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WO2024193739A1 - Procédé de production de protéines dans des cellules hôtes - Google Patents

Procédé de production de protéines dans des cellules hôtes Download PDF

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Publication number
WO2024193739A1
WO2024193739A1 PCT/CZ2023/050015 CZ2023050015W WO2024193739A1 WO 2024193739 A1 WO2024193739 A1 WO 2024193739A1 CZ 2023050015 W CZ2023050015 W CZ 2023050015W WO 2024193739 A1 WO2024193739 A1 WO 2024193739A1
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WO
WIPO (PCT)
Prior art keywords
protein
sequence
hairpin
nucleotide sequence
plasmid
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
Application number
PCT/CZ2023/050015
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English (en)
Inventor
Michal SIMICEK
Alexandr VDOVIN
Zdenek KORISTEK
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ostravska Univerzita
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Ostravska Univerzita
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Ostravska Univerzita filed Critical Ostravska Univerzita
Priority to PCT/CZ2023/050015 priority Critical patent/WO2024193739A1/fr
Publication of WO2024193739A1 publication Critical patent/WO2024193739A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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    • 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
    • C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline

Definitions

  • the present invention relates to improvements in methods for expression of proteins in host cells.
  • the present invention provides a method for producing a protein in a host cell, comprising the steps of:
  • the host cell is preferably an eukaryotic cell. In other embodiments, the host cell is preferably a prokaryotic cell.
  • the coding nucleotide sequence of the protein to be produced and the hairpin nucleotide sequence are introduced into the host cell in such a manner that both sequences are translated into mRNA as a single construct comprising both the sequence of the protein to be produced and the hairpin sequence.
  • the coding nucleotide sequence of the protein to be produced and the hairpin sequence are preferably introduced using a plasmid.
  • the coding nucleotide sequence of the protein to be produced is cloned into the plasmid, and the hairpin sequence is cloned into the plasmid downstream of the stop codon of the coding nucleotide sequence of the protein to be produced but before the poly-A sequence, i.e., in the non-coding region.
  • the hairpin sequence useful in combination with the MCP protein is MS2 hairpin sequence.
  • the MS2 hairpin sequence is a bacteriophage sequence from the Emesvirus zinderi bacteriophage genomic RNA. This nucleotide sequence creates spontaneously a defined 3D hairpin structure in the cytoplasm of the cell. Being an exogenous structure, this hairpin is not bound by cellular RNA-binding proteins, but exclusively by the bacteriophage protein MCP.
  • the MS2 hairpin sequence preferably contains a plurality of MS2 hairpins, for example 12 to 48 MS2 hairpins, preferably 24 to 36 MS2 hairpins.
  • the nucleotide sequence of one MS2 hairpin is: acatgaggatcacccatgt (SEQ ID NO: 1).
  • nucleotide sequence of 24xMS2 is: acatgaggatcacccatgtctgcaggtcgactctagaaaacatgaggatcacccatgtctgcagtattcccgggttcattagatcctaaggtacctaat tgcctagaaaacatgaggatcacccatgtctgcaggtcgactctagaaaacatgaggatcacccatgtctgcagtattcccgggtttcattagatcctaa ggtacctaattgcctagaaaacatgaggatcacccatgtctgcaggtcgactccagaaaacatgaggatcacccatgtctgcaggtcgactccagaaaacatgaggatcacccatgtctgcaggtcgactccagaaaacatgaggatcacccatgtctgcagg
  • the MCP protein needs to be co-expressed simultaneously with mRNA encoding the protein to be produced that is fused with MS2 hairpin in the same host cell.
  • it is introduced into the host cell using a plasmid containing the MCP nucleotide coding sequence.
  • the host cell may be a cell which has the MCP nucleotide coding sequence in the genome, controlled so that it is coexpressed with the protein to be produced and with the MS2 hairpin.
  • the nucleotide sequence of the MCP is: atgctagccgttaaaatggcttctaactttactcagttcgttctcgtcgacaatggcggaactggcgacgtgactgtcgccccaagcaacttcgctaac gggatcgctgaatggatcagctctaactcgcgttcacaggcttacaaagtaacctgtagcgttcgtcagagctctgcgcagaatcgcaaatacaccat caaagtcgaggtgcctaaaggcgcctggcgttcgtacttaaatatggaactaaccattccaatttttcgccacgaattccgactgcgagcttattgttaagcaatgcaaggtctcgactgcga
  • Protein sequence of the MCP is:
  • the inventors observed that in case of simultaneous expression of the MCP protein and mRNA containing the MS2 hairpin, there is a massive increase in the synthesis of the protein encoded by this mRNA. This effect was observed both for proteins synthesized in the cytosol and for proteins synthesized into the endoplasmic reticulum which are then secreted outside the cell.
  • the hairpin sequence useful in combination with the PCP protein is PP7 hairpin sequence.
  • the PP7 hairpin sequence is a bacteriophage sequence from Pseudomonas aeruginosa bacteriophage. This sequence of nucleotides in the cytoplasm of the cell spontaneously creates a defined 3D hairpin structure. Being an exogenous structure, this hairpin is not recognized by eukaryotic RNA- binding proteins, but exclusively by the bacteriophage protein PCP.
  • the PP7 hairpin sequence preferably contains a plurality of PP7 hairpins, for example 12 to 48 PP7 hairpins, preferably 24 to 36 PP7 hairpins.
  • the nucleotide sequence of two non-identical stem-loops is (in uppercase): taaggtacctaattgcctagaaaGGAGCAGACGATATGGCGTCGCTCCctgcaggtcgactctagaaa- CCAGCAGAGCATATGGGCTCGCTGGctgcagtattcccgggttcatt (SEQ ID NO:5).
  • the PCP protein needs to be co-expressed with the protein to be produced and with the PP7 hairpin in the same host cell.
  • it is introduced into the host cell using a plasmid containing the PCP nucleotide coding sequence.
  • the host cell may be a cell which has the PCP nucleotide coding sequence in the genome, controlled so that it is co-expressed with the protein to be produced and with the PP7 hairpin.
  • the PCP protein amino acid sequence is: ggsmsktivlsvgeatrtlteiqstadrqifeekvgplvgrlrltaslrqngaktayrvnlkldqadvvdsglpkvrytqvwshdvtivansteasrk slydltkslvatsqvedlvvnlvplgr (SEQ ID NO:6).
  • the step of expressing the protein to be produced from the coding nucleotide sequence extended by MS2 or PP7 hairpin in the 5 -UTR in the presence of MCP or PCP protein in the host cell includes cultivating the host cells under conditions suitable for expression of the proteins. Such conditions are known for each type of host cells or can be determined by routine experimentation.
  • the produced protein is harvested by known technologies, e.g., by collecting the cultivation medium (in case of excreted proteins) or by collecting and lysing the host cells.
  • the present invention further includes a plasmid kit comprising a first plasmid and a second plasmid, wherein the first plasmid comprises a nucleotide sequence coding MS2 or PP7 hairpin sequence positioned upstream of a poly-A sequence and the second plasmid comprises a nucleotide sequence coding the MCP or PCP protein.
  • the present invention includes a plasmid comprising a nucleotide sequence coding MS2 or PP7 hairpin sequence positioned upstream of a poly-A sequence, and a nucleotide sequence coding the MCP or PCP protein.
  • MS2 hairpin shall be used in combination with the MCP protein
  • PPZ hairpin shall be used in combination with the PCP protein
  • FIG. 1 is a schematic illustration of one embodiment of the system MS2/MCP according to the invention.
  • the MCP produced from a second plasmid containing the MCP coding sequence interacts with the MS2 hairpin in a way which increases expression but does not hinder the translation into protein. More particularly, the first plasmid contains the gene of interest and 24 repeats of MS2 hairpin in its 3’ UTR.
  • the second plasmid contains the gene encoding MCP protein, which binds to MS2 hairpins.
  • Figure 2 shows schematically the second plasmid (confining the MCP coding sequence) as used in the examples.
  • Figure 3 shows schematically the first plasmid, wherein the protein of interest is CFP.
  • Figure 4 shows schematically the first plasmid, wherein the protein of interest is immunoglobulin.
  • Figure 5 represents the Western blotting analysis of IgL protein production.
  • Hek293 cells were transfected with plasmid containing IgL sequence followed by 24xMS2 in 3 ’UTR.
  • Control sample was cotransfected with empty vector and test sample with plasmid, expressing MCP protein.
  • the membrane was stained for HA -peptide (MCP protein was tagged with HA peptide), PCNA as loading control, and IgL.
  • Figure 6 represents the quantification of IgL production when its coding sequence is followed by MS2 hairpin sequence in the presence of MCP vs. without the presence of MCP. Bands intensity was measured by densitometry, IgL amount was normalised to loading control.
  • Figure 7 shows flow cytometry analysis of CFP production.
  • Hek293 cells were transfected with plasmid containing CFP sequence followed by 24xMS2 in 3 ’UTR.
  • Control sample was cotransfected with empty vector and test sample with plasmid, expressing MCP protein.
  • the amount of CFP was measured by fluorescence intensity.
  • Y axis represents number of cells, x axis - fluorescence intensity.
  • 3.5 x 10 6 HEK293 cells were plated on a 10 cm culture dish in 10 mL of DMEM medium with 10% fetal bovine serum. The cells were cultivated at 37°C with 5% CO2. The next day, the cells were transfected with two plasmids.
  • the first plasmid contained the gene of interest (IgL or ECFP coding sequence) followed by 24 repeats of MS2 hairpin, while the second plasmid contained the sequence of MCP protein or an empty plasmid serving as a control sample.
  • the term termed of interest“ refers to the coding sequence of the protein to be produced.
  • the transfection procedure involved mixing 2.5 pg of each plasmid and 15 pL of 1 mg/mL polyethylenimine in 1 mL of Opti-MEMTM Reduced-Serum Medium (Gibco) for 15 min at room temperature. The mixture was added to the cell culture. The next day, the growth medium was changed to fresh medium. After two days post-transfection, the amount of the protein of interest was compared between the test and control samples. Sequence of the plasmid containing the MCP protein coding sequence (the MCP protein coding sequence is marked in bold; the plasmid is schematically shown in Fig.
  • the membrane was washed three times for 15 min in PBST and incubated for 1 hour in a secondary antibody solution (Peroxidase AffiniPure Goat Anti-Mouse, 211-032-171, Exbio; DONKEY ANTI-RABBIT IGG HRP-LINK WHOLE, NA934, Merck; GOAT ANTI-RAT IGG-HRP LINKED WHOLE AB, NA935, Merck).
  • the membrane was washed three times for 15 min in PBST and developed using PierceTM ECL Western Blotting Substrate.
  • the cyan fluorescent protein (CFP) was detected using flow cytometry.
  • Cells were detached by removing the culture medium from the dish, rinsing the cells with 10 mL of PBS, incubating the cells with 1 mL of Trypsin-EDTA (Gibco) for 5 min at 37°C, suspending the detached cells in 10 mL of PBS, and spinning the cells at 300g for 5 min. The cells were then resuspended in PBS at a concentration of

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Abstract

La présente invention concerne un procédé de production d'une protéine dans une cellule hôte, comprenant les étapes consistant à : introduire une séquence nucléotidique codante de la protéine à produire et une séquence nucléotidique en épingle à cheveux dans la cellule hôte, et exprimer la protéine à produire à partir de la séquence nucléotidique codante en présence d'une protéine MCP ou PCP dans la cellule hôte.
PCT/CZ2023/050015 2023-03-23 2023-03-23 Procédé de production de protéines dans des cellules hôtes Pending WO2024193739A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CZ2023/050015 WO2024193739A1 (fr) 2023-03-23 2023-03-23 Procédé de production de protéines dans des cellules hôtes

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CZ2023/050015 WO2024193739A1 (fr) 2023-03-23 2023-03-23 Procédé de production de protéines dans des cellules hôtes

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WO2024193739A1 true WO2024193739A1 (fr) 2024-09-26

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018033110A1 (fr) * 2016-08-19 2018-02-22 苏州兰希亚生物科技有限公司 Procédé de réparation d'une mutation ponctuelle génique
WO2018200653A1 (fr) * 2017-04-25 2018-11-01 The Johns Hopkins University Système d'interaction arn-protéine à deux hybrides de levure basé sur crispr-dcas9 catalytiquement inactivé
RU2690935C2 (ru) * 2013-06-04 2019-06-06 Президент Энд Фэллоуз Оф Харвард Коллидж Направляемая рнк регуляция транскрипции
WO2021046243A2 (fr) * 2019-09-03 2021-03-11 Myeloid Therapeutics, Inc. Méthodes et compositions d'intégration génomique
WO2021175289A1 (fr) * 2020-03-04 2021-09-10 中国科学院遗传与发育生物学研究所 Procédé et système d'édition de génome multiplex
WO2022241029A1 (fr) * 2021-05-11 2022-11-17 Myeloid Therapeutics, Inc. Procédés et compositions pour l'intégration génomique

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2690935C2 (ru) * 2013-06-04 2019-06-06 Президент Энд Фэллоуз Оф Харвард Коллидж Направляемая рнк регуляция транскрипции
WO2018033110A1 (fr) * 2016-08-19 2018-02-22 苏州兰希亚生物科技有限公司 Procédé de réparation d'une mutation ponctuelle génique
WO2018200653A1 (fr) * 2017-04-25 2018-11-01 The Johns Hopkins University Système d'interaction arn-protéine à deux hybrides de levure basé sur crispr-dcas9 catalytiquement inactivé
WO2021046243A2 (fr) * 2019-09-03 2021-03-11 Myeloid Therapeutics, Inc. Méthodes et compositions d'intégration génomique
WO2021175289A1 (fr) * 2020-03-04 2021-09-10 中国科学院遗传与发育生物学研究所 Procédé et système d'édition de génome multiplex
WO2022241029A1 (fr) * 2021-05-11 2022-11-17 Myeloid Therapeutics, Inc. Procédés et compositions pour l'intégration génomique

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BARREAU CARINE ET AL: "Protein expression is increased by a class III AU-rich element and tethered CUG-BP1", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ELSEVIER, AMSTERDAM NL, vol. 347, no. 3, 1 September 2006 (2006-09-01), pages 723 - 730, XP002509498, ISSN: 0006-291X, [retrieved on 20060710], DOI: 10.1016/J.BBRC.2006.06.177 *
ROBERT HOGG J ET AL: "RNA-based affinity purification reveals 7SK RNPs with distinct composition and regulation", vol. 13, no. 6, 1 June 2007 (2007-06-01), pages 868 - 880, XP002679502, ISSN: 1355-8382, Retrieved from the Internet <URL:http://rnajournal.cshlp.org/content/13/6/868> [retrieved on 20070424], DOI: 10.1261/RNA.565207 *
SILVANA KONERMANN ET AL: "Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex", NATURE, vol. 517, no. 7536, 1 January 2015 (2015-01-01), London, pages 583 - 588, XP055585957, ISSN: 0028-0836, DOI: 10.1038/nature14136 *

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