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BR-112018075010-B1 - Fusion protein and in vitro method to improve protein expression level.

BR112018075010B1BR 112018075010 B1BR112018075010 B1BR 112018075010B1BR-112018075010-B1

Abstract

[Problem to be solved] The objective of the present invention is to develop a method for regulating a target RNA. [Solution] A fusion protein is provided comprising a functional domain that enhances the level of protein expression from mRNA and a PPR protein that can bind to a target mRNA in an RNA base-selective or RNA base sequence-specific manner.

Inventors

  • Takahiro Nakamura
  • Yusuke Yagi

Assignees

  • KYUSHU UNIVERSITY, NATIONAL UNIVERSITY CORPORATION

Dates

Publication Date
20260310
Application Date
20170530
Priority Date
20160617

Claims (11)

  1. 1. Fusion protein to enhance a level of protein expression from a target mRNA, the fusion protein characterized in that it comprises: (A) one or more functional domains that enhance a level of protein expression from an mRNA; and (B) a polypeptide moiety that can bind to a target mRNA in a selective RNA base or RNA base sequence specific manner, wherein the polypeptide moiety (B) comprises one or more PPR motifs, each PPR motif comprising a polypeptide consisting of 30 to 38 amino acids in length and being represented by Formula 1: where Helix A is a portion consisting of 12 amino acids in length and can form an α-helix structure, and is represented by Formula 2: where A1 to A12 each independently represents an amino acid; X is not present, or is a portion consisting of 1 to 9 amino acids in length; Helix B is a portion consisting of 11 to 13 amino acids in length and may form an α-helix structure; L is a portion consisting of 2 to 7 amino acids in length and represented by Formula 3: where amino acids are numbered from the C-terminus as "i" (-1), "ii" (-2), ... and Liii to Lvii may not be present, and a combination of three amino acids A1, A4 and Lii or a combination of two amino acids A4 and Lii corresponds to a base or base sequence of the target mRNA, wherein one or more functional domains (A) are selected from the group consisting of a domain that guides the ribosome to the mRNA, a domain that initiates or promotes mRNA translation, a domain associated with mRNA nuclear export, a domain that binds to an endoplasmic reticulum membrane, a domain containing an endoplasmic reticulum retention signal sequence (ER retention signal) and a domain containing an endoplasmic reticulum signal sequence, wherein the domain that guides the ribosome to the mRNA is a domain containing all or a functional part of a polypeptide selected from the group consisting of DENR (density-regulated protein), MCT-1 (amplified T-cell sequence 1). The domain that initiates or promotes mRNA translation is a domain containing all or part of a functional polypeptide selected from the group consisting of eIF4E and eIF4G; the domain associated with nuclear export of mRNA is a domain containing all or part of a functional SLBP (stem-loop binding protein); the domain that binds to an endoplasmic reticulum membrane is a domain containing all or part of a functional polypeptide selected from the group consisting of SEC61B, TRAP-alpha (translocon-associated protein alpha), SR-alpha, Dia1 (cytochrome b5 reductase 3), and p180; the endoplasmic reticulum retention signal sequence (ER retention signal) is a signal sequence containing a KDEL (SEQ ID NO:20) or KEEL (SEQ ID NO:21) sequence, and/or the signal sequence of endoplasmic reticulum is a signal sequence containing MGWSCIILFLVATATGAHS (SEQ ID NO:22).
  2. 2. Fusion protein, according to claim 1, characterized in that the polypeptide portion (B) comprises a plurality of 2 to 30 PPR motifs and the plurality of PPR motifs is arranged so as to bind specifically to the target mRNA base sequence.
  3. 3. Fusion protein, according to claim 2, characterized in that the polypeptide portion (B) comprises a plurality of 5 to 25 PPR motifs.
  4. 4. Fusion protein, according to claim 1, characterized in that one or more functional domains (A) each bind to an N-terminus and/or C-terminus of the polypeptide portion (B).
  5. 5. Fusion protein, according to claim 1, characterized in that the combination of the three amino acids A1, A4 and Lii in each of the PPR motifs is: (valine, threonine, asparagine), (phenylalanine, serine, asparagine), (phenylalanine, threonine, asparagine), (isoleucine, asparagine, aspartic acid), or (threonine, threonine, asparagine) in the order of (A1, A4, Lii) when a target base for the PPR motif is A (adenine); (glutamic acid, glycine, aspartic acid), (valine, threonine, aspartic acid), (lysine, threonine, aspartic acid), or (leucine, threonine, aspartic acid) in the order of (A1, A4, Lii) when the target base for the PPR motif is G (guanine); (valine, asparagine, aspartic acid). (isoleucine, asparagine, asparagine), (isoleucine, asparagine, aspartic acid), (isoleucine, methionine, aspartic acid), (phenylalanine, proline, aspartic acid), or (tyrosine, proline, aspartic acid) in the order of (A1, A4, Lii) when the target base for the PPR motif is U (uracil); or (valine, asparagine, asparagine), (isoleucine, asparagine, asparagine), (valine, asparagine, serine), or (isoleucine, methionine, aspartic acid) in the order of (A1, A4, Lii) when the target base for the PPR motif is C (cytosine).
  6. 6. Fusion protein, according to claim 1, characterized in that the combination of the two amino acids A4 and Lii in each of the PPR motifs is: (threonine, asparagine), (serine, asparagine) or (glycine, asparagine) in the order of (A4, Lii) when a target base for the PPR motif is A (adenine); (threonine, aspartic acid) or (glycine, aspartic acid) in the order of (A4, Lii) when the target base for the PPR motif is G (guanine); (asparagine, aspartic acid), (proline, aspartic acid), (methionine, aspartic acid) or (valine, threonine) in the order of (A4, Lii) when the target base for the PPR motif is U (uracil); or (asparagine, asparagine), (asparagine, serine) or (leucine, aspartic acid) in the order of (A4, Lii) when the target base for the PPR motif is C (cytosine).
  7. 7. In vitro method for improving the expression level of a target mRNA protein within a cell, the method characterized by comprising: introducing the fusion protein, as defined in claim 1, or a nucleic acid encoding the fusion protein into a cell.
  8. 8. Method according to claim 7, characterized in that the cell is a eukaryotic cell.
  9. 9. Method according to claim 8, characterized in that the cell is an animal cell.
  10. 10. Method according to claim 9, characterized in that the animal cell is a human cell.
  11. 11. Method according to claim 7, characterized in that the fusion protein is encoded in a vector.

Description

[Technical field] [001] The present invention relates to fusion proteins for improving protein expression levels from target mRNA. [Previous technique] [002] Techniques for linking nucleic acid-binding protein factors revealed by a variety of analyses of sequences of interest have been established and used in recent years. The use of this sequence-specific linking allows the removal of a target DNA sequence or regulation (activation or inactivation) of the expression of a protein-coding gene present downstream of the target DNA sequence to some extent. [003] While zinc finger nuclease (ZFN), TAL effector nuclease (TALEN), Crispr-Cas9 and the like are known as techniques using protein factors that act on DNA, the development of techniques using protein factors that act specifically on RNA is still limited. [004] The present inventors have proposed a method for designing a protein that can specifically bind to a target RNA sequence using the properties of PPR proteins (protein with one or more pentatricopeptide repeat motifs (PPR)), which are proteins found mainly in plants (Patent Literature 1). [List of quotations] [Patent Literature] [005] [Patent Literature 1] WO 2013/058404 [Summary of the Invention] [Technical problem] [006] In the description according to Patent Literature 1, the amino acids that function when a PPR motif demonstrates RNA-binding properties were identified, and the relationship between the structure of the PPR motif and the target base was revealed, thus allowing the construction of proteins that have one or more PPR motifs and can bind to RNAs possessing any sequence and length. However, no method was found that actually regulates target RNAs using the techniques according to Patent Literature 1. [Solution to the Problem] [007] As a result of extensive research on a method for improving the protein expression level of a target mRNA using a PPR protein, the present inventors found that a fusion protein of a predetermined functional domain and a PPR protein improves the protein expression level of the target mRNA, and completed the present invention. [008] Specifically, one embodiment of the present invention relates to a fusion protein for enhancing the protein expression level of a target mRNA, the fusion protein comprising: (A) one or more functional domains that enhance the protein expression level from an mRNA; and (B) a polypeptide moiety that can bind to a target mRNA in an RNA base-selective or RNA base sequence-specific manner, wherein the polypeptide moiety (B) is a polypeptide moiety comprising one or more PPR motifs, each PPR motif comprising a polypeptide consisting of 30 to 38 amino acids in length and being represented by formula 1:[formula 1] where helix A is a portion consisting of 12 amino acids in length and can form an α-helix structure, and is represented by formula 2:[formula 2] where A1 to A12 each independently represents an amino acid; X is not present, or is a portion consisting of 1 to 9 amino acids in length; helix B is a portion consisting of 11 to 13 amino acids in length and may form an α-helix structure; L is a portion consisting of 2 to 7 amino acids in length and represented by formula 3:[formula 3] where amino acids are numbered from the C-terminus as "i" (-1), "ii" (-2), ... and Liii to Lvii may not be present, and a combination of three amino acids A1, A4 and Lii or a combination of two amino acids A4 and Lii corresponds to a base or base sequence of the target mRNA. [009] In one embodiment according to the present invention, the polypeptide portion (B) comprises 2 to 30 PPR motifs, and the plurality of PPR motifs is arranged so as to specifically bind to the target mRNA base sequence. [0010] Furthermore, in one embodiment according to the present invention, the polypeptide portion (B) comprises 5 to 25 PPR motifs. [0011] Furthermore, in one embodiment according to the present invention, each one or more functional domains (A) bind to an N-terminal side and/or a C-terminal side of the polypeptide portion (B). [0012] In addition, in one embodiment according to the present invention, one or more functional domains (A) are selected from the group consisting of a domain that guides the ribosome to mRNA, a domain associated with the initiation or promotion of mRNA translation, a domain associated with the nuclear export of mRNA, a domain associated with binding to an endoplasmic reticulum membrane, a domain containing an endoplasmic reticulum retention signal sequence (ER retention signal), and a domain containing an endoplasmic reticulum signaling sequence. [0013] Furthermore, in one embodiment according to the present invention, the domain that guides the ribosome to the mRNA is a domain containing all or part of the functional portion of a polypeptide selected from the group consisting of DENR (density-regulated protein), MCT-1 (malignant T-cell amplified sequence 1), TPT1 (translational tumor control protein), and Lerepo4 (CCCH domain in zinc finge