WO2017160118A2 - Nouveau peptide pour améliorer l'efficacité d'expression d'une protéine cible, et protéine de fusion le comprenant - Google Patents

Nouveau peptide pour améliorer l'efficacité d'expression d'une protéine cible, et protéine de fusion le comprenant Download PDF

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WO2017160118A2
WO2017160118A2 PCT/KR2017/002902 KR2017002902W WO2017160118A2 WO 2017160118 A2 WO2017160118 A2 WO 2017160118A2 KR 2017002902 W KR2017002902 W KR 2017002902W WO 2017160118 A2 WO2017160118 A2 WO 2017160118A2
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protein
target protein
peptide
seq
expression
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Korean (ko)
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WO2017160118A3 (fr
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성백린
권순빈
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Industry Academic Cooperation Foundation of Yonsei University
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Industry Academic Cooperation Foundation of Yonsei University
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Priority claimed from KR1020170033692A external-priority patent/KR101915740B1/ko
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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

Definitions

  • the present invention relates to novel peptides and fusion proteins comprising the same for enhancing the expression efficiency of the protein of interest. More specifically, the present invention relates to a peptide consisting of four amino acids derived from LysRS (Lysyl tRNA synthetase), and a fusion protein comprising hRBD (human LysRS RNA binding domain) as a fusion partner thereof.
  • LysRS Lysyl tRNA synthetase
  • hRBD human LysRS RNA binding domain
  • the present inventors while studying the water solubility and expression efficiency of the protein in order to solve the problems of the prior art, the peptide consisting of four amino acids derived from LysRS (Lysyl tRNa synthetase) plays a crucial role in enhancing the expression efficiency of the target protein
  • the fusion protein including the human LysRS RNA binding domain (hRBD) as a fusion partner of four amino acids derived from LysRS and its fusion partner can not only enhance the expression efficiency of the target protein but also increase water solubility.
  • an object of the present invention is to provide a peptide consisting of four amino acids derived from LysRS, an expression vector comprising the peptide, and a host cell transformed with the expression vector.
  • the present invention also provides a peptide comprising four amino acids derived from LysRS and a fusion protein comprising hRBD as its fusion partner, an expression vector comprising the fusion protein, and a host cell transformed with the expression vector. The purpose.
  • the present invention is to provide a peptide for enhancing the expression efficiency of the target protein consisting of the amino acid sequence of SEQ ID NO: 1.
  • aminoacyl tRNA synthetase binds a corresponding amino acid to a tRNA in a cell, and the aminoacyl tRNA thus formed is transferred to an elongation factor or ribosome. It is used for protein synthesis.
  • the binding specificity of aminoacyl tRNA synthetases to amino acids and tRNAs serves as an important determinant in maintaining the accuracy of the protein synthesis process.
  • Lysyl tRNA synthetase is a member of an aminoacyl tRNA synthetase and is used in some mammals to modulate the various functions of the proteins that make up the aminoacyl tRNA synthetase. It forms macromolecular complexes that act as molecular reservoirs.
  • target protein is a protein that a person skilled in the art intends to produce in large quantities, and means any protein that can be expressed in a host cell by inserting a polynucleotide encoding the protein into a recombinant expression vector. .
  • fusion protein refers to a protein in which another protein is linked to or added to another N-terminus or C-terminus of the original target protein sequence. Means.
  • amino acid sequence of SEQ ID NO: 1 may be derived from LysRS (Lysyl tRNA synthetase).
  • the peptide for enhancing the expression efficiency of the target protein may bind to the N-terminus of the target protein.
  • the target protein may be at least one selected from the group consisting of antigen, antibody, cell receptor, enzyme, structural protein, serum and cellular protein.
  • the present invention is to provide an expression vector comprising a gene sequence encoding a peptide for binding to the N-terminus of the target protein and consisting of the amino acid sequence of SEQ ID NO: 1 to enhance the expression efficiency of the target protein.
  • the expression vector is a polynucleotide encoding a target protein and a polynucleotide encoding a peptide represented by SEQ ID NO: 1 bound to the 5'-end of the polynucleotide encoding the target protein It is characterized by including.
  • expression vector is a linear or circular DNA molecule consisting of fragments encoding a polypeptide of interest operably linked to additional fragments provided for transcription of the expression vector. Such additional fragments include promoter and termination code sequences. Expression vectors also include one or more replication initiation points, one or more selection markers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or contain elements of both.
  • operably linked refers to the arrangement of fragments in a promoter to act to initiate transcription and proceed through an encoding sequence to an end code.
  • the expression vector may be a plasmid, viral vector, phage particle or genomic insert.
  • the expression vector may be transformed into a host cell and then replicated or integrated into the genome of the host cell irrespective of the genome of the host cell.
  • the present invention also provides a host cell transformed with an expression vector that binds to the N-terminus of the protein of interest and consists of the amino acid sequence of SEQ ID NO: 1 comprising a gene sequence encoding a peptide for enhancing the expression efficiency of the protein of interest. I would like to.
  • transformation means that DNA is introduced into a host such that the DNA is replicable as an extrachromosomal factor or by chromosomal integration completion.
  • Method for transforming the expression vector according to the present invention is electroporation (electrophoration), calcium phosphate (CaPO 4 ) method, calcium chloride (CaCl 2 ) method, microinjection (microinjection), polyethylene glycol (PEG) method, DEAE-dex It may include, but is not limited to, the Tran method, the cationic liposome method or the lithium acetate-DMSO method.
  • the host cell is preferably a high DNA introduction efficiency, a host cell having a high expression efficiency of the introduced DNA, and any microorganism including prokaryotic and eukaryotic may be used.
  • the host cell may be E. coli .
  • the present invention is to provide a fusion protein for enhancing the expression efficiency and water solubility of the target protein comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and hRBD (human LysRS RNA binding domain) as a fusion partner thereof.
  • human LysRS RNA binding domain refers to a unique N-terminal extension that is involved in the interaction between LysRS RNA and other proteins of human origin. it means.
  • amino acid sequence of SEQ ID NO: 1 may be derived from LysRS (Lysyl tRNA synthetase).
  • the peptide may bind to the N-terminus of the target protein.
  • the hRBD may be composed of the amino acid sequence of SEQ ID NO.
  • the target protein may be selected from at least one selected from the group consisting of antigen, antibody, cell receptor, enzyme, structural protein, serum and cellular protein. have.
  • the invention also enhances the expression efficiency and water solubility of a target protein having a fusion protein comprising a peptide that binds to the N-terminus of the target protein and consists of the amino acid sequence of SEQ ID NO: 1 and a polynucleotide encoding hRBD as its fusion partner.
  • a target protein having a fusion protein comprising a peptide that binds to the N-terminus of the target protein and consists of the amino acid sequence of SEQ ID NO: 1 and a polynucleotide encoding hRBD as its fusion partner.
  • the expression vector is a polynucleotide encoding a target protein and a polynucleotide encoding a fusion protein represented by SEQ ID NO: 5 bonded to the 5 '-end of the polynucleotide encoding the target protein It provides an expression vector comprising a.
  • the expression vector may be a plasmid, viral vector, phage particle or genomic insert.
  • the expression vector may be transformed into a host cell and then replicated or integrated into the genome of the host cell irrespective of the genome of the host cell.
  • the invention also enhances the expression efficiency and water solubility of a target protein having a fusion protein comprising a peptide that binds to the N-terminus of the target protein and consists of the amino acid sequence of SEQ ID NO: 1 and a polynucleotide encoding hRBD as its fusion partner.
  • a target protein having a fusion protein comprising a peptide that binds to the N-terminus of the target protein and consists of the amino acid sequence of SEQ ID NO: 1 and a polynucleotide encoding hRBD as its fusion partner.
  • the host cell is preferably a host cell having a high DNA introduction efficiency and a high expression efficiency of the introduced DNA, and any microorganism including prokaryotic and eukaryotic may be used.
  • the host cell may be E. coli .
  • the present invention also provides a method for producing a target protein having enhanced expression efficiency.
  • the production method of the target protein is:
  • (C) culturing the transformant to induce expression of the recombinant protein of interest may include obtaining the same.
  • the peptide to enhance the expression efficiency of the target protein may be derived from LysRS (Lysyl tRNA systhetase), preferably may be an amino acid sequence represented by SEQ ID NO: 1.
  • the peptide that enhances the expression efficiency of the target protein may be a fusion protein combined with a peptide sequence derived from LysRS (Lysyl tRNA systhetase) and hRBD, preferably SEQ ID NO: 5 It may be an amino acid sequence represented by.
  • Peptides consisting of four amino acids derived from LysRS according to the present invention can improve the expression efficiency of the target protein, and the peptide and the fusion protein comprising hRBD as its fusion partner is not only water-soluble but also the expression efficiency of the target protein By improving, it can be usefully used for the production of a recombinant target protein.
  • Example 1 is a graph showing the effect of the peptide of the present invention according to Example 1 on the expression efficiency of EGFP-HBx protein.
  • Figure 2 is a graph showing the effect of the peptide of the present invention according to Examples 2 and 3 and the fusion protein comprising the same on the expression efficiency and water solubility of CsTA381 and CsTA1953 protein.
  • Example 3 is SDS-PAGE showing the effect of the peptide of the present invention and the fusion protein comprising the same according to Example 4 on the expression efficiency and water solubility of CsTA37 and CsTA422 proteins.
  • Figure 4 is an experimental result comparing the effect of increasing the target protein expression efficiency of the peptide sequence according to the present invention compared to other peptide sequences.
  • PGE-LysRS expression vector was used as protein expression vector (Choi SI et al., Protein solubility and folding enhancement by interaction with RNA, PLoS ONE (2008), 3: e2677).
  • pGE-LysRS is regulated by the T7 promoter, and the LysRS gene is cut using one of the cleavage sites in Nde1 and MCS (Kpn1-BamH1-EcoRV-Sal1-Hind3), where EGFP-HBx or hRBD , Or other proteins were inserted.
  • the amino acid sequence of SEQ ID NO: 1 was inserted at the N-terminal position of the inserted protein.
  • the target proteins to be expressed were inserted using two enzyme sites in the prepared hRBD or MCS of the amino acid containing hRBD vector of SEQ ID NO: 1.
  • the prepared protein expression vector was transformed into BL21 (DE3) or BL21 * (DE3) -pLysS or BL21 (DE3) -pLysE competent cells and cultured. All transformed Escherichia coli were cultured in LB medium containing 50 ⁇ g / ml Ampicillin, and E. coli transformed with BL21 * (DE3) -pLysS or BL21 (DE3) -pLysE was 34 ⁇ g / ml chloramphenicol. It was cultured in the added medium. The culture temperature is different for each protein, it was incubated under the conditions of 25 ⁇ 37 °C. When the OD 600 value of E.
  • IPTG is added at a level of 0 ⁇ M to 1 mM to activate the T7 promoter, and 3 hours or 25 at 37 ° C after IPTG is added to allow sufficient protein production. C was incubated for about 5 hours. Fully cultured E. coli was stored after centrifugation to remove the supernatant. Then, 0.3 ml of PBS was added to the E. coli harvest corresponding to 5 ml of the LB medium, and ultrasonically pulverized to prepare a lysate. The lysate was centrifuged and divided into a soluble fraction and a pellet fraction. The total lysate, soluble fraction, and pellet fraction were divided into SDS-PAGE. Analyzed.
  • Samples to be analyzed were transferred directly to PVDF membranes after SDS-PAGE. Empty spaces in the PVDF membrane that were not filled with sample protein were blocked with 5% skim milk in TBST (pH 7.5 Tris-buffered saline, + 0.05% tween 20). Then, after washing the membrane three times with TBST, Anti-his tag (1: 20000) (QIAGEN, 34660) was treated to the membrane at 4 °C for 16 hours. After washing the membrane three times again with TBST, the membrane was treated with an anti-mouse antibody (Sigma, A4416) with HRP as a secondary antibody for 1 hour at room temperature and again with TBST three times. After washing, treated with WEST-ZOL (Intron, Korea) and confirmed with a film.
  • TBST pH 7.5 Tris-buffered saline, + 0.05% tween 20.
  • LysRS was isolated from the pGE LysRS plasmid using Nde1 and Hind3 restriction enzymes, and the EGFP-HBx gene sequence (SEQ ID NO: 2) was added to the N-terminus at the position. Two pieces of fusion form were put in each.
  • the vector is expressed by the T7 promoter, and promoter activation is regulated by IPTG in a form regulated by the lac operator.
  • Two recombinant plasmids were transformed into BL21 (DE3) -pLysE competent cells, and the proteins were expressed at 37 ° C. for 3 hours. At this time, IPTG was treated with four concentrations of 0, 10, 20 and 40 ⁇ M. As a result, the fusion form of the EGFP-HBx gene was confirmed that the expression was better than the direct form (Fig. 1A).
  • the pGE forms the direct and fusion forms of the two proteins using the same method as in Example 1.
  • Each was inserted into LysRS plasmid.
  • a total of four recombinant plasmids were transformed into BL21 * (DE3) -pLysS competent cells and expressed proteins at 37 ° C.
  • IPTG was treated at 1 mM concentration and incubated for 3 hours.
  • the expression level of the fusion form of the CsTA381 and CsTA1953 proteins increased compared to the direct form (FIGS. 2A and 2B).
  • Example 3 According to the present invention Peptide And hRBD A fusion protein comprising CsTA381 and CsTA1953 Effect on protein expression efficiency and water solubility
  • HRBD was used as the fusion partner to increase the water solubility of the CsTA381 and CsTA1953 proteins according to Example 2.
  • hRBD is represented by the amino acid sequence of SEQ ID NO: 3, the fusion protein (mseq-hRBD) in which the peptide sequence of SEQ ID NO: 1 to hRBD is shown as the amino acid sequence of SEQ ID NO: 5.
  • LysRS was cut out from pGE LysRS plasmid using Nde1 and Kpn1, and DNAs of SEQ ID NO: 4 and SEQ ID NO: 6, which are gene sequences encoding hRBD and mseq-hRBD, were inserted at the positions. Then, the CsTA381 gene and the CsTA1953 gene were inserted into the pGE hRBD plasmid and the pGE mseq-hRBD plasmid using BamH1 and Hind3, respectively.
  • the expression level of the mseq-hRBD binding form of the CsTA381 and CsTA1953 proteins was at least four times higher than that of the hRBD binding form. It was found to increase ( Figures 2E and 2F). And in the case of mseq-hRBD binding form, the expression level is much increased compared to the SEQ ID NO: 1 binding form, it was also confirmed that the synergistic effect of the hybrid form.
  • Example 4 Peptide And hRBD A fusion protein comprising CsTA37 and CsTA422 Effect on protein expression efficiency and water solubility
  • the LysRS is removed from the pGE LysRS plasmid using Nde1 and Hind3 restriction enzymes, and the gene sequence encoding the peptide having SEQ ID NO: 1 added to the N-terminus at the GFP gene sequence (SEQ ID NO: 2, fusion form) Mseq) and two gene sequences (SEQ ID NO: 8, fufusion form; Mseqhaq) encoding a peptide added at the N-terminus of 7 were respectively inserted.
  • the vector is expressed by the T7 promoter, and promoter activation is regulated by IPTG in a form regulated by the lac operator.
  • Each of the three recombinant plasmids was transformed into BL21 (DE3) -pLysE competent cells and expressed the protein for 3 hours at 37 ° C. At this time, IPTG was treated with four concentrations of 0, 10, 20, 40 ⁇ M.

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Abstract

La présente invention concerne un nouveau peptide permettant d'améliorer l'efficacité d'expression d'une protéine cible, et une protéine de fusion le comprenant. Le nouveau peptide de la présente invention peut améliorer l'efficacité d'expression d'une protéine cible, et une protéine de fusion contenant le peptide peut être utilisée pour produire une protéine cible recombinante en améliorant non seulement l'efficacité d'expression mais également la solubilité dans l'eau de la protéine cible.
PCT/KR2017/002902 2016-03-18 2017-03-17 Nouveau peptide pour améliorer l'efficacité d'expression d'une protéine cible, et protéine de fusion le comprenant Ceased WO2017160118A2 (fr)

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KR20160032451 2016-03-18
KR10-2016-0032451 2016-03-18
KR1020170033692A KR101915740B1 (ko) 2016-03-18 2017-03-17 목적 단백질의 발현 효율을 증진시키기 위한 신규한 펩타이드 및 이를 포함하는 융합 단백질
KR10-2017-0033692 2017-03-17

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US20230098002A1 (en) * 2019-02-14 2023-03-30 European Molecular Biology Laboratory Means and methods for preparing engineered target proteins by genetic code expansion in a target protein-selective manner

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KR100890579B1 (ko) * 2002-08-19 2009-04-27 프로테온 주식회사 Ｒｎａ 결합 단백질의 유전자를 융합파트너로 이용한재조합 단백질의 제조방법
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KR101360375B1 (ko) * 2011-08-19 2014-02-10 연세대학교 산학협력단 수용성 ｂｍｐ-2를 생산하는 재조합 대장균 및 이를 이용한 수용성 ｂｍｐ-2의 제조방법

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230098002A1 (en) * 2019-02-14 2023-03-30 European Molecular Biology Laboratory Means and methods for preparing engineered target proteins by genetic code expansion in a target protein-selective manner

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