator dye sensitive to Ca 2+ into cells, the indicator dye sensitive to free Ca 2+ is released by cleavage of cytoplasmic enzyme, and the intracellular calcium influx and the indicator dye are combined to emit stronger fluorescent signals, so that the blocking effect of drugs on channels at different concentrations reduces the calcium influx, the fluorescent signal intensity is reduced, and the blocking effect of the reactive drugs on the channels is strong or weak.
Table 1 shows the data relating to the inhibitor function experiments after screening of the 17AA bicyclic peptide library.
TABLE 1
| Sequence number | Name of the name | Polypeptide sequence | IC50(μM) |
| 1 | KP2007 | PACCEGNWCRWLRLDTCG | 0.1249 |
| 2 | KP2002 | MWCCTGPFCLPSLEHRCG | 0.8734 |
| 3 | KP2003 | EFCCNPFFCKMSQSGGCG | 0.2663 |
| 4 | KP2004 | RSCCLGHECPIPMFWLCG | 0.5013 |
| 5 | KP2005 | YECCMHPACAWLRSRECG | 0.4330 |
| 6 | KP2006 | HICCTHPACASIREDLCG | 3.3780 |
| 7 | KP2008 | AHCCKESWCLPAAAFLCG | 1.8530 |
| 8 | KP2009 | GRCCWGHGCLPAELWVCG | 4.2200 |
Comparative example 1:
Linear peptide library construction and screening experimental procedures:
(1) Constructing an AGX 9 linear peptide library, wherein the sequence skeleton of the polypeptide library is as follows from N end to C end: AGXXXXXXXXX, wherein X represents any amino acid.
1. Reagents, vectors, strains and instruments.
1.1. And (3) a reagent.
1.1.1. Enzyme: dpn1 (NEB: R0176S); bsa1 (NEB: R0535S); t4 DNA ligase (NEB: M0202T).
1.1.2.PrimeSTARGXL(Takara:R050A)
1.1.3. The kit comprises: plasmid miniprep kit; DNA gel recovery kit; DNA recovery kit.
1.2. A carrier.
And (3) a carrier: PCantab 5E' (mutation of S240 (TCT) to S240 (TCG))
Remarks: the BSA1 cleavage site of the vector was mutated.
1.3. Strains: TG1 electrotransformation competence.
1.4. Instrument: a PCR instrument; a DNA electrophoresis apparatus; and (5) an electrotransport converter.
2. And (5) designing a primer.
Forward primer:
TTTGGTCTCGGTGCGCCGGTGCCGTATCCGGATCCGCTG(SEQ ID NO:11)
reverse primer:
TTTGGTCTCAGCACCMNNMNNMNNMNNMNNMNNMNNMNNMN NACCGGCCATGGCCGGCTGGGCCGCATAGAAAGG, wherein N is any one of T/A/G/C bases and M is any one of T/G bases. (SEQ ID NO: 12)
Remarks: BSA1 cleavage site: GGTCTCN1; CCAGAGN.
(The following procedure for constructing phage library experiments is the same as in example 1)
PCR reaction.
PCR reaction (50. Mu.L).
Parameters of PCR reaction System
DNA gel identification of PCR products.
1% Agarose gel was added, 1×TAE buffer was added, and after mixing and heating, gelred was added.
The ultraviolet lamp 254 irradiates the display strip at a wavelength.
DNA gel recovery.
PCR product cleavage (DPN1+BSA1).
4.1. And (5) an enzyme digestion system.
Parameters of enzyme digestion system
| BSA1 | 1μL |
| Dpn1 | 1μL |
| DNA | 1μg |
| 10×Cutsmart | 5μL |
| Total | 50μL |
37 ℃,2H, water bath.
Sample was kept, and DNA gel (1% agarose gel) was run.
4.2. And (5) purifying DNA after enzyme digestion.
T4 dnase ligation.
5.1. The ligation system (20. Mu.L).
Connection system parameters
16℃Overnight.
DNA purification.
6. Preparation of electrotransformation competent cells and electrotransformation
200ML TG1 bacteria liquid electrocompetence preparation
6.1. E.coli TG1 was selected as competent cell for this experiment. TG1 was picked up and placed in 10mL of LB medium, and shaken at 37℃and 220rpm for 6h.
6.2. 2ML of the bacterial liquid was poured into 200mL of LB medium, and after shaking at 37℃and 220rpm for 2 hours, OD600 was measured. When the OD600 reaches 0.6-0.7, pouring the mixture into a bacterial harvesting bottle pre-cooled in advance, and carrying out ice bath for 15min. Centrifuging at 4deg.C for 10min at 5200g, and removing supernatant to obtain thallus.
6.3. Re-suspend with 5mL of pre-chilled double distilled water and pour into 40mL. Centrifuging at 4deg.C for 10min at 5200g, and removing supernatant to obtain thallus.
6.4. Resuspended with pre-chilled 10% glycerol 40 mL/tube. Centrifuging at 4deg.C for 10min at 5200g, and removing supernatant to obtain thallus.
6.5. Resuspended with 10 mL/tube of pre-chilled 10% glycerol. Converging into a tube. Centrifuging at 4deg.C for 10min at 5200g, and removing supernatant to obtain thallus.
6.6. Resuspended with 2mL of pre-chilled 10% glycerol. 100 mu L of the resuspended bacterial liquid is respectively packed in EP tubes, and immediately transformed by an electrotransformation instrument, or frozen and stored at-80 ℃ for a long time.
7. Electrotransport DNA into competence.
EC2 program with burle instrument (2.5 kv,0.2cm electric rotor)
7.1. Ice bath electric rotating cup
7.2. 1. Mu.g of DNA was taken, 100. Mu.L of competent cells was added, mixed well, added to an electric beaker, placed on ice and covered with a lid of the electric beaker.
7.3. Put into a clamping groove of an electrotransport device, immediately taken out after electric shock according to the PULSE, added with 1mL of SOC culture medium, poured into an EP tube after being uniformly mixed, and the temperature is 37 ℃ for 1h at 220rpm.
7.4. Pouring the bacterial liquid after electric transformation into 1L of LB liquid culture medium, uniformly mixing, taking out 1mL of bacterial liquid for later use, firstly taking out 100 mu L of coated plates (100), sequentially diluting the rest bacterial liquid with LB culture medium according to 10 times of gradients, and taking out 100 mu L of solid culture substrate coated with ampicillin antibiotics for each gradient. After 12h at 37℃the colonies were counted.
Peptide reservoir capacity = 10 x gradient x 10 3 pfu/mL.
7.5. The rest bacterial liquid is re-suspended by adding 40% glycerol after being subjected to 37 ℃,220rpm and 12 hours, and frozen to be stored at-80 ℃.
8. Phage preparation.
8.1. Taking out the phage glycerol bacteria library frozen at-80 ℃.
8.2. 1ML of glycerol bacteria was taken into 1L of freshly sterilized LB liquid medium, mixed well, and OD600 was measured such that OD600 = 0.1. Ampicillin (1:1000 addition, mother liquor concentration 100 mg/mL) was added, and the culture was carried out on a shaker at 37℃at 220rpm to achieve the logarithmic phase (OD 600 = 0.6-0.8). Adding auxiliary phage, standing at 37deg.C in incubator for 60min, and shake culturing for 60min.
8.3. Kanamycin (50 mg/mL mother liquor concentration) was added to each flask in a volume ratio of 1:1000. 30 ℃,220rpm, shaking overnight culture.
8.4.10000G, centrifuged at 4℃for 15min, the supernatant collected in a clean and sterilized large beaker, 1/5 volume of PEG/NaCl (20% (w/v) PEG-8000,2.5M NaCl) was added, and the ice bath was settled for 2h.
8.5. The above liquid was centrifuged in batches (10 min) at 4℃and 10000g with 50mL of sterilized centrifuge tube, and the supernatant was decanted to leave a pellet. After all centrifugation, phage pellet at the bottom of the centrifuge tube was resuspended in 10mL PBS and pooled in one tube after complete lysis. Centrifuge for 10min at 4℃with 10000g, and collect supernatant into a new centrifuge tube. The tube was filled with 1/5V volume of PEG/NaCl and ice-bath settled for 2h.
8.6.10000G, centrifuged at 4℃for 10min, the supernatant removed and the bottom off-white solid was the phage prepared.
8.7. Resuspension was performed with 10mL of PBS, after complete dissolution, 40% glycerol was added to the same volume, mixed well, sterilized by passing through a membrane, sub-packaged and frozen in a-80 ℃ refrigerator.
9. Titer determination experiments.
9.1. TG1 was picked and added to 10mL of LB liquid medium, cultured at 37 ℃ at 220rpm to logarithmic growth phase, at which time od600=0.6 to 0.8 was measured.
9.2. A1.5 mL centrifuge tube was prepared, and 90. Mu.L of bacterial liquid was added to each tube.
9.3. 10 Mu L of phage to be tested is added into a first centrifuge tube, after being uniformly mixed by a gun head, 10 mu L is sucked and added into a lower tube, gradient dilution is carried out by the method, and in the process, the gun head needs to be replaced cleanly in each step to prevent errors.
Incubate at 9.4.37 ℃for 40 min.
9.5. Mu.L of the liquid in each tube was spread on LB-ampicillin plates and incubated overnight (10 to 12 hours) at 37 ℃.
9.6. Titer was measured, titer = colony count on solid medium x fold of dilution x 100 = titer (pfu/mL).
(2) Solid phase screening was performed targeting α7 nachrs. (screening Experimental procedure same as in example 2)
1. The target protein was coated overnight.
Purified 10 μg α7nAChR protein was coated in polystyrene 96-well plates (coated protein from 10 μg in the first round to 5 μg in the second round to 2 μg in the third round as the number of screening increases) using binding buffer (pbs+0.025% gdn) to 100 μl volume per well, gently shaking at 4 ℃,60rpm, and incubating overnight.
2. And (5) sealing.
The liquid in the plate was drained and unbound protein was removed.
200. Mu.L of blocking buffer (1% BSA+0.025% GDN, PBS) was added to the wells and incubated at 60rpm for 2h at 4 ℃.
Mu.L phage (titer: 1X 10 12 pfu/mL) and 100. Mu.L blocking buffer were added to a 1.5mL EP tube and incubated at 4℃for 2h at 60 rpm.
3. Co-incubation.
The blocked phage were added to wells of a protein coated 96-well plate by removing the liquid from the plate.
Incubation was performed at 4℃at 60rpm for 2 h.
4. Washing the plate.
Unbound phage were removed by pouring and the plate was inverted to remove residual solution on a clean paper towel. 200. Mu.L of washing buffer (PBS+0.025% GDN++0.1% [ v/v ] Tween-20) was added, and the 96-well plate was gently shaken and the pipette was blotted off and repeated 10 times.
200. Mu.L of binding buffer (PBS+0.025% GDN) was added and the 96-well plate was gently shaken to drain the liquid out and repeated 3 times.
5. Eluting.
The remaining liquid in the wells was removed and 200. Mu.L of elution buffer (0.2M Glycine-HCl, pH 2.2) was added.
Gently shake at room temperature for 10min, aspirate the liquid and then add 20 μl 1M Tris-HCl (ph=9) to neutralize the eluate.
6. Titer was measured.
The colony of monoclonal TG1 was picked, and 15mL,4 ℃,220rpm,6h were added to a 50mL tube and incubated to logarithmic growth phase (od600=0.6 to 0.8).
6.1. Taking 10 mu L of the screened phage, adding 90 mu L of TG1 bacterial liquid, and diluting the phage in sequence according to the proportion for 5-6 orders of magnitude.
6.2. The diluted bacterial liquid is coated on a solid culture medium and cultured for 12 hours at 37 ℃.
6.3. Counting: colony count on solid medium x fold of dilution x 10 = titer (pfu/mL).
7. Amplifying the remaining phage.
7.1. Phage were added to 20mL of TG1 bacteria (OD 600 = 0.6-0.8), 37 ℃,220rpm,1h. Centrifuging, removing supernatant, adding LB liquid medium, re-suspending, spreading on solid medium, and culturing at 37deg.C for 12 hr. An appropriate amount of LB liquid medium was added, colonies were blown off, an equal volume of 40% glycerol was added, and the colonies were stored at-80 ℃.
7.2. Phage were amplified and phage peptide libraries were prepared for details reference.
8. Sequencing.
Colonies on the solid medium in step 6 were picked up and sequenced. And selecting a sequence with high abundance according to a sequencing result, and synthesizing.
Sequencing sequence enrichment results
(3) Solid phase synthesis of polypeptides
1. Swelling the resin.
0.2Mmol (512 mg) of the chlorinated resin was weighed and put into a polypeptide synthesis tube. To the polypeptide synthesis tube were added 5mL of DMF (N, N-dimethylformamide) and 5mL of DCM (dichloromethane), and the mixture was left at room temperature for 30min, the solvent was pumped down with an air pump, and after washing with 10mL of DMF, the solvent was pumped down.
Note that: DMF and DCM referred to in this item are both common reagents with 99.7% purity. The step of pumping the solvent is to pump the solvent in the polypeptide synthesis tube into a pumping filter flask by using an air pump.
2. Synthesis starts from the first amino acid on the right (C-terminal) side of the peptide stretch to the left (N-terminal).
2.1. 0.8Mmol Fmoc-Ala-OH (alanine) was weighed into a 10mL EP tube, 6mL DMF was added to the EP tube and dissolved, shaking was complete, and 1.6mmol (264. Mu.L) DIEA was added to the EP tube.
2.2. Transferring the mixed solution into a polypeptide synthesis tube, transferring the polypeptide synthesis tube into a shaking table at a constant temperature of 33 ℃ for shaking overnight, and taking out the polypeptide synthesis tube.
2.3. Cleaning: the resin was washed three times (10 mL each) with DMF and the solvent was drained; the resin was rinsed three times (10 mL each) with DCM and the solvent was drained; finally, the solvent was drained after three more DMF washes (10 mL each).
3. And (5) deprotection.
3.1. Adding 10mL of 20% piperidine solution to the polypeptide synthesis tube to submerge the resin, and transferring to a 33 ℃ constant temperature shaking table to oscillate for 5min;
3.2. taking the polypeptide synthesis tube out of the shaker;
3.3. cleaning: the above 2 washing steps 2.3 are repeated.
4. And a second amino acid.
4.1. 0.8Mmol Fmoc-Gly-OH (glycine) and 0.76mmol (314 mg) of condensing agent (HCTU) were weighed into a10 mL EP tube, 6mL DMF was added to the EP tube and dissolved, shaking thoroughly was performed, and 1.6mmol (264. Mu.L) of DIEA was added to the EP tube.
4.2. Transferring the mixed solution into a polypeptide synthesis tube, transferring the polypeptide synthesis tube into a shaking table at a constant temperature of 33 ℃ for 1h, and taking out the polypeptide synthesis tube.
4.3. Cleaning: the above 2 washing steps 2.3 are repeated.
5. Peptide chain extension: and (3) continuing to repeat the step (3) according to the polypeptide sequence, deprotecting, and synthesizing and prolonging the peptide chain by the amino acid in the step (4) until the synthesis of the last amino acid is finished.
6. Cleavage of crude peptide.
The polypeptide synthesis tube was removed and the resin was washed three times with DMF (10 mL each time), the solvent was drained after each washing, the resin was washed three times with DCM (10 mL each time), and the solvent was drained after each washing (drained to dry resin). After draining, the cleavage reagent was formulated in a volume ratio of TFA/H2O/phenol/Tips (10 mL/500. Mu.L/500 mg/500. Mu.L) in a 50mL EP tube. Transferring the cutting reagent into the polypeptide synthesis tube, placing the polypeptide synthesis tube into a shaking table at a constant temperature of 26 ℃ for oscillating reaction for 2.5 hours, and taking out the polypeptide synthesis tube, wherein the solution in the tube is the peptide chain lysate.
7. Blow-drying and flushing.
7.1. 10ML of the peptide chain lysate was transferred to a 50mL EP tube using an ear-washing ball, and the lysate was dried as much as possible to 5mL or less using nitrogen at room temperature.
7.2. Adding 40mL of glacial ethyl ether into 50mL of EP pipe, properly shaking the EP pipe, putting the EP pipe into a centrifuge, and centrifuging for 3min at 3500 revolutions; after centrifugation, the supernatant was decanted.
7.3. And (5) repeating.
7.4. Airing at room temperature, and mashing after airing.
8. Crude peptide chromatography and separation: the crude peptide was analyzed for correctness by HPLC and mass spectrometry of shimadzu. After verification of correctness, the correct product is isolated and lyophilized. Figure 6 shows HPLC purification and mass spectrometry profiles of KP1794, KP1795 and KP1796 polypeptides.
(4) And detecting the functions of the polypeptide inhibitor. (Experimental procedures are the same as in example 4)
1. Cells were cultured (HEK-293T).
1.1. The reagent used is as follows: PBS; trypsin; DMEM (medium).
1.2. 10 6 HEK-293T cells were added to a petri dish, 4mL of DMEM was added, and after gentle shaking, the cells were incubated at 37℃with 5% CO 2 for 48 hours.
1.3. The medium was removed, 1mL of PBS was added along the dish wall, and after gentle shaking, the PBS was removed.
1.4. Add 500. Mu.L of pancreatin, shake the dish gently, cover the bottom of the dish with pancreatin, digest for 30s, remove pancreatin.
1.5. 1ML of DMEM was added and the cells were resuspended.
1.6. Culture dishes were taken, 4mL of DMEM medium was added to each dish, 250. Mu.L of the cell resuspension was added, and after gentle shaking, the cells were dispersed evenly.
2. And (3) transfecting the plasmid.
2.1. The reagent used is as follows: OMEM; lipo3000; p3000; a master plasmid: alpha 7-PCDNA 3.1.1; helper plasmid: RIC-PCDNA 3.1.1; NACHO-PCDNA 3.1.1.
2.2. Preparing solution A: OMEM 100. Mu.L+lipo3000. Mu.L; and (2) liquid B: OMEM 100. Mu.L+p3000. Mu.L+2. Mu.g. Alpha.7-PCDNA 3.1.1+1. Mu.g RIC-PCDNA 3.1.1+1. Mu. g NACHO-PCDNA 3.1.1, and standing for 5min.
2.3. Adding the solution A into the solution B, and standing for 20min.
2.4. The mixture was added to a petri dish and incubated at 37℃for 24 hours with 5% CO 2.
3. Cells were plated.
The transfected cells were plated in 96-well plates with 6X 10 4 cells per well.
4. Calcium ion influx experiment using FLIPR instrument
4.1. Calcium flux reagent binding buffer (Hanks solution+0.1% bsa+2.5 μ M Probeacid, ph=7.4).
Staining reagent: 10mL of binding buffer+10. Mu.L of Fluo-4+5. Mu.L of 20% plu.
Nicotine agent: 1. Mu.M nicotine (binding buffer formulation reagent).
PNU reagent: 40. Mu.M PNU (binding buffer configuration reagent).
4.2. Polypeptide reagent is prepared, and polypeptide is diluted by the gradient of the binding buffer solution.
4.3. The 96-well plate was removed, 100. Mu.L of binding buffer was added to each well, and the cells were washed once to remove the liquid.
4.4. 50 Mu L of the prepared dye reagent is added into each hole, and the mixture is placed into an incubator to stand for 45min after being subjected to light-proof operation.
4.5. The 96-well plate was removed, the dye reagent was removed, 100. Mu.L of binding buffer was added, the cells were washed once, the liquid was removed, and the procedure was repeated twice.
4.6. The diluted polypeptide reagents are added one by one in sequence, and the diluted polypeptide reagents are slowly added by adherence.
4.7. Detection was performed using a FLIPR instrument.
Results
Phage display libraries of linear peptides were constructed and screened against the α7 acetylcholine receptor. The results showed that the optimal half-inhibitory concentration (IC 50) of the linear polypeptide obtained by screening was 8.19 μm for the α7-type acetylcholine receptor, which was much weaker in biological activity than the 17AA bicyclic peptide (KP 2007, IC 50 =0.12 μm) (fig. 7).
Table 2: inhibition performance comparison summary
Comparative example 2:
The experimental steps are as follows:
(1) Construction of bicyclic peptide libraries 16AA(GCX1CX2X3X4X5X6CX7X8X9X10X11C)
1. Library building primer
Forward primer: TTTGGTCTCGGTGCGCCGGTGCCGTATCCGGATCCGCTG (SEQ ID NO: 16)
Reverse primer:
TTTGGTCTCAGCACCTGCGGCCGCACAMNNMNNMNNMNNMNNGCAMNNMNNMNNMNNMNNGCAMNNGCAACCGGCCATGGCCGGCTGGGCCGC(SEQ ID NO:17), Wherein n=a/T/C/G; m=a/C.
2. And (3) a library building process: the library construction procedure was consistent with example 1, with a final library capacity of 7X 10 9
3. Sequencing results
10 Monoclonal antibodies were randomly picked and sequenced to check the accuracy of the peptide library.
Sequencing primer: CCATGATTACGCCAAGCTTTGGAGCC A
Sequencing was performed by Shanghai Biotechnology Co., ltd, and 1000bp was sequenced
The sequencing results translated into amino acid sequences are shown in the following table:
| Monoclonal antibodies | Sequence(s) | SEQ ID NO: |
| 1 | GCICGHLPRCEYVWDC | 18 |
| 2 | GCMCIGTGSCTPDGFC | 19 |
| 3 | GCPCLTRSNCRDAVTC | 20 |
| 4 | GCLCGTTYLCPIERGC | 21 |
| 5 | GCSCHMSFKCDMNLFC | 22 |
| 6 | GCTCHYDLPCSAPPTC | 23 |
| 7 | GCFCPVDLGCITSEAC | 24 |
| 8 | GCYCLGKIECITRVTC | 25 |
| 9 | GCFCMGPRTCRQSPPC | 26 |
| 10 | GCTCFVHSVCEYGCDC | 27 |
Sequencing results indicated that the library of 16AA was constructed correctly.
(2) Solid phase screening was performed targeting α7 nachrs. (screening Experimental procedure same as in example 2)
2.1. The target protein was coated overnight.
Purified 10 μg α7nAChR protein was coated in polystyrene 96-well plates (coated protein from 10 μg in the first round to 5 μg in the second round to 2 μg in the third round as the number of screening increases), the coated protein was added to 100 μl volume per well using binding buffer (pbs+0.025% gdn), gently shaken at 4 ℃, and incubated overnight.
2.2. And (5) sealing.
The liquid in the plate was drained and unbound protein was removed.
200. Mu.L of blocking buffer (1% BSA+0.025% GDN, PBS) was added to the wells and incubated at 60rpm for 2h at 4 ℃.
Mu.L phage (titer: 1X 10 12 pfu/mL) and 100. Mu.L blocking buffer were added to a 1.5mL EP tube and incubated at 4℃for 2h at 60 rpm.
2.3. Co-incubation.
The blocked phage were added to wells of a protein coated 96-well plate by removing the liquid from the plate.
Incubation was performed at 4℃at 60rpm for 2 h.
2.4. Washing the plate.
Unbound phage were removed by pouring and the plate was inverted to remove residual solution on a clean paper towel. 200. Mu.L of washing buffer (PBS+0.025% GDN++0.1% [ v/v ] Tween-20) was added, and the 96-well plate was gently shaken and the pipette was blotted off and repeated 10 times.
200. Mu.L of binding buffer (PBS+0.025% GDN) was added and the 96-well plate was gently shaken to drain the liquid out and repeated 3 times.
2.5. Eluting.
The remaining liquid in the wells was removed and 200. Mu.L of elution buffer (0.2M Glycine-HCl, pH 2.2) was added.
The mixture was gently shaken at room temperature for 10min, and after the liquid was aspirated, 20. Mu.L of 1M Tris-HCl (pH 9) was added to neutralize the eluate.
2.6. Titer was measured.
2.6.1. The colony of the monoclonal TG1 was picked, 15mL of the colony was added to a 50mL tube, and the colony was cultured at 220rpm for 6 hours at 4℃until the colony reached the logarithmic growth phase (OD 600 value: 0.6 to 0.8).
2.6.2. Taking 10 mu L of the screened phage, adding 90 mu L of TG1 bacterial liquid, and diluting the phage in sequence according to the proportion for 5-6 orders of magnitude.
2.6.3. The diluted bacterial liquid is coated on a solid culture medium and cultured for 12 hours at 37 ℃.
2.6.4. Counting: colony count on solid medium x fold of dilution x 10 = titer (pfu/mL).
2.7. Amplifying the remaining phage.
2.7.1. Phage were added to 20mL of TG1 bacteria (OD 600 value 0.6-0.8), 37℃at 220rpm,1h. Centrifuging, removing supernatant, adding LB liquid medium, re-suspending, spreading on solid medium, and culturing at 37deg.C for 12 hr. An appropriate amount of LB liquid medium was added, colonies were blown off, an equal volume of 40% glycerol was added, and the colonies were stored at-80 ℃.
2.7.2. Phage were amplified, see 1. Phage peptide libraries were prepared for details.
2.8. Sequencing.
Colonies on the solid medium in step 2.6 were picked up and sequenced. And selecting a sequence with high enrichment degree according to a sequencing result, and synthesizing.
(3) Solid phase synthesis of polypeptides
3.1. Swelling the resin.
3.1.1. 0.2Mmol (512 mg) of the chlorinated resin was weighed and put into a polypeptide synthesis tube. To the polypeptide synthesis tube were added 5mL of DMF (N, N-dimethylformamide) and 5mL of DCM (dichloromethane), and the mixture was left at room temperature for 30min, the solvent was pumped down with an air pump, and after washing with 10mL of DMF, the solvent was pumped down.
The DMF and DCM referred to in this example are both common reagents with 99.7% purity. The step of pumping the solvent is to pump the solvent in the polypeptide synthesis tube into a pumping filter flask by using an air pump.
3.2. Synthesis starts from the first amino acid on the right (C-terminal) side of the peptide stretch to the left (N-terminal).
3.2.1. 0.8Mmol Fmoc-Gly-OH (glycine) was weighed into a 10mL EP tube, 6mL DMF was added to the EP tube for dissolution, shaking thoroughly was performed, and 1.6mmol (264. Mu.L) DIEA was added to the EP tube.
3.2.2. Transferring the mixed solution into a polypeptide synthesis tube, transferring the polypeptide synthesis tube into a shaking table at a constant temperature of 33 ℃ for shaking overnight, and taking out the polypeptide synthesis tube.
3.2.3. Cleaning: the resin was washed three times (10 mL each) with DMF and the solvent was drained; the resin was rinsed three times (10 mL each) with DCM and the solvent was drained; finally, the solvent was drained after three more DMF washes (10 mL each).
3.3. And (5) deprotection.
3.3.1. 10ML of 20% piperidine solution was added to the polypeptide synthesis tube to submerge the resin, and the mixture was transferred to a 33℃constant temperature shaker for 5min.
3.3.2. The polypeptide synthesis tube was removed from the shaker.
3.3.3. Cleaning: the above 3.2. Washing step 3.2.1 was repeated.
3.4. And a second amino acid.
3.4.1. 0.8Mmol (468.5 mg) of Fmoc-Cys (Trt) -OH (cysteine) and 0.76mmol (314 mg) of condensing agent (HCTU) were weighed into a 10mL EP tube, 6mL of DMF was added to the EP tube and dissolved, thoroughly shaken, and 1.6mmol (264. Mu.L) of DIEA was added to the EP tube.
3.4.2. Transferring the mixed solution into a polypeptide synthesis tube, transferring the polypeptide synthesis tube into a shaking table at a constant temperature of 33 ℃ for 1h, and taking out the polypeptide synthesis tube.
3.4.3. Cleaning: the above 3.2. Washing step 3.2.1 was repeated.
3.5. Peptide chain extension: and (3) continuing to repeat the step 3.3 deprotection and the step 3.4 amino acid synthesis according to the polypeptide sequence until the synthesis of the last amino acid is finished.
3.6. Cleavage of crude peptide.
The polypeptide synthesis tube was removed and the resin was washed three times with DMF (10 mL each time), the solvent was drained after each washing, the resin was washed three times with DCM (10 mL each time), and the solvent was drained after each washing (drained to dry resin). After draining, the cleavage reagent was formulated in a volume ratio of TFA/H2O/phenol/Tips (10 mL/500. Mu.L/500 mg/500. Mu.L) in a 50mL EP tube. Transferring the cutting reagent into the polypeptide synthesis tube, placing the polypeptide synthesis tube into a shaking table at a constant temperature of 26 ℃ for oscillating reaction for 2.5 hours, and taking out the polypeptide synthesis tube, wherein the solution in the tube is the peptide chain lysate.
3.7. Blow-drying and flushing.
3.7.2. 10ML of the peptide chain lysate was transferred to a 50mL EP tube using an ear-washing ball, and the lysate was dried as much as possible to 5mL or less using nitrogen at room temperature.
3.7.3. Adding 40mL of glacial ethyl ether into 50mL of EP pipe, properly shaking the EP pipe, putting the EP pipe into a centrifuge, and centrifuging for 3min at 3500 rpm; after centrifugation, the supernatant was decanted.
3.7.4. And (5) repeating.
3.7.5. Airing at room temperature, and mashing after airing.
3.8. And (5) performing chromatographic analysis and separation on the crude peptide.
The crude peptide was analyzed for correctness by HPLC and mass spectrometry of shimadzu. After verification of correctness, the correct product is isolated and lyophilized.
3.9. And (5) polypeptide renaturation.
0.1M (1.6 g) Tris, 2M (12 g) urea, 1mM (24 mg) cystine and 1mM (10 mg) cysteine are weighed and added into a 500mL volumetric flask, 100mL purified water is weighed and added into the volumetric flask by using a cylinder, the pH is adjusted to 7.5-8.5, 10mg polypeptide is added, the reaction is carried out at room temperature, and the correctness of the crude peptide is analyzed by utilizing HPLC and mass spectrum of Shimadzu. After verification of correctness, the correct product is isolated and lyophilized.
(4) And detecting the functions of the polypeptide inhibitor. (Experimental procedure is the same as in example 4).
Results
Figure 8 shows HPLC purification and mass spectrometry detection patterns of KP1877 polypeptides. FIG. 9 shows concentration response curves for KP1877 polypeptide inhibiting the activity of the α7-type acetylcholine receptor. FIG. 10 shows nuclear magnetic resonance structural diagrams of KP1877 polypeptide. Phage display libraries of bicyclic peptides with a framework GCX1CX2X3X4X5X6CX7X8X9X10X11C(A)( in which the C-terminal a residue is a linker residue (16 AA bicyclic peptide library) were constructed and screened against the α7 type acetylcholine receptor. The results show that the optimal half-inhibitory concentration (IC 50) of the 16AA bicyclic peptide obtained by screening was 0.18 μm at the α7-type acetylcholine receptor, and the biological activity was weaker than that of the 17AA bicyclic peptide (KP 2007, IC 50 =0.12 μm).
Meanwhile, 8 bicyclic peptides (KP 2002-KP 2009) with inhibition effect on alpha 7 type acetylcholine receptors are obtained by screening by using a 17AA bicyclic peptide library, and 1 bicyclic peptide with inhibition effect is obtained by screening by using a 16AA bicyclic peptide library. The result shows that the screening success rate of the 17AA double-ring peptide library is better than that of the 16AA double-ring peptide aiming at the target protein of the alpha 7 type acetylcholine receptor.
Note that: NS, no effective activity data was detected
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (14)
1. A cyclic peptide library, wherein the cyclic peptide comprises an amino acid sequence shown by X pC1C2-Xn-C3-Xm-C4Xq, wherein p = 0-10, q = 0-10, n = 4 and m = 4-16, X is any of 20 natural amino acids, the cyclic peptide library comprising a cyclic peptide having two disulfide bonds comprising a disulfide bond formed by cysteine C 1 with cysteine C 3 and a disulfide bond formed by cysteine C 2 with cysteine C 4, and/or a disulfide bond formed by cysteine C 1 with cysteine C 4 and a disulfide bond formed by cysteine C 2 with cysteine C 3;
Preferably, wherein p=1-8 or 2-6; preferably, wherein q = 1-8 or 2-6; preferably, wherein m=5-9 or 6-8;
Preferably, the cyclic peptide library is a phage-displayed cyclic peptide library, preferably a phage-displayed cyclic peptide library is a single-chain filamentous phage display system, lambda phage display system, T4 phage display system, or T7 phage display system-displayed cyclic peptide library;
Preferably, the single-stranded filamentous phage display system is a PIII display system or PVIII display system;
preferably, the lambda phage display system is a PV display system or a protein D display system.
2. The cyclic peptide library according to claim 1, wherein the cyclic peptide amino acid sequence has a length of 12-50, 13-30, 14-20, or 15-18 amino acid sequences.
3. The cyclic peptide library according to claim 1 or 2, wherein the cyclic peptide library comprises an amino acid sequence shown at X1X2C1C2X3X4X5X6C3X7X8X9X10X1 1X12X13C4, X 1-X13 is any of 20 natural amino acids, preferably the cyclic peptide library comprises cyclic peptides wherein two disulfide bonds are formed between cysteine C 1 and cysteine C 3 and between cysteine C 2 and cysteine C 4, and/or two disulfide bonds are formed between cysteine C 1 and cysteine C 4 and between cysteine C 2 and cysteine C 3.
4. A cyclic peptide library according to any one of claims 1-3, wherein the amino acid sequence further has a linker, preferably a flexible or rigid linker, preferably a linker comprising 1-15, 1-12, 1-10 or 1-5 amino acid residues, preferably a linker comprising (G) n1 or (a) n2, wherein n 1 and n 2 are 1-15, 1-12, 1-10 or 1-5.
5. The cyclic peptide library of any one of claims 3-4, wherein X 1-X13 is any one of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, cysteine, tyrosine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine; preferably, the phage-displayed cyclic peptide library is generated by the PIII display system, preferably by the pcatab 5E phage display system.
6. The cyclic peptide library according to any one of claims 1-5, wherein the phage-displayed cyclic peptide library has one or more of the following properties: each of the plurality of cyclic peptides is present in the cyclic peptide library in an equal or substantially equal percentage; the cyclic peptide library capacity is at least 1X 10 9.
7. The cyclic peptide library according to any one of claims 1 to 6, which is prepared by a method comprising plasmid PCR and a self-ligation step; wherein plasmid PCR is performed on a phage vector using primers, and the PCR product is digested and ligated into a phage vector, wherein the phage vector is a pCANTAB 5E phage vector, preferably a pCANTAB 5E phage vector comprising an S240 site base mutation TCG;
Preferably, the primers are the primers of SEQ ID NO. 9 and SEQ ID NO. 10.
8. The cyclic peptide library according to claim 7, wherein the method comprises introducing a phage vector into a host cell, and adding helper phage when culturing the host cell comprising the phage vector to log phase, separating after culturing to obtain a supernatant, separating phage precipitate from the supernatant, preferably by adding PEG and NaCl to the supernatant, preferably such that 1/5 volume of PEG and NaCl is added; preferably the host cell is an E.coli cell.
9. A method of preparing a cyclic peptide library according to any one of claims 1-8, comprising displaying the cyclic peptide library by a phage display system;
Preferably, the phage-displayed cyclic peptide library is a single-chain filamentous phage display system, lambda phage display system, T4 phage display system, or T7 phage display system-displayed cyclic peptide library;
Preferably, the single-stranded filamentous phage display system is a PIII display system or PVIII display system;
preferably, the lambda phage display system is a PV display system or a protein D display system;
preferably, the PIII display system is the pcatab 5E display system;
Preferably, the phage display system uses a pcatab 5E phage vector, preferably a pcatab 5E phage vector comprising an S240 site base mutation TCG;
Preferably, the method comprises plasmid PCR and a self-ligation step; wherein plasmid PCR is performed on a phage vector using primers, and the PCR product is digested and ligated into a phage vector, wherein the phage vector is a pCANTAB 5E phage vector, preferably a pCANTAB 5E phage vector comprising an S240 site base mutation TCG; preferably, the primers are SEQ ID NO. 9 and SEQ ID NO. 10.
10. The method according to claim 9, further comprising introducing a phage vector into the host cell, and adding a helper phage when culturing the host cell comprising the phage vector to the logarithmic growth phase, separating after culturing to obtain a supernatant, separating phage precipitate from the supernatant, preferably by adding a solution of PEG and NaCl to the supernatant, preferably such that 1/5 volume of PEG and NaCl is added; optionally the method comprises treating the phage under conditions suitable for formation of cysteines to form disulfide bonds to promote disulfide bond formation.
11. A method of screening for cyclic peptides targeting a target protein comprising
(1) Adding a cyclic peptide library according to any one of claims 1-8 to a target protein coated well plate, washing the plate after incubation, and eluting phage that bind to the target protein;
(2) Optionally amplifying the eluted phage and contacting the amplified phage with a target protein coated well plate, washing the plate, and eluting phage that bind to the target protein;
(3) Optionally repeating steps (1) - (2) one or more times;
(4) Optionally sequencing the eluted phage surface displayed polypeptides, determining abundance according to sequencing results and ordering according to abundance, and selecting high abundance phages;
Preferably, the target protein is selected from the group consisting of ion channels, G Protein Coupled Receptors (GPCRs), transport proteins, α7 nicotinic acetylcholine receptor (α7 nAChR) proteins;
preferably, the well plate is a multi-well plate, such as a 96-well plate.
12. A cyclic peptide comprising one or more of the following sequences: 1-8 or 32-39 or a variant thereof, said variant comprising an amino acid sequence mutated by 1-4 amino acid residues compared to any of SEQ ID NOs 1-8 or 32-39 or an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of SEQ ID NOs 1-8 or 32-39, wherein the amino acids at positions corresponding to positions 3 and 9 and the amino acids at positions 4 and 17 are linked by a bond, or the amino acids at positions corresponding to positions 3 and 17 and the amino acids at positions 4 and 9 are linked by a bond, preferably covalently linked in a side chain to side chain manner, of any of SEQ ID NOs 1-8 or 32-39;
preferably, the bond comprises one or more atoms of carbon, sulfur, oxygen, nitrogen or selenium;
Preferably, the bond is -S-S-、-S-O-、-S-Se-、-O-Se-、-O-(CH2)n3-、-S-(CH2)n3-、-Se-(CH2)n3-、-(CH2)n3-、-O-(NH)n3-、-S-(NH)n3-、-Se-(NH)n3-、 or- (NH) n3 -, where n 3 =1-6, e.g. 1-3;
Preferably, the variant retains the original cysteine of any of SEQ ID NOs 1-8 or 32-39 and the mutated amino acid is not a cysteine residue, wherein two disulfide bonds are formed between the 1 st and 3 rd cysteines and between the 2 nd and 4 th cysteines from the N-terminus, or two disulfide bonds are formed between the 1 st and 4 th cysteines and between the 2 nd and 3 rd cysteines;
preferably, the mutation is a substitution, addition, deletion or insertion, preferably a conservative amino acid substitution;
Preferably, the amino acid sequence of the cyclic peptide is SEQ ID NO. 1or 32, wherein two disulfide bonds are formed between the cysteines at positions 3 and 17 and between the cysteines at positions 4 and 9.
13. A method of preparing the cyclic peptide of claim 12 comprising preparing a linear peptide from an amino acid sequence comprising SEQ ID NOs 1-8 or 32-39 or variants thereof and chemically cyclizing the linear peptide to the cyclic peptide of claim 12; alternatively, the polynucleotide encoding the amino acid sequence is introduced into a host cell and expressed, and then the cell supernatant or lysate comprising the cyclic peptide is harvested, optionally purifying the cyclic peptide.
14. Use of the cyclic peptide of claim 12 as an alpha 7-nAChR inhibitor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311346656.4A CN118324860A (en) | 2023-10-17 | 2023-10-17 | Construction of phage display cyclopeptide library based on natural cyclopeptide skeleton |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311346656.4A CN118324860A (en) | 2023-10-17 | 2023-10-17 | Construction of phage display cyclopeptide library based on natural cyclopeptide skeleton |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118324860A true CN118324860A (en) | 2024-07-12 |
Family
ID=91766727
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311346656.4A Pending CN118324860A (en) | 2023-10-17 | 2023-10-17 | Construction of phage display cyclopeptide library based on natural cyclopeptide skeleton |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118324860A (en) |
-
2023
- 2023-10-17 CN CN202311346656.4A patent/CN118324860A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Fang et al. | Review on biomimetic affinity chromatography with short peptide ligands and its application to protein purification | |
| CN111499692A (en) | Polypeptide of targeting novel coronavirus COVID-19 and application thereof | |
| JP2012131816A (en) | BINDING POLYPEPTIDE FOR B LYMPHOCYTE STIMULATOR PROTEIN (BLyS) | |
| AU2003288069B2 (en) | Template fixed beta-hairpin loop mimetics and their use in phage display | |
| JP2000502897A (en) | Methods for identifying compounds that bind to a target | |
| CN113248569B (en) | Leptin receptor affinity peptide and application thereof | |
| CN113461782B (en) | Malachite green antigen mimic epitope, preparation method and application thereof | |
| Barredo-Vacchelli et al. | Peptide affinity chromatography applied to therapeutic antibodies purification | |
| CN113372414B (en) | Fluorescent probe for detecting ochratoxin A and preparation method thereof | |
| CN110317241B (en) | Polypeptide molecule resisting Cry1Da protein and application thereof | |
| CN118324860A (en) | Construction of phage display cyclopeptide library based on natural cyclopeptide skeleton | |
| TW201309720A (en) | Peptide library | |
| CN116640790A (en) | Epitope mimic peptide aiming at ochratoxin A and taking camel-source nano antibody as target, and screening method and application thereof | |
| CN101412750B (en) | Bursin specific binding peptide and screening method thereof | |
| Kunys et al. | Specificity Profiling of Protein‐Binding Domains Using One‐Bead‐One‐Compound Peptide Libraries | |
| CN114891069A (en) | Specific targeting mCN alpha binary cyclic peptide ligand and obtaining method thereof | |
| CN112920277B (en) | VHH-ELISA kit for analyzing cyantraniliprole and chlorantraniliprole residues and application thereof | |
| CN110317242B (en) | Polypeptide molecule capable of specifically binding Cry1Da protein and application thereof | |
| CN114989257A (en) | Amantadine antigen mimic epitope and application thereof in magnetic particle enzymatic chemiluminescence homogeneous immunoassay method | |
| CN108358996B (en) | Antigen epitope peptide simulating diethylstilbestrol, preparation method and application thereof | |
| CN107446021B (en) | Folate receptor alpha specific binding peptide 5 and application thereof | |
| CN110294790B (en) | Polypeptide ligand molecule of Cry1Da protein and application thereof | |
| CN110283232B (en) | Polypeptide molecule combined with Cry1Da protein and application thereof | |
| CN112521452B (en) | Polypeptide of targeted interferon gamma and application thereof | |
| CN113105525B (en) | Material with function of enriching nitrotyrosine peptide fragments and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination |