CN116376867A - 改造型DNA聚合酶φ及其应用 - Google Patents
改造型DNA聚合酶φ及其应用 Download PDFInfo
- Publication number
- CN116376867A CN116376867A CN202211063781.XA CN202211063781A CN116376867A CN 116376867 A CN116376867 A CN 116376867A CN 202211063781 A CN202211063781 A CN 202211063781A CN 116376867 A CN116376867 A CN 116376867A
- Authority
- CN
- China
- Prior art keywords
- amino acid
- dna polymerase
- acid sequence
- seq
- modified dna
- 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
- 101710197907 rDNA transcriptional regulator pol5 Proteins 0.000 title abstract description 3
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 88
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 82
- 108020004414 DNA Proteins 0.000 claims abstract description 64
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 53
- 230000000694 effects Effects 0.000 claims abstract description 22
- 230000004568 DNA-binding Effects 0.000 claims abstract description 14
- 238000012216 screening Methods 0.000 claims abstract description 9
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 8
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 8
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 8
- 238000009510 drug design Methods 0.000 claims abstract description 7
- 238000012916 structural analysis Methods 0.000 claims abstract description 7
- 238000007877 drug screening Methods 0.000 claims abstract description 5
- 150000003384 small molecules Chemical class 0.000 claims abstract description 5
- 238000004458 analytical method Methods 0.000 claims abstract description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 44
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 44
- 210000004027 cell Anatomy 0.000 claims description 43
- 108091033319 polynucleotide Proteins 0.000 claims description 23
- 102000040430 polynucleotide Human genes 0.000 claims description 23
- 239000002157 polynucleotide Substances 0.000 claims description 22
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 17
- 239000004365 Protease Substances 0.000 claims description 12
- 108091005804 Peptidases Proteins 0.000 claims description 11
- 238000003776 cleavage reaction Methods 0.000 claims description 10
- 230000007017 scission Effects 0.000 claims description 10
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 8
- 239000013604 expression vector Substances 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 8
- 125000000539 amino acid group Chemical group 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- 210000004899 c-terminal region Anatomy 0.000 claims description 4
- 238000002405 diagnostic procedure Methods 0.000 claims description 2
- 201000010099 disease Diseases 0.000 claims description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 2
- 230000001225 therapeutic effect Effects 0.000 claims description 2
- 238000002560 therapeutic procedure Methods 0.000 claims description 2
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 21
- 150000001413 amino acids Chemical class 0.000 description 18
- 230000014509 gene expression Effects 0.000 description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 239000000523 sample Substances 0.000 description 12
- 238000006467 substitution reaction Methods 0.000 description 12
- 239000000872 buffer Substances 0.000 description 11
- 238000005516 engineering process Methods 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 9
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 9
- 108010052968 leupeptin Proteins 0.000 description 9
- 241000701447 unidentified baculovirus Species 0.000 description 9
- 108060004795 Methyltransferase Proteins 0.000 description 8
- 206010028980 Neoplasm Diseases 0.000 description 8
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 230000008439 repair process Effects 0.000 description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- 241000238631 Hexapoda Species 0.000 description 6
- 230000005782 double-strand break Effects 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 238000009396 hybridization Methods 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 229920001184 polypeptide Polymers 0.000 description 6
- 102000004196 processed proteins & peptides Human genes 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 5
- 239000007995 HEPES buffer Substances 0.000 description 5
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 5
- 230000004071 biological effect Effects 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 230000037361 pathway Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 238000001042 affinity chromatography Methods 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000011559 double-strand break repair via nonhomologous end joining Effects 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000005457 optimization Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 102000053602 DNA Human genes 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 102000035195 Peptidases Human genes 0.000 description 3
- 230000027455 binding Effects 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 239000012520 frozen sample Substances 0.000 description 3
- 108020001507 fusion proteins Proteins 0.000 description 3
- 102000037865 fusion proteins Human genes 0.000 description 3
- 239000010931 gold Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 229910052759 nickel Inorganic materials 0.000 description 3
- 230000006780 non-homologous end joining Effects 0.000 description 3
- 238000012856 packing Methods 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- -1 small molecule compound Chemical class 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 229930182566 Gentamicin Natural products 0.000 description 2
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 2
- 241000606768 Haemophilus influenzae Species 0.000 description 2
- 241000235058 Komagataella pastoris Species 0.000 description 2
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 2
- 241000607142 Salmonella Species 0.000 description 2
- 241000194017 Streptococcus Species 0.000 description 2
- 102100036407 Thioredoxin Human genes 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 125000004429 atom Chemical group 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 238000009509 drug development Methods 0.000 description 2
- 101150015101 dsbC gene Proteins 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 229940047650 haemophilus influenzae Drugs 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 229940042040 innovative drug Drugs 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion 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
- 238000011068 loading method Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 108091005573 modified proteins Proteins 0.000 description 2
- 102000035118 modified proteins Human genes 0.000 description 2
- 229940126586 small molecule drug Drugs 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 108060008226 thioredoxin Proteins 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 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 1
- 108010091324 3C proteases Proteins 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- XZWXFWBHYRFLEF-FSPLSTOPSA-N Ala-His Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 XZWXFWBHYRFLEF-FSPLSTOPSA-N 0.000 description 1
- IPWKGIFRRBGCJO-IMJSIDKUSA-N Ala-Ser Chemical compound C[C@H]([NH3+])C(=O)N[C@@H](CO)C([O-])=O IPWKGIFRRBGCJO-IMJSIDKUSA-N 0.000 description 1
- TWXZVVXRRRRSLT-IMJSIDKUSA-N Asn-Cys Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CS)C(O)=O TWXZVVXRRRRSLT-IMJSIDKUSA-N 0.000 description 1
- FRYULLIZUDQONW-IMJSIDKUSA-N Asp-Asp Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(O)=O FRYULLIZUDQONW-IMJSIDKUSA-N 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 101000741396 Chlamydia muridarum (strain MoPn / Nigg) Probable oxidoreductase TC_0900 Proteins 0.000 description 1
- 101000741399 Chlamydia pneumoniae Probable oxidoreductase CPn_0761/CP_1111/CPj0761/CpB0789 Proteins 0.000 description 1
- 101000741400 Chlamydia trachomatis (strain D/UW-3/Cx) Probable oxidoreductase CT_610 Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical group [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- 102100029995 DNA ligase 1 Human genes 0.000 description 1
- 102100033688 DNA ligase 3 Human genes 0.000 description 1
- 108010093204 DNA polymerase theta Proteins 0.000 description 1
- 102100029766 DNA polymerase theta Human genes 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 101100388059 Drosophila melanogaster PolQ gene Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000588921 Enterobacteriaceae Species 0.000 description 1
- OTMSDBZUPAUEDD-UHFFFAOYSA-N Ethane Chemical compound CC OTMSDBZUPAUEDD-UHFFFAOYSA-N 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- JEFZIKRIDLHOIF-BYPYZUCNSA-N Gln-Gly Chemical compound NC(=O)CC[C@H](N)C(=O)NCC(O)=O JEFZIKRIDLHOIF-BYPYZUCNSA-N 0.000 description 1
- XITLYYAIPBBHPX-ZKWXMUAHSA-N Gln-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CCC(N)=O XITLYYAIPBBHPX-ZKWXMUAHSA-N 0.000 description 1
- KOSRFJWDECSPRO-WDSKDSINSA-N Glu-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(O)=O KOSRFJWDECSPRO-WDSKDSINSA-N 0.000 description 1
- 102000005720 Glutathione transferase Human genes 0.000 description 1
- 108010070675 Glutathione transferase Proteins 0.000 description 1
- WSDOHRLQDGAOGU-BQBZGAKWSA-N His-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CN=CN1 WSDOHRLQDGAOGU-BQBZGAKWSA-N 0.000 description 1
- 101000863770 Homo sapiens DNA ligase 1 Proteins 0.000 description 1
- 101000927847 Homo sapiens DNA ligase 3 Proteins 0.000 description 1
- 101000619640 Homo sapiens Leucine-rich repeats and immunoglobulin-like domains protein 1 Proteins 0.000 description 1
- 241000430519 Human rhinovirus sp. Species 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- VTJUNIYRYIAIHF-IUCAKERBSA-N Leu-Pro Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(O)=O VTJUNIYRYIAIHF-IUCAKERBSA-N 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101000708578 Milk vetch dwarf virus (isolate N) Para-Rep C3 Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 241001195348 Nusa Species 0.000 description 1
- 239000012661 PARP inhibitor Substances 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- FSXRLASFHBWESK-HOTGVXAUSA-N Phe-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 FSXRLASFHBWESK-HOTGVXAUSA-N 0.000 description 1
- 229940121906 Poly ADP ribose polymerase inhibitor Drugs 0.000 description 1
- 102000006010 Protein Disulfide-Isomerase Human genes 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- RZEQTVHJZCIUBT-WDSKDSINSA-N Ser-Arg Chemical compound OC[C@H](N)C(=O)N[C@H](C(O)=O)CCCNC(N)=N RZEQTVHJZCIUBT-WDSKDSINSA-N 0.000 description 1
- LZLREEUGSYITMX-JQWIXIFHSA-N Ser-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CO)N)C(O)=O)=CNC2=C1 LZLREEUGSYITMX-JQWIXIFHSA-N 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 241000519995 Stachys sylvatica Species 0.000 description 1
- 240000005572 Syzygium cordatum Species 0.000 description 1
- 235000006650 Syzygium cordatum Nutrition 0.000 description 1
- DSGIVWSDDRDJIO-ZXXMMSQZSA-N Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O DSGIVWSDDRDJIO-ZXXMMSQZSA-N 0.000 description 1
- 108020004566 Transfer RNA Proteins 0.000 description 1
- CGWAPUBOXJWXMS-HOTGVXAUSA-N Tyr-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 CGWAPUBOXJWXMS-HOTGVXAUSA-N 0.000 description 1
- 108090000848 Ubiquitin Proteins 0.000 description 1
- 102000044159 Ubiquitin Human genes 0.000 description 1
- XXDVDTMEVBYRPK-XPUUQOCRSA-N Val-Gln Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O XXDVDTMEVBYRPK-XPUUQOCRSA-N 0.000 description 1
- JKHXYJKMNSSFFL-IUCAKERBSA-N Val-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(O)=O)CCCCN JKHXYJKMNSSFFL-IUCAKERBSA-N 0.000 description 1
- ABUBSBSOTTXVPV-UHFFFAOYSA-H [U+6].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O Chemical compound [U+6].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O ABUBSBSOTTXVPV-UHFFFAOYSA-H 0.000 description 1
- 239000012932 acetate dye Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 108010070944 alanylhistidine Proteins 0.000 description 1
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 1
- 238000011021 bench scale process Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- FSXRLASFHBWESK-UHFFFAOYSA-N dipeptide phenylalanyl-tyrosine Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FSXRLASFHBWESK-UHFFFAOYSA-N 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 125000005842 heteroatom Chemical group 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 150000002611 lead compounds Chemical class 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 108091005763 multidomain proteins Proteins 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000010248 power generation Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 108020003519 protein disulfide isomerase Proteins 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 108020004418 ribosomal RNA Proteins 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000003007 single stranded DNA break Effects 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229940094937 thioredoxin Drugs 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0601—Invertebrate cells or tissues, e.g. insect cells; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/07—Nucleotidyltransferases (2.7.7)
- C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14041—Use of virus, viral particle or viral elements as a vector
- C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
Description
技术领域
背景技术
维持基因组的稳定性是对于细胞的正常生长至关重要的。尽管细胞有着一套精密的DNA复制以及修复系统来维持其稳定性,但仍不可避免地会产生各种各样的DNA损伤,例如DNA双链断裂(DSBs,Double-strand breaks)。引起DSBs的因素也多种多样,如各种辐射、化学药剂等,DSBs产生的有害后果,包括基因组重排和细胞死亡等。双链DNA断裂作为真核细胞中常见的事件,其主要具有三种主要的修复途径:同源重组(homologousrecombination,HR),非同源DNA末端连接(nonhomologous DNA end joining,NHEJ)以及DNA聚合酶介导的末端连接(DNA polymerase theta mediated end-joining,TMEJ)1,2。NHEJ是发生在复制之外的主要DSB修复途径。在没有NHEJ的情况下,断裂的末端被核酸酶切除,然后暴露的单链DNA尾部被HR或TMEJ修复。TMEJ是主要DSB修复途径(HR和NHEJ)的一个重要替代途径,近年来在越来越多的癌症模型研究中备受关注3。研究表明,DNA聚合酶/>介导的TMEJ活性对于BRCA突变的癌细胞的存活至关重要4。DNA聚合酶/>在多个不同的肿瘤中呈现上调现象,并且它的过表达与肿瘤预后不佳相关5–7。研究表明,在HR缺陷的背景下敲除DNA聚合酶/>被证明会损害细胞的活力,可能的机制是DNA聚合酶/>和HR因子之间的合成致死关系8。由于DNA聚合酶/>在TMEJ中的活性,HR缺陷的癌细胞存活依赖于DNA聚合酶/>(或简称为PolQ)。癌细胞的持续增殖导致慢性复制压力,并且当出错的复制叉没有得到解决时,DSBs的负荷增加。虽然这种DSBs在健康细胞中会被HR修复,但HR缺陷的癌细胞却要依靠TMEJ来修复它们9。因此,作用于TMEJ通路的LIG3和LIG1的抑制剂,在人类乳腺癌细胞系中与PARP抑制剂协同作用。由于这些原因,目前多个生物技术公司和实验室正在开发靶向DNA聚合酶/>的抑制剂,这也为个性化的肿瘤治疗提供了新的思路。
全长的DNA聚合酶是一个由2590个氨基酸组成的多结构域蛋白。它的N端具有一个保守的SF2超家族解旋酶样结构域(简称为解旋酶结构域),C端具有一个A家族DNA聚合酶结构域,两个结构域由一个无二级结构的中间区域分开10。根据AlphaFold结构预测,DNA聚合酶/>蛋白中间的无序区约有近900个氨基酸,这为表达、纯化和制备DNA聚合酶/>蛋白并用于药物筛选研究设置了障碍。研究表明,DNA聚合酶/>的N端和C端两个结构域都是潜在的药物靶点,分别针对两个结构域开发小分子或大分子抑制剂具有重要的临床价值10。结构和功能分析表明,DNA聚合酶/>的N端的解旋酶结构域在DSB修复过程中挥了核心作用11。
2017年,诺贝尔化学奖授予三位在冷冻电镜技术开发过程中作出卓越贡献的科学家12,这标志着以冷冻电镜为主导的结构生物学领域进入一个新时代13。基于靶点结构的药物设计是对传统新药研发方案的革新14,15。在AI赋能创新药研发的时代,如何利用蛋白质的序列信息进行结构解析工作,并基于计算机辅助的药物设计(computer-aided drugdesign,CADD)或基于分子结构的药物设计(structure-based drug design,SBDD)思路,进行活性小分子药物的设计和筛选,亟需创新性的新思维和新方案。同时,DNA编码的小分子化合物库(DNA Encoded Library,DEL)筛选的小分子药苗头化合物的技术名声鹊起。越来越多的大药企开始投资建立自己的DEL技术平台,例如葛兰素史克成功运用DEL技术找到两个可成药的分子并快速推进至临床。
发明内容
发明要解决的问题
用于解决问题的方案
(i)如SEQ ID NO:2所示的氨基酸序列;
(ii)与SEQ ID NO:2所示的氨基酸序列具有至少80%、82%、85%、87%、90%、92%、95%、96%、97%、98%或99%同一性的氨基酸序列,并且其保留如SEQ ID NO:2所示的氨基酸序列的DNA结合活性和蛋白结构;
(iii)在SEQ ID NO:2所示的氨基酸序列中添加、取代、缺失或插入1个或多个氨基酸残基的氨基酸序列,并且其保留如SEQ ID NO:2所示的氨基酸序列的DNA结合活性和蛋白结构;或者,
(iv)由核苷酸序列编码的氨基酸序列,所述核苷酸序列与编码如SEQ ID NO:2所示的氨基酸序列的多核苷酸序列在严格条件下杂交,并且所述氨基酸序列保留以SEQ IDNO:2所示的氨基酸序列的DNA结合活性和蛋白结构,所述严格条件是中等严格条件,中-高严格条件,高严格条件或非常高严格条件。
(i)如SEQ ID NO:3所示的氨基酸序列;
(ii)与SEQ ID NO:3所示的氨基酸序列具有至少80%、82%、85%、87%、90%、92%、95%、96%、97%、98%或99%同一性的氨基酸序列,并且其保留如SEQ ID NO:3所示的氨基酸序列的DNA结合活性和蛋白结构;
(iii)在SEQ ID NO:3所示的氨基酸序列中添加、取代、缺失或插入1个或多个氨基酸残基的氨基酸序列,并且其保留如SEQ ID NO:3所示的氨基酸序列的DNA结合活性和蛋白结构;或者,
(iv)由核苷酸序列编码的氨基酸序列,所述核苷酸序列与编码如SEQ ID NO:3所示的氨基酸序列的多核苷酸序列在严格条件下杂交,并且所述氨基酸序列保留以SEQ IDNO:3所示的氨基酸序列的DNA结合活性和蛋白结构,所述严格条件是中等严格条件,中-高严格条件,高严格条件或非常高严格条件。
在一些具体的实施方案中,所述多核苷酸的序列如SEQ ID NO:4所示。
本发明的第三方面提供了一种表达载体,其包含如本发明第二方面所述的多核苷酸。
本发明的第四方面提供了一种宿主细胞,其包含如本发明第三方面所述的表达载体。
本发明的第五方面提供了如本发明第一方面所述的改造型DNA聚合酶如本发明第二方面所述的多核苷酸、如本发明第三方面所述的表达载体或如本发明第四方面所述的宿主细胞在用于DNA聚合酶/>结构解析、DNA聚合酶/>活性分析、核酸编码小分子库筛选、计算机辅助的药物设计和药物筛选中的用途;所述用途为非疾病治疗或诊断方法。
发明的效果
附图说明
具体实施方式
为了更容易理解本发明,以下具体定义了某些技术和科学术语。除非在本文中另有明确定义,本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
本说明书中,使用“数值A~数值B”表示的数值范围是指包含端点数值A、B的范围。
本说明书中,使用“基本上”或“实质上”表示与理论模型或理论数据的标准偏差在5%、优选为3%、更优选为1%范围以内。
本说明书中,使用“可以”表示的含义包括了进行某种处理以及不进行某种处理两方面的含义。
本说明书中,“任选的”或“任选地”是指接下来描述的事件或情况可发生或可不发生,并且该描述包括该事件发生的情况和该事件不发生的情况。
本说明书中,所提及的“一些具体/优选的实施方案”、“另一些具体/优选的实施方案”、“实施方案”等是指所描述的与该实施方案有关的特定要素(例如,特征、结构、性质和/或特性)包括在此处所述的至少一种实施方案中,并且可存在于其它实施方案中或者可不存在于其它实施方案中。另外,应理解,所述要素可以任何合适的方式组合在各种实施方案中。
根据本发明,术语“多肽”、“蛋白质”、“肽”在本文中可互换的使用,指任何长度的氨基酸的聚合形态,可包括编码的和非编码的氨基酸,化学或生物化学修饰的或衍生的氨基酸,和具有相似的肽骨架的多肽。
根据本发明,术语“核酸分子”、“多核苷酸”、“多聚核酸”、“核酸”可互换的使用,指任何长度的核苷酸的聚合形态,不论是脱氧核糖核苷酸或核糖核苷酸,或其类似物。多核苷酸可具有任何三维结构,可实施任何已知或未知的功能。多核苷酸的非限制例子包括基因、基因片段、外显子、内含子、信使RNA(mRNA)、转运RNA、核糖体RNA、核酶、cDNA、重组多核苷酸、分支多核苷酸、质粒、载体、任何序列的分离的DNA、控制区、任何序列的分离的RNA、核酸探针和引物。核酸分子可以是线性或环状的。
根据本发明,所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
根据本发明,术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员,例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株;芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis);肺炎球菌(Pneumococcus);链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。
根据本发明,氨基酸“添加”指在氨基酸序列的C端或N端添加氨基酸。根据本发明,氨基酸“缺失”指可以从氨基酸序列中删除1、2或3个以上氨基酸。根据本发明,氨基酸“插入”指在氨基酸序列中的适当位置插入氨基酸残基,插入的氨基酸残基也可以全部或部分彼此相邻,或插入的氨基酸之间都不彼此相邻。
根据本发明,氨基酸“取代”指在氨基酸序列中的某个位置的某个氨基酸残基被其他氨基酸残基替代;其中,“取代”可以是保守氨基酸取代。
根据本发明,“保守修饰”、“保守取代”或“保守置换”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸,使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓,一般而言,多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页,(第4版))。另外,结构或功能类似的氨基酸的置换不大可能破坏生物学活性。示例性保守取代于以下“示例性氨基酸保守取代”中陈述。
示例性氨基酸保守取代
原始残基 | 保守取代 |
Ala(A) | Gly;Ser |
Arg(R) | Lys;His |
Asn(N) | Gln;His;Asp |
Asp(D) | Glu;Asn |
Cys(C) | Ser;Ala;Val |
Gln(Q) | Asn;Glu |
Glu(E) | Asp;Gln |
Gly(G) | Ala |
His(H) | Asn;Gln |
Ile(I) | Leu;Val |
Leu(L) | Ile;Val |
Lys(K) | Arg;His |
Met(M) | Leu;Ile;Tyr |
Phe(F) | Tyr;Met;Leu |
Pro(P) | Ala |
Ser(S) | Thr |
Thr(T) | Ser |
Trp(W) | Tyr;Phe |
Tyr(Y) | Trp;Phe |
Val(V) | Ile;Leu |
根据本发明,“中等至非常高等严格条件”包括“中等严格条件”,“中-高严格条件”,“高严格条件”或“非常高严格条件”,其描述了核酸杂交和洗涤的条件。进行杂交反应的指导参见Current Protocols in Molecular Biology,John Wiley&Sons,N.Y.(1989),6.3.1-6.3.6,其通过引用并入本文。在该文献中描述了含水的和非含水的方法,且可以使用任一种。例如,具体的杂交条件如下:(1)低严格性杂交条件在6×氯化钠/柠檬酸钠(SSC)中,在约45℃,然后在至少50℃,在0.2×SSC,0.1%SDS中洗涤2次(对于低严格性条件,可以将洗涤温度升高到55℃);(2)中等严格性杂交条件在6×SSC,在约45℃,然后在60℃,在0.2×SSC,0.1%SDS中洗涤1次或多次;(3)高严格性杂交条件在6×SSC,在约45℃,然后在65℃,在0.2×SSC,0.1%SDS中洗涤1次或多次且优选;(4)非常高的严格性杂交条件是0.5M磷酸钠,7%SDS,在65℃,然后在65℃,在0.2×SSC,1%SDS中洗涤1次或多次。
根据本发明,“同源性”或“同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时,例如如果两个DNA分子的每一个位置都被腺嘌呤占据时,那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如,在序列最佳比对时,如果两个序列中的10个位置有6个匹配或同源,那么两个序列为60%同源;如果两个序列中的100个位置有95个匹配或同源,那么两个序列为95%同源。通常,当比对两个序列时进行比较以给出最大百分比同源性。例如,可以通过BLAST算法执行比较,其中选择算法的参数以在各个参考序列的整个长度上给出各个序列之间的最大匹配。以下参考文献涉及经常用于序列分析的BLAST算法:BLAST算法(BLAST ALGORITHMS):Altschul,S.F.等人,(1990)J.Mol.Biol.215:403-410;Gish,W.等人,(1993)NatureGenet.3:266-272;Madden,T.L.等人,(1996)Meth.Enzymol.266:131-141;Altschul,S.F.等人,(1997)Nucleic Acids Res.25:3389-3402;Zhang,J.等人,(1997)Genome Res.7:649-656。其他如NCBI BLAST提供的常规BLAST算法也为本领域技术人员所熟知。
根据本发明,术语“密码子优化”是指编码多肽的核苷酸序列已被配置为包含宿主细胞或生物体优选的密码子,以改善宿主细胞或生物体中的基因表达并提高翻译效率。
根据本发明,术语“标签”是指这样的短肽,其与目的蛋白(例如本发明的改造型DNA聚合酶)融合或连接,并由此促进重组蛋白的可溶性表达、检测和/或纯化。标签可融合或连接至目的蛋白的N端和/或C端(任选地通过接头或蛋白酶切割位点)。此类标签是本领域技术人员熟知的,并且已在现有技术文献中进行了详细描述。例如,此类标签包括但不限于,组氨酸标签(Sockolosky,J.T.and F.C.Szoka(2013).Protein Expr Purif 87(2):129-135)、谷胱甘肽转移酶(GST)标签(Hayashi,K.and C.Kojima(2008).ProteinExprPurif62(1):120-127)、麦芽糖结合蛋白(MBP)标签(Bataille,L.,W.Dieryck,A.Hocquellet,C.Cabanne,K.Bathany,S.Lecommandoux,B.Garbay and E.Garanger,Protein Expression and Purification Volume 110,June 2015,Pages165-171)、硫氧还蛋白(Trx)标签(Tomala,M.,A.Lavrentieva,P.Moretti,U.Rinas,C.Kasper,F.Stahl,A.Schambach,E.Warlich,U.Martin,T.Cantz and T.Scheper,2010,Protein ExprPurif73(1):51-57)、NusA标签(Li,K.,T.Jiang,B.Yu,L.Wang,C.Gao,C.Ma,P.Xu and Y.Ma(2013).Sci Rep3:2347)、二硫键异构酶DsbA标签(Zhang,Y.,D.R.Olsen,K.B.Nguyen,P.S.Olson,E.T.Rhodes and D.Mascarenhas(1998).Protein Expr Purif 12(2):159-165)、DsbC标签(Kurokawa,Y.,H.Yanagi and T.Yura(2001).J Biol Chem 276(17):14393-14399)、SUMO标签(Marblestone,J.G.,S.C.Edavettal,Y.Lim,P.Lim,X.Zuo and T.R.Butt(2006).Protein Sci 15(1):182-189)、msyB标签(Zou,Z.,L.Cao,P.Zhou,Y.Su,Y.Sun andW.Li(2008).J Biotechnol 135(4):333-339)、TF标签、引发因子标签(Kim,E.K.,J.C.Moon,J.M.Lee,M.S.Jeong,C.Oh,S.M.Ahn,Y.J.Yoo and H.H.Jang(2012).Protein ExprPurif86(1):53-57)、泛素标签(Sabin,E.A.,Lee-Ng,Chun Ting,Shuster,Jeffrey R.,Barr,Philip J.(1989).Nature Biotechnology7(7):705-709)、Myc标签、Flag标签、荧光蛋白(例如GFP)标签(Pedelacq,J.D.,S.Cabantous,T.Tran,T.C.Terwilliger and G.S.Waldo(2006).Nat Biotechnol24(1):79-88)、Twin-strep标签、生物素标签、以及亲和素标签。
根据本发明,术语“蛋白酶切割位点”是指,能够被蛋白酶特异性识别并切割的位点。各种特异性蛋白酶及其识别位点是本领域技术人员所熟知的,并见于许多现有技术文献中。本领域技术人员可根据实际情况,在融合蛋白中使用合适的蛋白酶切割位点,并用相应的蛋白酶进行切割。蛋白酶切割位点的使用可以是有利的,例如,其可用于从融合蛋白中切除信号肽和/或标签,从而获得具有目的活性的成熟蛋白。
根据本发明,术语“肽接头”或“为人工连接序列”是指用于连接两个分子(例如蛋白)的短肽。通常,通过将编码该短肽的多核苷酸序列引入(例如,通过PCR扩增或连接酶)分别编码所要连接的两种目的蛋白的两个DNA片段之间,并进行蛋白质表达来获得融合蛋白,例如目的蛋白1-肽接头-目的蛋白2。或者,通过将编码该短肽的多核苷酸序列引入分别编码所要连接的目的蛋白以及蛋白酶切割位点和/或标签序列之间,例如目的蛋白-肽接头-蛋白酶切割位点-标签序列。
根据本发明,术语“载体”是指,可将多核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸所编码的蛋白获得表达时,载体称为表达载体。载体可以通过转化,转导或者转染导入宿主细胞,使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的,包括但不限于:质粒、噬菌体、柯斯质粒等等。
根据本发明,术语“细胞”、“细胞系”和“细胞培养物”可互换使用,并且所有这类名称都包括后代。因此,术语“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物,而不考虑转移数目。还应当理解的是,由于故意或非有意的突变,所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下,其由上下文清楚可见。
以下结合附图,通过实施例进一步说明本发明,但不作为对本发明的限制。以下提供了本发明实施方案中所使用的具体材料及其来源。但是,应当理解的是,这些仅仅是示例性的,并不意图限制本发明,与如下试剂和仪器的类型、型号、品质、性质或功能相同或相似的材料均可以用于实施本发明。下述实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
1、序列优化
在本实施例中,所采用的预测蛋白结构软件为AlphaFold本地版(v2.1.0)。
针对野生型DNA聚合酶的解旋酶结构域具有较长的无序区,在蛋白的表达过程中,容易被蛋白酶降解,并且存在的较长的无序区,不适合蛋白质结构解析。本实施例中,根据AlphaFold结构预测,对全长的DNA聚合酶/>进行截断,保留解旋酶结构域。
野生型DNA聚合酶的氨基酸序列(SEQ ID NO:1):MNLLRRSGKRRRSESGSDSFSGSGGDSSASPQFLSGSVLSPPPGLGRCLKAAAAGECKPTVPDYERDKLLLANWGLPKAVLEKYHSFGVKKMFEWQAECLLLG QVLEGKNLVYSAPTSAGKTLVAELLILKRVLEMRKKALFILPFVSVAKEKKYYLQSLFQEVGIKVDGYMGSTSPSRH FSSLDIAVCTIERANGLINRLIEENKMDLLGMVVVDELHMLGDSHRGYLLELLLTKICYITRKSASCQADLASSLSN AVQIVGMSATLPNLELVASWLNAELYHTDFRPVPLLESVKVGNSIYDSSMKLVREFEPMLQVKGDEDHVVSLCYETI CDNHSVLLFCPSKKWCEKLADIIAREFYNLHHQAEGLVKPSECPPVILEQKELLEVMDQLRRLPSGLDSVLQKTVPW GVAFHHAGLTFEERDIIEGAFRQGLIRVLAATSTLSSGVNLPARRVIIRTPIFGGRPLDILTYKQMVGRAGRKGVDT VGESILICKNSEKSKGIALLQGSLKPVRSCLQRREGEEVTGSMIRAILEIIVGGVASTSQDMHTYAACTFLAASMKE GKQGIQRNQESVQLGAIEACVMWLLENEFIQSTEASDGTEGKVYHPTHLGSATLSSSLSPADTLDIFADLQRAMKGF VLENDLHILYLVTPMFEDWTTIDWYRFFCLWEKLPTSMKRVAELVGVEEGFLARCVKGKVVARTERQHRQMAIHKRF FTSLVLLDLISEVPLREINQKYGCNRGQIQSLQQSAAVYAGMITVFSNRLGWHNMELLLSQFQKRLTFGIQRELCDL VRVSLLNAQRARVLYASGFHTVADLARANIVEVEVILKNAVPFKSARKAVDEEEEAVEERRNMRTIWVTGRKGLTER EAAALIVEEARMILQQDLVEMGVQWNPCALLHSSTCSLTHSESEVKEHTFISQTKSSYKKLTSKNKSNTIFSDSYIKHSPNIVQDLNKSREHTSSFNCNFQNGNQEHQTCSIFRARKRASLDINKEKPGASQNEGKTSDKKVVQTFSQKTKKAPLNFNSEKMSRSFRSWKRRKHLKRSRDSSPLKDSGACRIHLQGQTLSNPSLCEDPFTLDEKKTEFRNSGPFAKNVSLSGKEKDNKTSFPLQIKQNCSWNITLTNDNFVEHIVTGSQSKNVTCQATSVVSEKGRGVAVEAEKINEVLIQNGSKNQNVYMKHHDIHPINQYLRKQSHEQTSTITKQKNIIERQMPCEAVSSYINRDSNVTINCERIKLNTEENKPSHFQALGDDISRTVIPSEVLPSAGAFSKSEGQHENFLNISRLQEKTGTYTTNKTKNNHVSDLGLVLCDFEDSFYLDTQSEKIIQQMATENAKLGAKDTNLAAGIMQKSLVQQNSMNSFQKECHIPFPAEQHPLGATKIDHLDLKTVGTMKQSSDSHGVDILTPESPIFHSPILLEENGLFLKKNEVSVTDSQLNSFLQGYQTQETVKPVILLIPQKRTPTGVEGECLPVPETSLNMSDSLLFDSFSDDYLVKEQLPDMQMKEPLPSEVTSNHFSDSLCLQEDLIKKSNVNENQDTHQQLTCSNDESIIFSEMDSVQMVEALDNVDIFPVQEKNHTVVSPRALELSDPVLDEHHQGDQDGGDQDERAEKSKLTGTRQNHSFIWSGASFDLSPGLQRILDKVSSPLENEKLKSMTINFSSLNRKNTELNEEQEVISNLETKQVQGISFSSNNEVKSKIEMLENNANHDETSSLLPRKESNIVDDNGLIPPTPIPTSASKLTFPGILETPVNPWKTNNVLQPGESYLFGSPSDIKNHDLSPGSRNGFKDNSPISDTSFSLQLSQDGLQLTPASSSSESLSIIDVASDQNLFQTFIKEWRCKKRFSISLACEKIRSLTSSKTATIGSRFKQASSPQEIPIRDDGFPIKGCDDTLVVGLAVCWGGRDAYYFSLQKEQKHSEISASLVPPSLDPSLTLKDRMWYLQSCLRKESDKECSVVIYDFIQSYKILLLSCGISLEQSYEDPKVACWLLDPDSQEPTLHSIVTSFLPHELPLLEGMETSQGIQSLGLNAGSEHSGRYRASVESILIFNSMNQLNSLLQKENLQDVFRKVEMPSQYCLALLELNGIGFSTAECESQKHIMQAKLDAIETQAYQLAGHSFSFTSSDDIAEVLFLELKLPPNREMKNQGSKKTLGSTRRGIDNGRKLRLGRQFSTSKDVLNKLKALHPLPGLILEWRRITNAITKVVFPLQREKCLNPFLGMERIYPVSQSHTATGRITFTEPNIQNVPRDFEIKMPTLVGESPPSQAVGKGLLPMGRGKYKKGFSVNPRCQAQMEERAADRGMPFSISMRHAFVPFPGGSILAADYSQLELRILAHLSHDRRLIQVLNTGADVFRSIAAEWKMIEPESVGDDLRQQAKQICYGIIYGMGAKSLGEQMGIKENDAACYIDSFKSRYTGINQFMTETVKNCKRDGFVQTILGRRRYLPGIKDNNPYRKAHAERQAINTIVQGSAADIVKIATVNIQKQLETFHSTFKSHGHREGMLQSDQTGLSRKRKLQGMFCPIRGGFFILQLHDELLYEVAEEDVVQVAQIVKNEMESAVKLSVKLKVKVKIGASWGELKDFDV
DKLLLANWGLPKAVLEKYHSFGVKKMFEWQAECLLLGQVLEGKNLVYSAPTSAGKTLVAELLILKRVLEMRKKALFILPFVSVAKEKKYYLQSLFQEVGIKVDGYMGSTSPSRHFSSLDIAVCTIERANGLINRLIEENKMDLLGMVVVDELHMLGDSHRGYLLELLLTKICYITRKSASCQADLASSLSNAVQIVGMSATLPNLELVASWLNAELYHTDFRPVPLLESVKVGNSIYDSSMKLVREFEPMLQVKGDEDHVVSLCYETICDNHSVLLFCPSKKWCEKLADIIAREFYNLHHQAEGLVKPSECPPVILEQKELLEVMDQLRRLPSGLDSVLQKTVPWGVAFHHAGLTFEERDIIEGAFRQGLIRVLAATSTLSSGVNLPARRVIIRTPIFGGRPLDILTYKQMVGRAGRKGVDTVGESILICKNSEKSKGIALLQGSLKPVRSCLQRREGEEVTGSMIRAILEIIVGGVASTSQDMHTYAACTFLAASMKEGKQGIQRNQESVQLGAIEACVMWLLENEFIQSTEASDGTEGKVYHPTHLGSATLSSSLSPADTLDIFADLQRAMKGFVLENDLHILYLVTPMFEDWTTIDWYRFFCLWEKLPTSMKRVAELVGVEEGFLARCVKGKVVARTERQHRQMAIHKRFFTSLVLLDLISEVPLREINQKYGCNRGQIQSLQQSAAVYAGMITVFSNRLGWHNMELLLSQFQKRLTFGIQRELCDLVRVSLLNAQRARVLYASGFHTVADLARANIVEVEVILKNAVPFKSARKAVDEEEEAVEERRNMRTIWVTGRKGLTEREAAALIVEEARMILQQDLVEMGVQWNPCALLHSSTCSLTHSESEVKEHTFISQTKSSYKKLTSKNKS
2、基因合成及质粒构建
ATGCACCACCACCATCATCACCACCACGACAAACTCCTCCTTGCAAATTGGGGCCTCCCTAAAGCCGTGCTTGAGAAGTACCATTCGTTCGGTGTAAAGAAAATGTTCGAGTGGCAGGCGGAATGCCTGTTGCTCGGTCAGGTCCTCGAAGGTAAAAATCTTGTCTATAGCGCTCCGACATCTGCGGGTAAAACCCTTGTGGCAGAGTTGTTGATTCTCAAGCGTGTCCTGGAAATGCGCAAGAAAGCTCTCTTCATTCTTCCCTTCGTCAGTGTGGCTAAGGAGAAGAAGTACTACTTGCAGAGCCTCTTTCAGGAAGTGGGCATAAAAGTTGATGGCTATATGGGAAGTACGTCGCCATCGAGACACTTTAGTTCACTGGACATCGCGGTGTGTACCATAGAGAGAGCTAACGGTCTGATTAACCGCCTCATTGAGGAGAATAAAATGGACCTCCTCGGAATGGTCGTAGTTGACGAATTGCACATGCTTGGAGATTCACATCGCGGATATTTGCTCGAACTGTTGCTCACCAAGATTTGCTATATCACTCGCAAATCTGCCTCTTGCCAAGCAGATCTTGCAAGCAGTCTCTCAAATGCCGTACAGATCGTAGGAATGTCAGCAACGCTGCCAAACTTGGAGCTGGTGGCTTCTTGGTTGAACGCTGAGCTTTATCATACAGACTTTAGGCCGGTTCCACTGCTGGAGTCGGTCAAAGTAGGAAATTCTATCTACGACAGCAGCATGAAGTTGGTAAGAGAGTTCGAACCTATGCTGCAGGTCAAGGGAGACGAGGACCATGTTGTCAGTCTGTGTTATGAGACGATTTGTGACAATCATTCTGTCCTTTTGTTCTGTCCGTCTAAAAAGTGGTGTGAGAAGTTGGCCGATATTATAGCTCGTGAATTTTATAACCTGCACCACCAGGCAGAAGGATTGGTGAAACCGTCAGAGTGTCCCCCCGTTATTCTTGAACAGAAGGAATTGTTGGAAGTCATGGACCAACTTCGCCGTTTGCCTAGTGGCCTGGACTCAGTCCTTCAGAAGACAGTTCCGTGGGGCGTAGCATTCCACCACGCAGGACTCACGTTTGAAGAACGCGACATAATCGAAGGCGCCTTTAGACAAGGTCTCATTCGCGTTCTTGCCGCTACGAGTACACTGTCAAGTGGAGTGAACCTGCCTGCAAGAAGAGTCATAATAAGAACACCTATATTCGGCGGAAGACCGCTCGATATATTGACCTACAAACAAATGGTCGGACGCGCAGGACGCAAGGGCGTCGACACCGTCGGAGAATCTATTCTTATTTGCAAGAATTCCGAAAAGAGTAAGGGTATTGCCCTTCTGCAAGGATCGCTGAAGCCCGTAAGAAGTTGTTTGCAGAGAAGAGAAGGCGAAGAGGTGACCGGATCTATGATACGTGCTATCCTTGAGATTATAGTGGGCGGTGTCGCATCGACGTCTCAGGATATGCATACTTACGCGGCTTGTACCTTTCTGGCCGCTAGTATGAAGGAGGGTAAACAGGGTATACAGCGCAATCAGGAATCTGTGCAATTGGGAGCGATAGAGGCGTGTGTGATGTGGCTTCTCGAAAATGAGTTTATACAATCAACCGAAGCGTCCGACGGTACGGAAGGTAAAGTATATCATCCCACTCACCTCGGCAGCGCAACACTCTCGTCATCACTGTCGCCTGCCGACACTTTGGACATATTCGCCGATCTTCAAAGGGCCATGAAAGGCTTTGTCCTCGAAAATGATCTTCACATTCTTTACTTGGTTACCCCCATGTTCGAAGACTGGACTACTATTGATTGGTATCGTTTTTTTTGCCTCTGGGAAAAGCTTCCCACGTCGATGAAACGCGTGGCGGAGTTGGTCGGAGTTGAGGAGGGATTCCTCGCGCGTTGCGTTAAGGGCAAAGTTGTGGCCCGTACAGAGCGTCAGCACCGTCAGATGGCCATCCACAAGCGCTTCTTCACGAGCTTGGTGCTCCTTGATTTGATTTCCGAAGTTCCCCTCAGGGAGATCAATCAAAAATATGGTTGCAACCGCGGACAGATCCAGTCACTTCAACAAAGCGCAGCCGTGTACGCTGGCATGATTACAGTCTTTTCAAATAGGCTCGGTTGGCATAATATGGAATTGCTTCTGTCGCAGTTCCAAAAAAGGCTGACATTCGGTATTCAGCGTGAGTTGTGCGATCTGGTTAGGGTAAGTCTGTTGAATGCGCAGCGCGCCCGTGTTCTGTATGCGAGCGGCTTCCACACGGTTGCCGATTTGGCACGTGCGAATATAGTAGAGGTAGAGGTGATACTGAAGAACGCAGTACCATTTAAAAGCGCGAGAAAAGCGGTCGACGAAGAGGAGGAGGCGGTTGAAGAGCGCCGCAATATGAGAACAATTTGGGTGACCGGCAGAAAGGGTCTCACAGAGCGTGAAGCAGCAGCTTTGATAGTTGAAGAAGCACGCATGATCCTCCAGCAAGACCTGGTAGAGATGGGTGTACAATGGAATCCCTGTGCTTTGCTGCATTCCAGTACCTGCTCACTCACGCATTCAGAATCAGAAGTGAAGGAACACACTTTCATCTCCCAAACAAAGTCATCGTATAAAAAGTTGACCAGTAAGAATAAGAGTGGCAGCGGTAGCGGATCTGGATCGGGTTCTGAAGTTCTGTTTCAGGGTCCGGGCTCGGCCTGGTCTCACCCCCAATTTGAAAAGGGCGGTGGCAGCGGAGGCGGTGGTTCTGGAGGCAGTGCGTGGAGCCACCCGCAGTTCGAGAAAtaa
3、重组杆状病毒的制备
3.1通过热激转化,将含有目的基因的重组pFastBac-1质粒导入到大肠杆菌DH10Bac感受态细胞(博迈德生物)中,在含有50μg/mL卡那霉素(BioBomei)、7μg/mL庆大霉素(BioBomei)、10μg/mL四环霉素(BioBomei)、200μg/mL X-gal(inalco)、40μg/mL IPTG(inalco)的LB固体培养基中,于37℃培养48小时。挑选均匀的白斑至3mL含有三种抗生素(50μg/mL卡那霉素、7μg/mL庆大霉素、10μg/mL四环霉素)的LB液体培养基中,37℃,200rpm条件下培养过夜,待菌液OD600约为0.6时,抽提重组杆状病毒质粒。
3.2取1mL昆虫细胞培养基(Insect Medium,Graces)加入15μL转染试剂(FuGENE)、5μg重组杆状病毒质粒室温下孵育15分钟,用此混合物溶液重悬10-12×106sf9昆虫细胞(购自Expression System公司,该昆虫细胞由细胞培养液在室温,500rpm的条件下离心10分钟获得),于27℃,200rpm的条件下培养4个小时,之后加入5mL ESF921昆虫细胞培养基(Expression Systems),于27℃,200rpm的条件下继续培养48小时。将培养了48小时的sf9细胞转移100mL锥形瓶中,27℃,110rpm条件下培养至细胞密度达2-4×106/mL时,室温下2500rpm离心10分钟上清即为P1代重组杆状病毒。
3.3取P1代重组杆状病毒按照1:10000的比例转染100mL细胞密度为1.5×106/mLsf9昆虫细胞,27℃,110rpm的条件下进行培养,待细胞密度达6×106/mL左右,细胞体积膨大且较为均匀时,室温下,2500rpm离心10分钟,上清液用0.22μm针头滤器过滤后即为P2代重组杆状病毒。
4、蛋白纯化小试
取P2代重组杆状病毒,按照1:50、1:100、1:150和1:200四个不同比例转染20mL密度为4×106/mL sf9昆虫细胞,27℃,110rpm条件下培养细胞48小时。培养结束后,取样进行Western blot检测以确定目的蛋白的表达情况。实验结果如图1所示,不同病毒比例蛋白表达量呈现明显的差异,其中1:50的转染比例目标蛋白的表达量最高。另外,Western blot结果显示,改造型DNA聚合酶在40kD左右存在降解条带。因此,为了获得全长的改造型DNA聚合酶/>串联使用N端和C端的标签以进行双步骤纯化。
5、蛋白大量表达与纯化
取P2代重组杆状病毒按照1:50的比例转染1L密度为4×106/mL sf9昆虫细胞,27℃,110rpm条件下培养细胞48小时。
细胞培养结束后,4℃,4000rpm离心20分钟,收集细胞。用Buffer A重悬细胞沉淀,于冰上用匀浆器对细胞悬液进行匀浆,以破碎细胞。匀浆完成后,于4℃,18000rpm的条件下离心1小时,离心后将上清液加载到镍亲和层析填料(Ni柱料)孵育1小时。之后用Buffer B洗脱杂蛋白,用Buffer C洗脱目的蛋白。将含有目的蛋白的洗脱液加载到Strep亲和层析柱(Strep柱料)上,用Buffer D洗脱杂蛋白,Buffer E洗脱目的蛋白,之后用50KDa的超滤管对目的蛋白进行浓缩,浓缩至体积为800μL左右,浓度为1.01mg/mL。最后,使用SDS-PAGE凝胶电泳检测目的蛋白的含量与纯度。
其中,上述缓冲液(Buffer)A-E具体成分如下:
Buffer A:50mM HEPES pH 7.5,150mM NaCl,10mM咪唑(imidazole),0.5mM TCEP,10μM亮肽素(leupeptin);
Buffer B:50mM HEPES pH 7.5,150mM NaCl,30mM咪唑(imidazole),0.5mM TCEP,10μM亮肽素(leupeptin),即图2中的30mM咪唑Buffer;
Buffer C:50mM HEPES pH 7.5,150mM NaCl,250mM咪唑(imidazole),0.5mMTCEP,10μM亮肽素(leupeptin),即图2中的250mM咪唑Buffer;
Buffer D:50mM HEPES pH 7.5,150mM NaCl,0.5mM TCEP,10μM亮肽素(leupeptin);
Buffer E:50mM HEPES pH 7.5,150mM NaCl,0.5mM TCEP,10μM亮肽素(leupeptin),50mM生物素(biotin)。
“沉淀”是指:匀浆破碎后的细胞裂解液经离心得到的沉淀;
“上清”是指:匀浆破碎后的细胞裂解液经离心得到的上清;
“镍流穿”是指:与镍亲和层析填料孵育后得到的流穿液;
“30ml咪唑Buffer洗杂”是指:用Buffer B洗脱杂蛋白所得样品;
“250mM咪唑Buffer洗脱”是指:用Buffer C洗脱目的蛋白所得样品;
“Strep流穿”是指:与Strep亲和层析填料孵育后得到的流穿液;
“Strep洗杂”是指:用Buffer D洗脱杂蛋白所得样品;
“Strep洗脱”是指:用Buffer E洗脱目标蛋白所得样品。
测试例
为验证改造型DNA聚合酶蛋白对于不同序列的DNA的结合活性,本测试例中合成两种序列存在差异的ssDNA序列:ssDNA-PN1(CTCTCTCTCTCTCTCTCTCTCTCTCTCTCT;SEQ IDNO:5)和ssDNA-PN2(CCAGTGAATTGTTGCTCGGTACCTGCTAAC;SEQ ID NO:6)。
首先,配制检测缓冲液Buffer F并用其配置400nM的实施例中制备的改造型DNA聚合酶蛋白储液,随后按2.5×梯度稀释蛋白(5个不同浓度梯度)。为减小实验误差,直接在384OptiPlate板进行反应体系混合及孵育。向板中加入2.5μL稀释蛋白,1000rpm离心10s,同时用Buffer F配置底物,包含200μM ATP(反应浓度100μM)和1200nM ssDNA-PN1或ssDNA-PN2(反应浓度600nM)。向384OptiPlate每孔中加入2.5μL底物,1000rpm离心10s,封板,震荡20s,1000rpm离心10s,23℃孵育120min。随后,向384OptiPlate每孔中加入5μL ADP-Glo(购自Promega公司),1000rpm离心10s,封板,震荡20s,1000rpm离心10s,23℃孵育40min。孵育后,向384OptiPlate每孔中加入10μL检测试剂(Detection reagent,购自Promega公司),1000rpm离心10s,封板,震荡20s,1000rpm离心10s,23℃孵育60min。最后,在Ensight酶标仪上检测冷光(Luminescence)信号,并使用GraphPad Prism 7分析和处理数据。
Buffer F:40mM Tris-HCl pH 7.5,20mM MgCl2,0.01%Triton X-100,0.01%牛血清白蛋白(Bovine albumin,BSA),1mM二硫苏糖醇(Dithiothreitol,DTT)。
实验结果如图3显示,荧光信号(图3中的纵坐标,相对光单位(relative lightunit,RLU)随着蛋白浓度(图3中的横坐标,PolQ-N表示改造型DNA聚合酶)的提高而提高,确认改造型DNA聚合酶/>蛋白具有DNA结合活性。另外,针对不同序列的DNA蛋白的荧光信号有明显的强度差异,其中采用ssDNA-PN1的实验窗口比ssDNA-PN2稍高些,说明改造型DNA聚合酶/>蛋白对不同的ssDNA序列结合活性具有一定的差异,但均具有结合活性。
为了进一步的利用改造型DNA聚合酶蛋白进行小分子药物筛选和小分子药物的靶向性活性口袋分析,本测试例利用改造型DNA聚合酶/>蛋白进行无配体结合状态下的靶点蛋白结构解析工作。本测试例将实施例中制备、纯化后的改造型DNA聚合酶/>进行负染评估和单颗粒冷冻结构研究。
1、负染制备步骤:
将样品稀释到合适浓度;做负染所用的超薄碳膜做亲水化处理后辉光放电;把稀释好的样品滴在载网上静置1min,滤纸吸掉样品,超纯水洗样三次后乙酸铀染液染色三次,最后一次染液静置45s,滤纸吸去多余染液,自然干涸并形成梯度;将制备好的载网放入负染样品储存盒中,并标记其放置位置;使用120kV Talos L120C TEM扫描/透射电子显微镜(购自赛默飞世尔科技公司)观察制备好的样品。
2、冷冻制样步骤:
打开Vitrobot冷冻电镜样品制备系统(购自赛默飞世尔科技公司),将温度和湿度分别设置为8℃和100%;将样品稀释到冻样所需浓度;
在泡沫杯内充满液氮,中间的铜环内充满液态乙烷;对金载网(Au quantifoilR1.2/1.3,购自德国Quantifoil公司)和GraFutureTM-GO载网(来自水木未来(北京)科技有限公司,http://shuimubio.uunn.cn/technology/1)和做亲水化处理后辉光发电;设置Vitrobot冷冻电镜样品制备系统(购自赛默飞世尔科技公司)冻样条件,制备冷冻样品,上样量为4μL;制备好的载网在300kv Krios G4冷冻电子显微镜(购自赛默飞世尔科技公司)上用Falcon 4相机观察并采集数据。
制备好的金载网和GraFutureTM-GO载网在300kv Krios G4冷冻电子显微镜(购自赛默飞世尔科技公司)上用Falcon 4相机采集数据,使用漂移矫正软件(MotionCor2)将原始TIFF格式数据(653张金载网显微照片(micrographs),4853张GraFutureTM-GO载网显微照片(micrographs))压缩、对齐、剂量加权,最终得到多帧图像合成为像素大小为的单帧照片;使用CTFFIND-4.1软件测定衬度传递函数(contrast transfer function,CTF)的参数;利用cryoSPARC软件分别对两种载网的原始数据进行颗粒挑选、挖颗粒、二维分类,将两种载网的二维分类结果整合在一起做三维分类及三维重构,最终得到分辨率为/>的密度图。
如图5和图6所示,改造型DNA聚合酶蛋白的二维分类有数种取向,三维分类及重构后得到的密度图密度较为完整,可以完成模型搭建。如图7所示,改造型DNA聚合酶/>蛋白与野生型DNA聚合酶/>蛋白(PDB:5A9J)做比对,依据RMSD值判断,改造型DNA聚合酶/>蛋白构象与野生型DNA聚合酶/>蛋白构象差别较小,说明改造后的不影响DNA聚合酶/>的结构。具体地,627个精简的Cα原子对之间的RMSD为0.962埃;所有的722个Cα原子:1.379。
参考文献
1.Lieber,M.R.The Mechanism of Double-Strand DNA Break Repair by theNonhomologous DNA End-Joining Pathway.Annu Rev Biochem 79,181–211(2010).
2.Hwang,T.et al.Defining the mutation signatures of DNA polymeraseθincancer genomes.Nar Cancer 2,zcaa017-(2020).
3.Schrempf,A.,Slyskova,J.&Loizou,J.I.Targeting the DNA Repair EnzymePolymeraseθin Cancer Therapy.Trends Cancer 7,98–111(2021).
4.Mateos-Gomez,P.A.et al.Mammalian polymeraseθpromotes alternativeNHEJ and suppresses recombination.Nature 518,254–257(2015).
5.Kawamura,K.et al.DNA polymeraseθis preferentially expressed inlymphoid tissues and upregulated in human cancers.Int J Cancer 109,9–16(2004).
6.Lemée,F.et al.DNA polymeraseθup-regulation is associated with poorsurvival in breast cancer,perturbs DNA replication,and promotes geneticinstability.Proc National Acad Sci 107,13390–13395(2010).
7.Higgins,G.S.et al.Overexpression of POLQ Confers a Poor Prognosisin Early Breast Cancer Patients.Oncotarget 1,175–184(2010).
8.Dai,C.-H.et al.Co-inhibition of polθand HR genes efficientlysynergize with cisplatin to suppress cisplatin-resistant lung cancer cellssurvival.Oncotarget 7,65157–65170(2016).
9.Tobin,L.A.et al.Targeting Abnormal DNA Repair in Therapy-ResistantBreast Cancers.Mol Cancer Res 10,96–107(2012).
10.Newman,J.A.,Cooper,C.D.O.,Aitkenhead,H.&Gileadi,O.Structure of theHelicase Domain of DNA Polymerase Theta Reveals a Possible Role in theMicrohomology-Mediated End-Joining Pathway.Struct Lond Engl 1993 23,2319–2330(2015).
11.Mateos-Gomez,P.A.et al.The helicase domain of Polθcounteracts RPAto promote alt-NHEJ.Nat Struct Mol Biol 24,1116–1123(2017).
12.Shen,P.S.The 2017 Nobel Prize in Chemistry:cryo-EM comes ofage.Anal Bioanal Chem 410,2053–2057(2018).
13.Callaway,E.Revolutionary cryo-EM is taking over structuralbiology.Nature 578,201–201(2020).
14.Schneider,G.&Fechner,U.Computer-based de novo design of drug-likemolecules.Nat Rev Drug Discov 4,649–663(2005).
15.Renaud,J.-P.et al.Cryo-EM in drug discovery:achievements,limitations and prospects.Nat Rev Drug Discov 17,471–492(2018).
Claims (10)
(i)如SEQ ID NO:2所示的氨基酸序列;
(ii)与SEQ ID NO:2所示的氨基酸序列具有至少80%、82%、85%、87%、90%、92%、95%、96%、97%、98%或99%同一性的氨基酸序列,并且其保留如SEQ ID NO:2所示的氨基酸序列的DNA结合活性和蛋白结构;
(iii)在SEQ ID NO:2所示的氨基酸序列中添加、取代、缺失或插入1个或多个氨基酸残基的氨基酸序列,并且其保留如SEQ ID NO:2所示的氨基酸序列的DNA结合活性和蛋白结构;或者,
(iv)由核苷酸序列编码的氨基酸序列,所述核苷酸序列与编码如SEQ ID NO:2所示的氨基酸序列的多核苷酸序列在严格条件下杂交,并且所述氨基酸序列保留以SEQ ID NO:2所示的氨基酸序列的DNA结合活性和蛋白结构,所述严格条件是中等严格条件,中-高严格条件,高严格条件或非常高严格条件。
(i)如SEQ ID NO:3所示的氨基酸序列;
(ii)与SEQ ID NO:3所示的氨基酸序列具有至少80%、82%、85%、87%、90%、92%、95%、96%、97%、98%或99%同一性的氨基酸序列,并且其保留如SEQ ID NO:3所示的氨基酸序列的DNA结合活性和蛋白结构;
(iii)在SEQ ID NO:3所示的氨基酸序列中添加、取代、缺失或插入1个或多个氨基酸残基的氨基酸序列,并且其保留如SEQ ID NO:3所示的氨基酸序列的DNA结合活性和蛋白结构;或者,
(iv)由核苷酸序列编码的氨基酸序列,所述核苷酸序列与编码如SEQ ID NO:3所示的氨基酸序列的多核苷酸序列在严格条件下杂交,并且所述氨基酸序列保留以SEQ ID NO:3所示的氨基酸序列的DNA结合活性和蛋白结构,所述严格条件是中等严格条件,中-高严格条件,高严格条件或非常高严格条件。
7.根据权利要求6所述的多核苷酸,其序列如SEQ ID NO:4所示。
8.一种表达载体,其包含权利要求6或7所述的多核苷酸。
9.一种宿主细胞,其包含权利要求8所述的表达载体。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211063781.XA CN116376867A (zh) | 2022-08-31 | 2022-08-31 | 改造型DNA聚合酶φ及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211063781.XA CN116376867A (zh) | 2022-08-31 | 2022-08-31 | 改造型DNA聚合酶φ及其应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116376867A true CN116376867A (zh) | 2023-07-04 |
Family
ID=86975565
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202211063781.XA Pending CN116376867A (zh) | 2022-08-31 | 2022-08-31 | 改造型DNA聚合酶φ及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116376867A (zh) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104145030A (zh) * | 2012-01-05 | 2014-11-12 | 国家科学研究中心 | 用于诊断肺癌侵袭性和遗传不稳定性的标记 |
WO2019057835A1 (en) * | 2017-09-20 | 2019-03-28 | Institut Pasteur | THETA POLYMERASE DNA MUTANTS, METHODS FOR PRODUCING THESE MUTANTS AND USES THEREOF |
-
2022
- 2022-08-31 CN CN202211063781.XA patent/CN116376867A/zh active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104145030A (zh) * | 2012-01-05 | 2014-11-12 | 国家科学研究中心 | 用于诊断肺癌侵袭性和遗传不稳定性的标记 |
WO2019057835A1 (en) * | 2017-09-20 | 2019-03-28 | Institut Pasteur | THETA POLYMERASE DNA MUTANTS, METHODS FOR PRODUCING THESE MUTANTS AND USES THEREOF |
Non-Patent Citations (3)
Title |
---|
SCOTT VANSON, ET AL: "Probing the structure and function of polymerase θ helicase-like domain", DNA REPAIR, vol. 116, pages 1 - 10 * |
SEKI, M., ET AL: "DNA polymerase theta [Homo sapiens]", pages 08421, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/protein/AAR08421.2?report=genbank&log$=prottop&blast_rank=1&RID=BZ424YA3013> * |
STRAUSBERG, R.L., ET AL: "Synthetic construct Homo sapiens clone IMAGE:100068983, MGC:198994 polymerase (DNA directed), theta (POLQ) mRNA, encodes complete protein", pages 172289, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/nucleotide/BC172289.1?report=genbank&log$=nuclalign&blast_rank=1&RID=CT5KRNV7013> * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN112375784B (zh) | 制备重组新型冠状病毒Spike蛋白的方法 | |
CN109627344B (zh) | cAMP荧光探针及其应用 | |
US20150026840A1 (en) | Constructs and systems and methods for producing microcompartments | |
CN111320684B (zh) | 人源N-甲基-D-天冬氨酸受体的GluN1/GluN2A四聚体的表达及其应用 | |
Chen et al. | Allosteric transcription stimulation by RNA polymerase II super elongation complex | |
EP3868882A1 (en) | Archaeal pyrrolysyl trna synthetases for orthogonal use | |
CN113354745B (zh) | 一种组合物及规模化生产成纤维细胞生长因子的方法 | |
CN114438054A (zh) | 一种突变型RNase R及其制备方法和应用 | |
CN116376867A (zh) | 改造型DNA聚合酶φ及其应用 | |
CN111575251B (zh) | 一种用于达托霉素生物合成的dptC1突变体的构建 | |
CN114057861A (zh) | 一种靶向UBE2C的bio-PROTAC人工蛋白 | |
CN113024674B (zh) | 荧光亮度宽幅变化的环磷酸腺苷荧光探针 | |
CN110093361B (zh) | 一种增强基因表达的增强子多肽及其应用 | |
Fagiewicz et al. | In vitro characterization of the full-length human dynein-1 cargo adaptor BicD2 | |
Ichikawa et al. | An asymmetric centromeric nucleosome | |
CN108753819B (zh) | 真核表达载体、真核表达系统、二者的制备方法和应用及gdf11蛋白 | |
CN110794129B (zh) | 细胞内检测生物分子间相互作用及其调控因子的方法与所用试剂 | |
Han et al. | Crystal structure of human calmodulin-like protein: insights into its functional role | |
CN113454211A (zh) | 氨酰基-tRNA合成酶及其用途 | |
CN113480666B (zh) | Ca153融合蛋白及其制备方法和ca153检测质控品或校准品 | |
CN114591987B (zh) | 一种用于检测活细胞中mTORC1活性的遗传编码荧光生物传感器及其构建方法 | |
CN116926034A (zh) | 重组肌酸激酶同工酶及其制备方法和应用 | |
Prizant et al. | Factors dictating the pseudocatalytic efficiency of avidins | |
WO2015184690A1 (zh) | 融合表达离子通道蛋白及运输蛋白的方法及其使用的蛋白片段 | |
CN116084026A (zh) | 一种筛选小鼠精子发生相关互作蛋白的方法及应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |