WO2017209122A1 - PROTÉINE DE FUSION POUR AMÉLIORER L'EXPRESSION DE PROTÉINES À PARTIR D'ARNm CIBLE - Google Patents

PROTÉINE DE FUSION POUR AMÉLIORER L'EXPRESSION DE PROTÉINES À PARTIR D'ARNm CIBLE Download PDF

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WO2017209122A1
WO2017209122A1 PCT/JP2017/020076 JP2017020076W WO2017209122A1 WO 2017209122 A1 WO2017209122 A1 WO 2017209122A1 JP 2017020076 W JP2017020076 W JP 2017020076W WO 2017209122 A1 WO2017209122 A1 WO 2017209122A1
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Prior art keywords
asparagine
fusion protein
amino acids
aspartic acid
ppr
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PCT/JP2017/020076
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English (en)
Japanese (ja)
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崇裕 中村
祐介 八木
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国立大学法人九州大学
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Priority to ES17806675T priority Critical patent/ES2874230T3/es
Priority to JP2018520924A priority patent/JP6928386B2/ja
Priority to SG11201810606TA priority patent/SG11201810606TA/en
Priority to US16/305,080 priority patent/US11136361B2/en
Priority to CN201780041619.9A priority patent/CN109415446B/zh
Priority to BR112018075010-7A priority patent/BR112018075010A2/pt
Priority to DK17806675.9T priority patent/DK3466978T3/da
Priority to CA3026340A priority patent/CA3026340A1/fr
Priority to AU2017275184A priority patent/AU2017275184B2/en
Priority to EP17806675.9A priority patent/EP3466978B1/fr
Priority to KR1020187037821A priority patent/KR102407776B1/ko
Publication of WO2017209122A1 publication Critical patent/WO2017209122A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif

Definitions

  • the present invention relates to a fusion protein for improving the protein expression level from a target mRNA.
  • Zinc finger nuclease ZFN
  • TALEN TAL Effector Nuclease
  • Crispr-cas9 etc. are known as techniques using protein factors that act on DNA, but protein factors that act specifically on RNA were used. Technology development is still limited.
  • PPR protein protein having a pentatripeptide repeat (PPR) motif
  • PPR pentatripeptide repeat
  • Patent Document 1 an amino acid that functions when the PPR motif exerts RNA binding properties is identified, and the relationship between the structure of the PPR motif and the target base has been clarified. It was possible to construct a protein having a PPR motif capable of binding to the RNA having it. However, a method for actually controlling a target RNA using the technique described in Patent Document 1 has not been found so far.
  • the present inventors have found that a fusion protein of a predetermined functional domain and the PPR protein is capable of expressing the protein from the target mRNA.
  • the inventors have found that the amount can be improved and have completed the present invention.
  • the present invention in one embodiment, is a fusion protein for improving the protein expression level from the target mRNA
  • the fusion protein is (A) one or a plurality of functional domains that improve protein expression from mRNA, and (B) a polypeptide portion that can bind to a target mRNA in an RNA base-selective or RNA base sequence-specific manner, Including
  • the polypeptide part (B) is a polypeptide part comprising at least one PPR motif consisting of a 30-38 amino acid polypeptide represented by Formula 1 (Where: Helix A is a 12 amino acid long portion capable of forming an ⁇ -helix structure, and is represented by Formula 2.
  • a 1 to A 12 each independently represent an amino acid; X is absent or is a moiety consisting of 1 to 9 amino acids in length; Helix B is a part capable of forming an ⁇ -helix structure consisting of 11 to 13 amino acids in length; L is a moiety represented by Formula 3 that is 2 to 7 amino acids long;
  • each amino acid is numbered from the C-terminal side as “i” ( ⁇ 1), “ii” ( ⁇ 2), However, L iii to L vii may not exist.
  • a combination of three amino acids of A 1 , A 4 and L ii , or a combination of two amino acids of A 4 and L ii corresponds to the base or base sequence of the target mRNA, Fusion protein.
  • the polypeptide part (B) includes 2 to 30 PPR motifs, and the plurality of PPR motifs specifically bind to a base sequence of a target mRNA. It is characterized by being arranged in.
  • the polypeptide part (B) includes 5 to 25 PPR motifs.
  • the functional domain (A) is bound to the N-terminal side and / or the C-terminal side of the polypeptide part (B).
  • the functional domain of (A) is related to a domain that induces ribosomes to mRNA, a domain that is related to mRNA translation initiation or promotion, and mRNA transport to the nucleus. It is selected from the group consisting of a domain, a domain related to binding to the endoplasmic reticulum membrane, a domain containing an ER retention signal sequence, and a domain containing an endoplasmic reticulum signal sequence.
  • the domain that induces ribosomes to mRNA is a density-regulated protein (DENR), an MCT-1 (Malignant T-cell amplified sequence 1), or a TPT1 (Translationally-controlled).
  • DER density-regulated protein
  • MCT-1 Malignant T-cell amplified sequence 1
  • TPT1 Translationally-controlled
  • the domain involved in translation initiation or translation promotion of mRNA is a domain comprising all or a functional part of a polypeptide selected from the group consisting of eIF4E and eIF4G
  • the domain related to transport of the mRNA to the outside of the nucleus is a domain containing all or a functional part of SLBP (Stem-loop binding protein);
  • the domain related to the binding to the endoplasmic reticulum membrane is a polypeptide selected from the group consisting of SEC61B, TRAP-alpha (Translocator associated protein alpha), SR-alpha, Dia1 (Cytochrom b5 reductase 3), and p180.
  • the endoplasmic reticulum retention signal sequence is a signal sequence comprising a KDEL (KEEL) sequence, or
  • the endoplasmic reticulum signal sequence is a signal sequence containing MGWSCIILFLVATATGAHS (SEQ ID NO: 22).
  • a combination of three amino acids of A 1 , A 4 , and L ii in each of the above PPR motifs When bases targeted for PPR motif is A (adenine), a combination of three amino acids of A 1, A 4, and L ii is in the order of (A 1, A 4, L ii), ( valine , Threonine, asparagine), (phenylalanine, serine, asparagine), (phenylalanine, threonine, asparagine), (isoleucine, asparagine, aspartic acid), or (threonine, threonine, asparagine); When bases targeted for PPR motif is G (guanine), a combination of three amino acids of A 1, A 4, and L ii is in the order of (A 1, A 4, L ii), ( glutamic acid , Glycine, aspartic acid), (valine, threonine, aspartic acid), (lysine, threonine,
  • the combination of two amino acids of A 4 and L ii in each of the aforementioned PPR motifs is as follows: When bases targeted for PPR motif is A (adenine), a combination of two amino acids of A 4 and L ii is in the order of (A 4, L ii), (threonine, asparagine), (Serine, asparagine) or (glycine, asparagine); When the target base of the PPR motif is G (guanine), the combination of the two amino acids A 4 and L ii is (A 4 , L ii ) in the order (threonine, aspartic acid).
  • glycine, aspartic acid When bases targeted for PPR motif is U (uracil), a combination of two amino acids of A 4 and L ii is in the order of (A 4, L ii), (asparagine, aspartic acid), (proline , Aspartic acid), (methionine, aspartic acid), or (valine, threonine); When the target base of the PPR motif is C (cytosine), the combination of two amino acids of A 4 and L ii is (A 4 , L ii ) in the order (asparagine, asparagine), (Asparagine, serine) or (leucine, aspartic acid).
  • the present invention also relates to a nucleic acid encoding the fusion protein of the present invention.
  • the present invention relates to a vector (preferably an expression vector) containing the nucleic acid of the present invention.
  • the present invention provides a method for improving the protein expression level from a target mRNA in a cell, Providing the fusion protein of the present invention as described above or the vector of the present invention as described above; and Introducing the fusion protein or vector into a cell, Method.
  • the cell is a eukaryotic cell.
  • the present invention is characterized in that the cell is an animal cell.
  • the animal cell is a human cell.
  • FIG. 1 shows a schematic diagram of an effector plasmid and a reporter plasmid used in Examples, and a schematic diagram of an experimental outline.
  • FIG. 1A shows a schematic diagram of an effector plasmid and a reporter plasmid used in this Example. From the effector plasmid, a protein in which the PPR motif and eIF4G are fused is expressed. In this example, CRR4 protein, which has been well studied for target sequences, was used. From the reporter plasmid, Renilla luciferase (RLuc) and firefly luciferase (FLuc) are transcribed in the form of dicistronic mRNA.
  • RLuc Renilla luciferase
  • FLuc firefly luciferase
  • FIG. 1B shows a schematic diagram of the experimental outline of this example.
  • RLuc is translated at similar levels with or without PPR binding sequences. Therefore, the activity value of RLuc can be treated as a control for transfection with this reporter system.
  • Translation of Fluc is initiated only when PPR-eIF4G binds to the PPR binding sequence and the translation factor can be attracted by the effect of eIF4G.
  • FIG. 2 shows an experimental procedure for a reporter assay using HEK293T cells.
  • FIG. 3 shows the experimental results of Example 1.
  • FIG. 4 shows an outline of the experiment of Example 2.
  • FIG. 5 shows the results of the experiment of Example 2 and explanation of the function of each domain.
  • FIG. 6 shows the result of the experiment of Example 2 and the explanation of the function of each domain.
  • PPR motif refers to the case where the E value obtained with PF01535 in Pfam and PS51375 in Prosite is less than a predetermined value when the amino acid sequence is analyzed with a protein domain search program on the Web, unless otherwise specified.
  • the position number of the amino acid constituting the PPR motif defined in the present invention is almost the same as that of PF01535, but the number obtained by subtracting 2 from the amino acid position of PS51375 (eg, number 1 of the present invention ⁇ number 3 of PS51375) Equivalent to.
  • the conserved amino acid sequence of the PPR motif has low conservation at the amino acid level, but the two ⁇ -helices are well conserved on the secondary structure.
  • a typical PPR motif is composed of 35 amino acids, but its length is variable from 30 to 38 amino acids.
  • the PPR motif referred to in the present invention consists of a polypeptide having a length of 30 to 38 amino acids represented by Formula 1.
  • Helix A is a 12 amino acid long portion capable of forming an ⁇ -helix structure, and is represented by Formula 2.
  • a 1 to A 12 each independently represent an amino acid;
  • X is absent or is a moiety consisting of 1 to 9 amino acids in length;
  • Helix B is a part capable of forming an ⁇ -helix structure consisting of 11 to 13 amino acids in length;
  • L is a moiety represented by Formula 3 that is 2 to 7 amino acids long;
  • each amino acid is numbered from the C-terminal side as “i” ( ⁇ 1), “ii” ( ⁇ 2), However, L iii to L vii may not exist.
  • PPR protein refers to a PPR protein having one or more, preferably two or more PPR motifs, unless otherwise specified.
  • protein refers to all substances consisting of polypeptides (chains in which a plurality of amino acids are peptide-bound) unless otherwise specified, and includes those consisting of relatively low molecular weight polypeptides.
  • amino acid may refer to a normal amino acid molecule and may refer to an amino acid residue constituting a peptide chain. Which one is pointed out will be apparent to the skilled person from the context.
  • the binding property of the PPR motif to the RNA base when it is referred to as “selective”, the binding activity to any one of the RNA bases is higher than the binding activity to other bases unless otherwise specified. It's expensive. This selectivity can be determined by a person skilled in the art by planning an experiment, and can also be obtained by calculation by a person skilled in the art.
  • RNA base refers to a base of a ribonucleotide that constitutes RNA, unless otherwise specified.
  • adenine (A), guanine (G), cytosine (C), or uracil ( U) PPR protein may have selectivity for bases in RNA, but does not bind to nucleic acid monomers.
  • PPR protein is abundant in plants, and 500 proteins and about 5000 motifs can be found in Arabidopsis thaliana. There are also various PPR motifs and PPR proteins of various amino acid sequences in terrestrial plants such as rice, poplar, and flax.
  • a PPR motif and a PPR protein that exist in nature may be used, or a PPR motif and a PPR protein designed based on the method disclosed in, for example, WO2013 / 058404 may be used.
  • a desired PPR motif and PPR protein can be designed based on the following information disclosed in WO2013 / 058404.
  • the present invention can utilize the knowledge regarding the combination of three amino acids A1, A4 and Lii and / or the combination of two amino acids A4 and Lii disclosed in WO2013 / 058404.
  • the combination of the three amino acids A1, A4, and Lii is, in order, valine, asparagine, and aspartic acid.
  • the PPR motif has a selective RNA base binding ability of binding strongly to U, then to C, and then to A or G.
  • the combination of three amino acids A1, A4, and Lii is, in order, valine, threonine, and asparagine, the PPR motif binds strongly to A, then to G, then to C It has a selective RNA base binding ability that binds to but does not bind to U.
  • PPR motif and PPR protein Identification and design: One PPR motif can recognize a specific base of RNA. Based on the present invention, it is possible to select or design a PPR motif selective for each of A, U, G, and C by making the amino acid at a specific position appropriate. A protein containing the appropriate sequence of can recognize the corresponding specific sequence. Furthermore, based on the above findings, a protein having a PPR motif that can selectively bind to a desired RNA base and a plurality of PPR motifs that can bind to a desired RNA in a sequence-specific manner can be designed. At the time of design, the sequence information of the natural PPR motif may be referred to for portions other than the amino acid at an important position in the PPR motif.
  • the amino acid may be designed by substituting only the amino acids at the important positions.
  • the number of repetitions of the PPR motif can be appropriately determined depending on the target sequence, but can be 2 or more, for example, 2 to 30.
  • the PPR motif or PPR protein designed as described above can be prepared by methods well known to those skilled in the art. For example, from the designed PPR motif or PPR protein amino acid sequence, the nucleic acid sequence encoding it is determined and cloned to produce a transformant (expression vector, etc.) that produces the desired PPR motif or PPR protein. be able to.
  • the present invention relates to the above-described PPR motif or PPR protein (that is, a polypeptide capable of binding to a target mRNA in an RNA base-selective or RNA base sequence-specific manner) and the protein expression level from the mRNA. It relates to a fusion protein with one or more functional domains to be improved.
  • the “functional domain that improves the protein expression level from mRNA” that can be used in the present invention is, for example, all or a functional domain of a protein that is known to directly or indirectly promote translation of mRNA. It may be a functional part. More specifically, functional domains that can be used in the present invention include, for example, a domain that induces ribosomes to mRNA, a domain that is related to mRNA translation initiation or translation promotion, and a domain that is related to mRNA export to the nucleus. , A domain associated with binding to the endoplasmic reticulum membrane, a domain containing an ER retention signal sequence, or a domain containing an endoplasmic reticulum signal sequence.
  • the domains for inducing ribosomes into the above mRNAs are: DENR (Density-regulated protein), MCT-1 (Malignant T-cell amplified sequence 1), TPT1 (Translationally-controlled pilot, and TPT1).
  • DENR Density-regulated protein
  • MCT-1 Malignant T-cell amplified sequence 1
  • TPT1 Translationally-controlled pilot
  • TPT1 Translationally-controlled pilot
  • TPT1 Translationally-controlled pilot
  • the domain related to the transport of mRNA to the outside of the nucleus may be a domain including all or a functional part of SLBP (Stem-loop binding protein).
  • the domain related to the binding to the endoplasmic reticulum membrane is selected from the group consisting of SEC61B, TRAP-alpha (Translocated protein alpha), SR-alpha, Dia1 (Cytochrome b5 reductase 3), and p180. It may be a domain that contains all or part of a polypeptide.
  • the endoplasmic reticulum retention signal (ER retention signal) sequence may be a signal sequence including a KDEL (KEEL) sequence.
  • the endoplasmic reticulum signal sequence may be a signal sequence including MGWSCIILFLVATATGAHS (SEQ ID NO: 22).
  • the functional domain may be fused to the N-terminal side of the PPR protein, may be fused to the C-terminal side, or may be fused to both the N-terminal side and the C-terminal side.
  • the fusion protein of the present invention may contain a plurality of functional domains (for example, 2 to 5 functional domains).
  • the functional domain and the PPR protein may be indirectly fused via a linker or the like.
  • the present invention also relates to a nucleic acid encoding the fusion protein described above and a vector (for example, an expression vector) containing the nucleic acid.
  • an expression vector means, for example, a vector comprising a DNA having a promoter sequence, a DNA encoding a desired protein, and a DNA having a terminator sequence from upstream, as long as the desired function is exhibited. They are not necessarily arranged in this order.
  • various expression vectors commonly used by those skilled in the art can be used.
  • the fusion protein of the present invention utilizes the eukaryotic RNA translation mechanism, it can function in cells of eukaryotes (eg, animals, plants, microorganisms (yeasts, etc.), protists).
  • the fusion protein of the present invention can particularly function in an animal cell (in vitro or in vivo).
  • animal cells into which the fusion protein of the present invention or a vector expressing the fusion protein of the present invention can be introduced include cells derived from humans, monkeys, pigs, cows, horses, dogs, cats, mice, and rats. be able to.
  • cultured cells into which the fusion protein of the present invention or a vector expressing the fusion protein of the present invention can be introduced include, for example, Chinese hamster ovary (CHO) cells, COS-1 cells, COS-7 cells, VERO ( ATCC CCL-81) cells, BHK cells, canine kidney-derived MDCK cells, hamster AV-12-664 cells, HeLa cells, WI38 cells, 293 cells, 293T cells, PER. C6 cells can be mentioned, but are not limited thereto.
  • Example 1 Improvement of protein expression level from target mRNA by fusion protein of PPR motif and eIF4G
  • Cell culture HEK293T cell line
  • Dulbecco's modified Eagle's medium DMEM, high glucose
  • FBS Fetal bovine serum
  • EDTA-NaCl solution 10 mM EDTA and 0.85% (w / v) NaCl, pH adjusted to 7.2-7.4, autoclaved, stored at room temperature 100 ⁇ 20 mm cell culture petri dish (Greiner bio one, Frickenhausen , Germany) ⁇ 10mL disposable sterile pipette ⁇ 15mL and 50mL plastic centrifuge tube ⁇ 1.8mL cryotube (Nunc; Thermo Fisher Scientific, Waltham, MA, USA) ⁇ Freeze container (Nalgene; Thermo Fisher Scientific, Waltham, MA, USA) ⁇ Banbanker (Lymphotec, Tokyo, Japan)
  • the luciferase gene is inserted into the expression cassette, and the PPR binding sequence is inserted into its 5′-UTR (100 ng / ⁇ L) Poly-L-lysine coated 96-well plate (AGC Techno glass, Shizuoka, Japan) 1 ⁇ phosphate-buffered saline, PBS ( ⁇ ): 1.47 mM KH 2 PO 4 , 8.1 mM Na 2 HPO 4 , 137 mM NaCl, and 2.7 mM KCl.
  • the reporter assay requires an effector plasmid and a reporter plasmid, both of which are constructed based on pcDNA3.1.
  • the effector plasmid side contains a PPR protein and a fusion gene encoding a partial domain (SEQ ID NO: 1) of human eIF4G (FIG. 1A).
  • CPR4 SEQ ID NO: 2 was used as the PPR protein portion.
  • the reporter plasmid contains two open reading frames (ORF), Renilla luciferase (RLuc) and firefly luciferase (FLuc), which are transcribed dicistronically (FIG. 1A).
  • the RLuc gene is located 5 ′ of the FLuc gene and was used as a control for gene expression.
  • the PPR binding region is inserted into the 5'UTR of the FLuc ORF and from 3 repeats of the CRR4 recognition sequence (5'-UAUCUUGUCUUA-3 ') (SEQ ID NO: 3) interrupted by the 4-base sequence (ATCG and GATC) Obviously.
  • the cytomegalovirus promoter (CMV) and the bovine growth hormone gene-derived polyadenylation signal were used for gene expression.
  • an effector plasmid without eIF4G was constructed by fusing a FLAG epitope tag to PPR.
  • a control reporter plasmid having no PPR binding region was constructed.
  • FIG. 1 The outline of the procedure from cell culture to reporter assay in this example is depicted in FIG.
  • Cell culture from frozen stock This process is performed aseptically. In advance, all instruments are disinfected with 70% ethanol. 1. Place 9 mL of DMEM medium in a 15 mL centrifuge tube (sterilized). 2. Thaw rapidly by incubating 1 mL of HEK293T frozen cells in a cryotube in a 37 ° C. water bath. 3. The cells are added to a 15 mL centrifuge tube containing 9 mL DMEM. 4). Centrifuge at 1100 ⁇ g for 2 minutes at room temperature and remove the supernatant. 5. The cells are resuspended in 10 mL DMEM (FBS added to a final concentration of 10%). 6).
  • the suspended cells were transferred to a 100 mm petri dish.
  • the petri dish was allowed to stand in an incubator under conditions of 37 ° C. and 5% CO 2 .
  • the cultured cells were passaged after 24 hours.
  • the cell density on the petri dish surface is maintained between 10% and 80%. Passaging is basically performed every 3 days (twice a week) or according to the cell growth rate. In addition, cells are re-cultured from frozen stock once a month to keep passage times low. Keeping passage times low and keeping cells healthy is important for efficient DNA transfection.
  • Cell cryopreservation Frozen stocks are made using Bambanker reagent and ⁇ 50% cell density cultured cells in logarithmic growth. By using Bambanker, a high recovery rate and long-term storage can be easily performed. 1. The cells on the second day after passage are detached according to the passage procedure. Add 5-10 mL of DMEM and collect cells in a 50 mL centrifuge tube. 2. Centrifuge at 1100 ⁇ g for 2 minutes at room temperature and remove the supernatant. 3. Add 1 mL of Bambanker per petri dish and suspend. 4). Dispense the suspended cells quickly into a cryotube and close the lid. 5. Place in a special freeze container and let stand at -80 ° C for 12 hours (see note 7 ). 6). Transfer to a normal sample box and store at -80 ° C or in liquid nitrogen.
  • Transient gene transfer Transfection
  • 1. Before starting, prepare the required number of petri dishes containing the cells on the second day after passage and check whether the cells are healthy (see note 8 ). With a rough estimate, 96 assays can be performed in one dish. 2.
  • the cells on the second day after passage are detached according to the passage procedure, and the suspended cells are transferred to a 50 mL centrifuge tube. 3. Centrifuge at 1100 ⁇ g for 2 minutes at room temperature and remove the supernatant. 4. Disperse the cell mass completely in 10 mL DMEM (FBS added to a final concentration of 10%). 5. The number of cells is counted using a hemocytometer and an inverted microscope.
  • the dual luciferase assay is performed using the Dual-Glo Luciferase Assay System, following the manufacturer's instructions with minor modifications. 1. 24 hours after transfection, the medium of each well is replaced with 40 ⁇ L of 1 ⁇ PBS ( ⁇ ). 2. Add 40 ⁇ L of Dual-Glo luciferase reagent to each well and mix well by pipetting. 3. Allow to stand at room temperature for 10 minutes and transfer the entire volume to a 96-well luminometer plate. 4). Luminescence by firefly luciferase for FLuc gene expression is measured with a plate reader. 5. Dilute the Stop & Glo substrate 100-fold with Dual-Glo Stop & Glo buffer. Add 40 ⁇ L of the diluted solution to each well. 6). Let stand for at least 10 minutes at room temperature, then measure luminescence by Renilla luciferase for RLuc gene expression.
  • FIG. 3 The results of the luciferase assay are shown in FIG. As shown in FIG. 3, a 2.75-fold translational activity was specifically observed in the presence of both PPR-eIF4G and a PPR binding sequence. That is, it was shown that a fusion protein of a PPR protein and a functional domain that improves the protein expression level from mRNA improves the protein expression level from the target mRNA.
  • HEK293T is a human fetal kidney cell line expressing SV40 large T antigen. This cell line is easy to culture and can be transfected efficiently with various methods. HEK293T cells are available at RIKEN BRC (ja.brc.riken.jp) or ATCC (www.atcc.org).
  • DMEM adds 1 ⁇ penicillin-streptomycin solution to avoid microbial contamination.
  • the FBS is immobilized at 56 ° C for 30 minutes and stored at 4 ° C.
  • Plasmid purity is critical to transfection efficiency. Plasmids should be isolated using transfection grade kits.
  • the dedicated freeze container is a box with adjustable freezing speed (at -80 ° C, -1 ° C per minute), and can be stored frozen in a non-programmed -80 ° C freezer. is there. (Note 8) Use cells at a culture density of 50-80% for transfection. However, the appropriate cell density depends on the transfection reagent.
  • transfection reagent ⁇ L
  • plasmid DNA ⁇ g
  • Example 2 Improvement of protein expression from target mRNA by fusion protein of PPR and other functional domains
  • the final protein synthesis amount is determined by gene insertion position, mRNA transcription amount, post-transcriptional control (control at the RNA level), post-translational modification, and the like. Therefore, a method for enhancing translation of mRNA using the fact that PPR protein binds to a target RNA molecule in a sequence-specific manner was devised (FIG. 4).
  • translation of mRNA is initiated by the translation initiation factor (eIF) intervening in the mRNA, and as a result, ribosomes are recruited near the translation initiation point.
  • eIF translation initiation factor
  • a reporter assay system using cultured animal cells was created (except that the functional domains used were different, and the experiment was performed in the same manner as described in Example 1). Performed).
  • a system was constructed using a CRR4 protein (one of the Arabidopsis PPR proteins) known to bind to a specific RNA sequence (UAUCUUGUCUUUA) (SEQ ID NO: 3).
  • CRR4 protein one of the Arabidopsis PPR proteins
  • UAUCUUGUCUUUA SEQ ID NO: 3
  • Candidate domains include (a) eIF proteins (eIF4E, eIF4G), (b) ribosome binding proteins (DENR, MCT-1, TPT1, Lelepo4), (c) Histon that facilitates transport of transcribed mRNA from the nucleus to the cytoplasm (DBP), (d) ER anchor protein (SEC61B, TRAP-alpha, SR-alpha, Dia1, p180), (e) ER retention signal (KDEL), (f) ER signal peptide .
  • the fusion protein was cloned to be expressed in the form of HA-CRR4-XX or XX-CRR4-HA (HA: epitope tag (SEQ ID NO: 4); XX: candidate domain).
  • the reporter plasmid was loaded with an expression cassette in which Renilla luciferase (RLuc) and firefly luciferase (Fluc) are transcribed in the form of dicistronic mRNA under the control of the CMV promoter.
  • RLuc Renilla luciferase
  • Fluc firefly luciferase
  • Three PPR binding sequences UAUCUUGUCUUUA) (SEQ ID NO: 3) are inserted on the 5 ′ side of Fluc.
  • the effector plasmid and the reporter plasmid were transfected into HEK293T cells, and the luminescence levels of RLUC and FLUC were measured.
  • the RLUC luminescence amount was treated as a transfection control, and the FLUC luminescence amount / RLUC luminescence amount value was treated as a translational activity amount.

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Abstract

Le problème décrit par la présente invention est de développer un procédé de régulation d'un ARN cible. La solution selon l'invention porte sur une protéine de fusion contenant : un domaine fonctionnel qui améliore l'expression de protéine à partir de l'ARNm; et une protéine PPR apte à se lier sélectivement à des bases d'ARN ou à se lier spécifiquement à une séquence de base d'ARN, par rapport à un ARNm cible.
PCT/JP2017/020076 2016-06-03 2017-05-30 PROTÉINE DE FUSION POUR AMÉLIORER L'EXPRESSION DE PROTÉINES À PARTIR D'ARNm CIBLE WO2017209122A1 (fr)

Priority Applications (11)

Application Number Priority Date Filing Date Title
ES17806675T ES2874230T3 (es) 2016-06-03 2017-05-30 Proteína de fusión para mejorar la expresión de proteínas a partir de ARNm diana
JP2018520924A JP6928386B2 (ja) 2016-06-03 2017-05-30 標的ｍＲＮＡからのタンパク質発現量を向上させるための融合タンパク質
SG11201810606TA SG11201810606TA (en) 2016-06-03 2017-05-30 FUSION PROTEIN FOR IMPROVING PROTEIN EXPRESSION FROM TARGET mRNA
US16/305,080 US11136361B2 (en) 2016-06-03 2017-05-30 Fusion protein for improving protein expression from target mRNA
CN201780041619.9A CN109415446B (zh) 2016-06-03 2017-05-30 用于提高靶mRNA的蛋白质表达水平的融合蛋白
BR112018075010-7A BR112018075010A2 (pt) 2016-06-03 2017-05-30 proteína de fusão para melhorar o nível de expressão de proteína a partir de rnam alvo
DK17806675.9T DK3466978T3 (da) 2016-06-03 2017-05-30 Fusionsprotein til forbedring af proteinekspression fra mål-mRNA
CA3026340A CA3026340A1 (fr) 2016-06-03 2017-05-30 Proteine de fusion pour ameliorer l'expression de proteines a partir d'arnm cible
AU2017275184A AU2017275184B2 (en) 2016-06-03 2017-05-30 Fusion protein for improving protein expression from target mRNA
EP17806675.9A EP3466978B1 (fr) 2016-06-03 2017-05-30 Protéine de fusion pour améliorer l'expression de protéines à partir d'arnm cible
KR1020187037821A KR102407776B1 (ko) 2016-06-03 2017-05-30 표적 mRNA로부터의 단백질 발현량을 향상시키기 위한 융합 단백질

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WO2020241876A1 (fr) 2019-05-29 2020-12-03 エディットフォース株式会社 Procédé de production de protéine ppr efficace et son utilisation
WO2020241877A1 (fr) 2019-05-29 2020-12-03 エディットフォース株式会社 Protéine ppr présentant une agrégation moindre et utilisation associée

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Publication number Priority date Publication date Assignee Title
WO2020241876A1 (fr) 2019-05-29 2020-12-03 エディットフォース株式会社 Procédé de production de protéine ppr efficace et son utilisation
WO2020241877A1 (fr) 2019-05-29 2020-12-03 エディットフォース株式会社 Protéine ppr présentant une agrégation moindre et utilisation associée

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AU2017275184A1 (en) 2018-12-13
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