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WO2020197518A1 - Chromosome artificiel 2 de tétrahyména thermophila (ttac2) et son utilisation dans la production de protéines recombinantes - Google Patents

Chromosome artificiel 2 de tétrahyména thermophila (ttac2) et son utilisation dans la production de protéines recombinantes Download PDF

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WO2020197518A1
WO2020197518A1 PCT/TR2019/051165 TR2019051165W WO2020197518A1 WO 2020197518 A1 WO2020197518 A1 WO 2020197518A1 TR 2019051165 W TR2019051165 W TR 2019051165W WO 2020197518 A1 WO2020197518 A1 WO 2020197518A1
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tetrahymena thermophila
artificial chromosome
sequence
nts
seq
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Muhittin ARSLANYOLU
Ayca Fulya USTUNTANIR DEDE
Mehmet CALISEKI
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Anadolu Universitesi
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Anadolu Universitesi
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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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • 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
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • 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

Definitions

  • Tetrahymena thermophila artificial chromosome having a nucleotide sequence that is at least 80% homologous to SEQ ID NO: 4 and comprising a modified HSP70.2 promoter having SEQ ID NO: 1, 3'NTS sequence having SEQ ID NO: 2, "5 'NTS containing C3 origin, rDNA genes and 3'NTS” sequence having SEQ ID NO: 3.
  • Tetrahymena thermophila artificial chromosome which is formed by biomimetics of the Tetrahymena thermophila macronucleus rDNA minichromosome further comprises a telomere sequence, Neo4 cassette and TtsfGFP marker gene cassette.
  • the present invention relates to a Tetrahymena thermophila artificial chromosome which can be used in linear or circular forms by virtue of the telomeric sequence it contains, including"5'NTS containing C3 origin, rDNA genes and 3'NTS” sequence on one arm, and 3'NTS sequence, Neo4 cassette and a marker gene cassette on the other arm, and which can be used in recombinant protein production by means of heat-shock induction owing to modified HSP70.2 promoter it contains.
  • Biomimetics refers to the imitation of models and systems in nature for the purpose of solving a problem or improving an existing system. By the virtue of this field, many technologies inspired by biological solutions at the macro or nano level have been developed. By constructing expression vectors with the principles of biomimetics, it is possible to obtain vectors with high transformation efficiency and intracellular stability. The aim of biomimetics applications in the field of molecular biology is to form vectors that are similar to the cell’s chromosome, therefore cells cannot distinguish between their wild-type structures and biomimetics products.
  • Tetrahymena thermophila as a host for recombinant protein production in a way that is productive and efficient.
  • various methods used for transforming the organism with the gene(s) of interest including microinjection, electroporation and biolistic bombardment (Cassidy-Hanley, D., et. al. 1997. Germline and somatic transformation of mating Tetrahymena thermophila by particle bombardment. Genetics, 146(1), 135-147).
  • Tetrahymena cells with a batch tank, a fed-batch tank and continuous feed bioreactors by approaching to values of a cell density of approximately 2xl0 7 cells/ml, a dry cell weight of 80 g/L and a tank scale-up volume of 1000 L.
  • a typical reactor time takes about 3-4 days from inoculation to harvest.
  • Tetrahymena ribosomal RNA genes are one of the best characterized eukaryotic genes (Yu, G. L, Hasson, M., & Blackburn, £. H., 1988. Circular ribosomal DNA plasmids transform Tetrahymena thermophila by homologous recombination with endogenous macronuclear ribosomal DNA. Proceedings of the National Academy of Sciences of the United States of America, 85(14), 5151- 5).
  • a cloning vector can be defined as a fragment of DNA that can be used as a tool to carry another fragment of DNA into a host organism.
  • Cloning vectors can be formed by combining the DNA sequences that are obtained from a virus, a prokaryotic bacterial cell or a cell of a higher organism.
  • Cloning vectors are intermediary DNA sequences used to transfer a nucleic acid sequence to the host organism and to quantitatively increase the DNA sequence in the host.
  • an ideal cloning vector possesses could be regarded as: not containing DNA elements that can cause unwanted changes by recombination or mutation in the host organism, containing a gene for selection after transformation, being designed as to contain a region that a DNA fragment can be cloned, having an origin region that enables controlled and stable copy numbers, etc.
  • Artificial chromosomes are used both as a cloning vector and expression vector. They can carry DNA inserts larger than the capacity of plasmids or lambda-phage-derived vectors. Artificial chromosomes, like other cloning vectors, contain the nucleic acid elements that are necessary for replication and stability of the vector and its product in the host cell. These elements ensure the continuity of the vector copy number in the daughter cells upon cell division. Artificial chromosomes are ideal vectors for stable, controlled and high-level production of proteins that necessitate the coordinated expression of several genes or that are encoded by large genes. US 8,288,610 B2 patent document relates to preparation of plant cell lines that comprise artificial chromosomes.
  • Antibiotic resistance cassettes are important tools in cloning and vector constitution. Antibiotic resistance allows the positive transformants to be selected easily and effectively. The transformant cells that contain the vector can grow in the presence of selected antibiotic, which proves that the transformation was successful and vector functions in the cell.
  • Tetrahymena thermophila Recombinant protein production in Tetrahymena thermophila has been widely used. Numerous proteins from different species, including functional human enzymes were produced using Tetrahymena as an expression system ( Weide T., Herrmann L, Bockau U., NieburN., Aldag /., Laroy W. , Contreras R., Tiedtke A., Hartmann M. W. (2006). Secretion of functional human enzymes by Tetrahymena thermophila. BMC Biotechnol. 6:19). Therefore, various vectors and promoters specific to the organism have been developed. However, current techniques and tools has its downsides along with the advantages described above. First of all, vectors used are mostly circular.
  • MTT1 is a inducible- repressible promoter for driving high-level expression of heterologous or homologous genes in Tetrahymena thermophila. It belongs to a Cd-inducible metallothionein gene (MTT1) of Tetrahymena thermophila.
  • MTT1 Cd-inducible metallothionein gene
  • the activation and deactivation of the promoter are achieved by simply adding or depleting cadmium.
  • Cd is a heavy metal. Heavy metals are toxic and restrain the cell growth. Also, the disposal of contaminated media takes much time and resource, since heavy metals are a threat to environment and to human health. Use of a different promoter with less negative impact on the host, humans and environment would be favourable.
  • Heat shock proteins are stress response proteins that are found in all eukaryotic organisms studied to date. The genes that code these proteins are inducible by heat, as well as other stress factors or external agents. That means the cells respond to a stress condition, such as high growth temperature, by producing high levels of HSPs. Regulation of HSPs has been studied extensively and HSP promoters were found to be strong and effective promoters. Also, the production process can be simply activated and deactivated by the changes in temperature, without a need for chemical agents. Therefore, heat shock promoters are suitable for recombinant studies and can be used for controlled heterologous gene expression at high levels.
  • WO 2007/006812 A1 relates to the use of heat-inducible HSP90 promoter that belongs to the family of Tetrahymena thermophila heat-shock proteins.
  • the use of the promoter for the expression of homologous and/or heterologous proteins in the ciliate Tetrahymena thermophila is also claimed within the scope of WO 2007/006812 A1 patent document.
  • CN 101586119 A, HSP70.2 promoter sequence is used in the expression of GFP marker gene.
  • HSP70.2 that is defined in this patent application consists of 1132 bp sequence.
  • Said sequence contains 1105 bp HSP70.2 promoter and 5'UTR region of the mRNA of the gene, 21 bp sequence of HSP70.2 gene that codes protein starting from ATG start codon and 6 bp BamHI restriction enzyme cloning site.
  • FIG. 1 shows the elements and sequences that construct TtAC2, the vectors that these elements are derived, and circular form of TtACl.
  • fluorescence microscopy analysis of the expression of the TtsfGFP gene in cells having TtAC2 at 5th month post-selection E. fluorescence microscopy analysis of the expression of the TtsfGFP gene in cells having TtAC2 at 24th month post-selection.
  • Said artificial chromosome was designed for unicellular eukaryotic organism Tetrahymena thermophila and other ciliate host cells of the Tetrahymenidae family.
  • the object of the invention is to produce recombinant proteins by forming an artificial chromosome that maintains intracellular stability for a long time and to increase the efficiency of Tetrahymena thermophila as an expression system.
  • This artificial chromosome (TtAC2) of the present invention was developed by performing biomimetics of the Tetrahymena thermophila rDNA minichromosome.
  • Tetrahymena thermophila is a well studied organism in the field of biology. Reaching high cell densities in short reproduction time, being able to grow easily and inexpensively in the laboratory and performing post- translational modifications in the produced proteins are some of the reasons for the widespread use of this organism. This latter feature is particularly necessary in the production of proteins of eukaryotic origin, since post-translational modifications play an important role in protein function in eukaryotes. Therefore, eukaryotic organism Tetrahymena thermophila, provides some advantages in recombinant protein production over prokaryotic host organisms.
  • Vector backbone is then added the gene sequence of protein or enzyme of interest.
  • vectors used as source of the backbone elements are preferred as pUC19 (Yanisch-Perron C, Vieira J Messing ]., 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33:103- 119) and pNeo4 (Mochizuki, K., 2008. High efficiency transformation ofTetrahymena using a codon- optimized neomycin resistance gene. Gene , 425(1), 79-83).
  • the artificial chromosome of the present invention comprises a modified HSP70.2 promoter having SEQ ID NO: 1, a 3’NTS sequence having SEQ ID NO: 2 and "5 'NTS containing C3 origin, rDNA genes and 3'NTS” sequence having SEQ ID NO: 3.
  • a specific HSP70.2 promoter of the invention is contained in the TtsfGFP cassette and has the nucleotide sequence set forth in SEQ ID NO: 1.
  • G (guanine) base of the ATG codon of the gene region encoding the artificial 11 amino acid HSP70 protein sequence which is involuntarily added to the N-terminus of the recombinant proteins, is changed by mutation with T (thymine) base, so the ATG codon was transformed to ATT, resulting in modification of the 11 amino acids encoding sequence into a non-coding form.
  • an A (adenine) base was added prior to the Pmel enzyme restriction sequence at the 3' end of the HSP70.2 promoter sequence.
  • MTT1 promoter which belongs to Cd-inducible metallothionein gene of Tetrahymena thermophila is widely used in recombinant protein production.
  • Cd is a heavy metal and the induction with Cd harms both the organism and environment Therefore Cd-inducible promoters are not favorable in the terms of good manufacturing practices.
  • Heat shock promoters on the other hand, are induced by heat or other stress conditions. They belong to heat shock genes, which are found in all eukaryotic cells. The induction and termination of transcription can simply be carried out by increasing or decreasing the growth temperature. Therefore, specific modified HSP70.2 promoter of the present invention is important and advantageous in that it can be induced by increasing the growth temperature in line with good manufacturing practices.
  • the artificial chromosome of the present invention produced for Tetrahymena thermophila comprises a 3'NTS sequence (SEQ ID NO: 2)
  • said 3'NTS sequence can be amplified with specific primers from the genomic DNA of the Tetrahymena thermophila SB210 strain and can be inserted into a vector backbone sequence to obtain the artificial chromosome.
  • the specific 3'NTS sequence of the invention comprises subtelomeric sequences of the rDNA minichromosome in Tetrahymena thermophila and enables the stable operation of telomeres and telomeric genes. Thus, it helps to increase the life time of the artificial chromosome in the cell.
  • the 3'NTS sequence is located between the gene cassettes and the telomere sequences necessary for the production of recombinant protein, thereby preventing possible telomere pressure in the transcription of the genes.
  • the Tetrahymena thermophila artificial chromosome of the present invention further comprises "5'NTS containing C3 origin, rDNA genes and 3'NTS” sequence (SEQ ID NO: 3).
  • the specific rDNA locus and C3 ori sequence of the present invention can be amplified with specific primers from the rDNA minichromosome of Tetrahymena thermophila C3.368.1 strain and can be inserted into the vector backbone of the present invention.
  • the rDNA locus and C3 ori sequence, which form an arm of the TtAC2 artificial chromosome, are important for the biomimetics of the Tetrahymena thermophila rDNA minichromosome and for long-term biological maintenance after transformation.
  • said vector backbone is pUC19 vector.
  • the specific vector backbone of the invention is advantageous in that it has diversity of restriction enzymes in the cloning site, the vector size is small (2.6 kbp) and it has an intermediate copy origin (100+).
  • artificial chromosome for Tetrahymena thermophila further comprises a telomere sequence.
  • said telomere sequence can be cut from a pPXV-GFP vector, amplified and inserted into a backbone vector sequence in order to obtain the artificial chromosome.
  • the telomere cassette of the invention is important and advantageous in that it enables the use of TtAC2 artificial chromosome in circular or linear form.
  • the use of linear vector presents the advantage of removing the limitation about the size of the insertion. Therefore, the linear form of the artifical chromosome enables vector to be usable when more gene cassettea are inserted.
  • the Tetrahymena thermophila artificial chromosome of the present invention further comprises a Neo4 neomycin resistance cassette.
  • the specific Neo4 cassette of the present invention can be amplified with specific primers from the pNeo4 vector and be inserted into pUC19 vector backbone of the present invention.
  • Neo4 cassette which is CdCl 2 -inducible via the MTT1 promoter is suitable for enabling efficient selection of positive transformants with increased antibiotic suppression and reduced cadmium induction by the Tetrahymena thermophila codon-optimized neomycin resistance cassette it contains.
  • the Tetrahymena thermophila artificial chromosome of the present invention further comprises a marker gene cassette.
  • the expression cassette containing the TtsfGFP-6xHis gene was taken from the pVTtsfGFP vector and inserted into TtAC2 as a marker gene with some sequence changes ( Yilmaz , G., & Arslanyolu, M. 2015. Efficient expression of codon- adapted affinity tagged superfolders for fluorescent protein localization and affinity purification studies in Tetrahymena thermophila BMC biotechnology ⁇ , 15 ( 1 ), 22).
  • the modified HSP70.2 promoter (SEQ ID NO: 1) contained in the cassette is heat-inducible, thereby enabling a favorable production by the production of the marker gene without the need for chemicals.
  • Tetrahymena thermophila artificial chromosome of the present invention (TtAC2) to produce TtsfGFP-6xHis protein proves that chromosome construction and transformation is successful and that TtAC2 works effectively in the cell.
  • the sfGFP protein emits fluorescent light, making it easier to trace.
  • the marker gene used in the invention may be replaced by a DNA sequence encoding homologous and/or heterologous genomic DNA, RNA and/or proteins and/or derivatives thereof.
  • the artificial chromosome of the present invention can be used for recombinant production of any desired sequence in Tetrahymena thermophila.
  • the artificial chromosome of the present invention is constructed as to contain "5 'NTS containing C3 origin, rDNA genes and 3'NTS” sequence (SEQ ID NO: 3) on one arm and a 3'NTS sequence (SEQ ID NO: 2), Neo4 cassette and TtsfGFP marker gene cassette on the other arm.
  • the direction of the transcription of the gene cassettes on both arms is configured from the center to the telomeres, thereby reducing the potential encounter of DNA replication and transcription complexes with biomimetics.
  • Tetrahymena thermophila artificial chromosome of the present invention has a nucleotide sequence that is at least 80% homologous to SEQ ID NO: 4.
  • the Tetrahymena thermophila artificial chromosome of the present invention may optionally be in circular or linear form by virtue of the telomere cassette it contains.
  • the specific telomere cassette sequence of the present invention comprises a fragment sequence that can be cut at both ends with the Sfil enzyme and released to convert the circular chromosome into a linear chromosome.
  • the conservation of the telomere sequences at the ends of the artificial chromosome in linear form is ensured by 3 'NTS sequences.
  • the present invention provides a method for producing the Tetrahymena thermophila artificial chromosome (TtAC2) described above, comprising the following steps:
  • said method further comprises the step of inserting the TtsfGFP marker gene cassette or a DNA sequence encoding homologous and/or heterologous genomic DNA, RNA and/or proteins and/or derivatives thereof.
  • said vector backbone is pUC19, so that the Tetrahymena thermophila artificial chromosome obtained by the method has a nucleotide sequence that is at least 80% homologous to SEQ ID NO: 4.
  • the present invention provides a method for recombinantly producing homologous and/or heterologous genomic DNA, RNA and/or proteins and/or derivatives thereof by using the Tetrahymena thermophila artificial chromosome as a production system/platform. Said method comprises the following steps:
  • Tetrahymena thermophila artificial chromosome TtAC2
  • Tetrahymena thermophila or Tetrahymenidae family member containing a recessed allele other than the C3 origin allele
  • Tetrahymena thermophila artificial chromosome by Tetrahymena thermophila or by a ciliate host cell member of the Tetrahymenidae family.
  • Microinjection, electroporation, and biolistic bombardment methods are usually preferred for transforming the Tetrahymena cells with the newly created vector.
  • biolistic gun method is used for the transformation of the artificial chromosome.
  • Tetrahymena thermophila artificial chromosome can be directed to the macronucleus of Tetrahymena thermophila or Tetrahymenidae family member ciliate host cell by biolistic gun method.
  • said Tetrahymena thermophila or Tetrahymenidae family member ciliate host cell is a cultured vegetative ciliate cell or two conjugating ciliate cells.
  • the present invention provides a transformant Tetrahymenidae family member ciliate host cell that is formed by transformation of TtAC2 bearing at least one homologous and/or heterologous sequence which is obtained as a result of recombinant production method of homologous and/or heterologous genomic DNA, RNA and/or proteins and/or derivatives thereof by using the Tetrahymena thermophila artificial chromosome of the invention as a production system /platform.
  • the present invention provides the use of the Tetrahymena thermophila artificial chromosome as a production system/platform for recombinantly producing homologous and/or heterologous genomic DNA, RNA and/or proteins and/or derivatives thereof.
  • the present invention is performed by inserting a modified HSP70.2 promoter (SEQ ID NO: 1), a 3'NTS sequence (SEQ ID NO: 2), and "5 'NTS containing C3 origin, rDNA genes and 3 'NTS” sequence (SEQ NO: 3) into the pUC19 vector backbone.
  • the vector backbone was also added with a telomere sequence, Neo4 neomycin resistance cassette and TtsfGFP marker gene cassette.
  • the present invention is a novel artificial chromosome vector that can be used for the production of recombinant protein in Tetrahymena thermophila or in a cilate host cell of the Tetrahymenidae family member.
  • Said Tetrahymena thermophila artificial chromosome shows at least 80% homology with SEQ ID NO: 4.
  • TtAC2 Tetrahymena Thermophila Artificial Chromosome
  • the vector pUC19 was used as the backbone vector for £. coli cloning ( Yanisch-Perron , C., Vieira, J., & Messing, ]. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpI8 and pUC19 vectors, Gene, 33 (1 ), 103-119). This vector is chosen because of the restriction enzyme diversity in the cloning region, the small size of the vector (2.6 kbp), and the intermediate copy origin (100+).
  • the wild-type Tetrahymena rDNA chromosome does not contain centromere, similarly TtAC2 artificial chromosome does not contain centromere.
  • the direction of transcription is configured from the center to the telomeres.
  • an artificial chromosome was obtained with the rDNA locus on one arm and the marker gene (TtsfGFP) and Neo4 cassettes on the other arm.
  • the TtAC2 artificial chromosome which can be used in circular form, can also be used in linear form by cutting from the Sfil cutting region located between the telomere regions.
  • the circular and linear forms of the artificial chromosome TtAC2 and the conversion of the circular form to the linear form are shown in Figure 2 -A.
  • the Tetrahymena thermophila macronucleus rDNA chromosomes have a 3'NTS region as a copy in the region between the telomere and the rDNA gene sequence at both ends.
  • the 3'NTS region contains the telomere associated DNA sequence.
  • One of the tasks of these subtelomeric sites is to ensure replication and maintenance of the end regions of chromosomes containing the DNA locus (Challoner, PU, Amine , AA, Pearlman, RE & Blackburn, EH 1985. conserveed arrangements of repeated DNA sequences in nontranscribed spacers of ciliate ribosomal RNA genes: evidence for molecular coevolution, Nucleic acids research, 13 (7), 2661-2680).
  • Another task is to include elements consisting of repetitive transcription and translation terminating DNA sequences. Finally, these regions are responsible for the formation of nucleosomes in the telomere region and help to pack the chromosome into the DNA-protein complex (Budarf ML, Blackburn, EH 1986. Chromatin structure of the telomeric region and 3'-nontranscribed spacer of Tetrahymena ribosomal RNA genes. Biological Chemistry, 261 (1), 363-369).
  • Purified genomic DNA from the Tetrahymena thermophila SB210 strain was used as the source DNA to produce the 1.8 kbp long 3'NTS sequence by Polymerase Chain Reaction (PCR)
  • the 3'NTS region was generated using the relevant forward and reverse primers.
  • the pUC19 vector was cut as to have sticky ends by restriction with the restriction enzymes Kpnl (Thermo, #ER0521) and BamHII (Thermo, #ER0052)
  • the cut pUC19 vector and 3'NTS sequence containing DNA bands were visualized on a 0.6% agarose gel and cut from the gel under a UV cabinet at 365 nm wavelength.
  • the respective sequence and vector were then purified using gel purification kit (Thermo, Genejet Gel Extraction Kit, #K0691)
  • the sticky end 3’NTS sequence and pUC19 vector were ligated by incubating with T4 DNA ligase (NEB, M0202S) at 16 °C for 16 hours and transformed into E. coli XLlBlue competent cells with heat shock of 42 °C. After incubation at 37 °C for 16 hours, the master plate (containing ampicillin-LB agar) was made from transformant competent cells and the selected colonies were screened by PCR. PCR products were run on 1% agarose gel and positive colonies were detected by imaging.
  • the Neo4 cassette contains the paramomycin-resistant Neo4 gene, which contains a heavy metal cadmium-inducible MTT1 promoter and a BTU2 termination sequence. This cassette is added to the artificial chromosome structure for the selection of positive Tetrahymena thermophila cells containing the vector.
  • pNeo4 vector (Mochizuki, K., 2008. High efficiency transformation of Tetrahymena using a codon-optimized neomycin resistance gene. Gene , 425(1 ), 79-83) was used as the source DNA and 2 kbp long DNA was amplified by the PCR reaction. Said 2 kbp long DNA band was purified from the agarose gel.
  • the vector pUC19-3'NTS (TtAC2.1) and the Neo4 gene cassette were made to become sticky-ended using restriction enzymes Sail (Thermo, #ER0642) and BamHII (Thermo, #ER0052) and then ligated. The resulting vector was transformed into E.
  • the GFP gene cassette was used as a marker gene to demonstrate the reproducibility of a recombinant protein, therapeutic drug or enzymes using the Tetrahymena thermophila artificial chromosome TtAC2.
  • the use of toxic substances or heavy metals as promoter stimulators in the production of recombinant proteins is not in line with the "Good Manufacturing Practices” (GMP) principles. Therefore, the modified HSP70.2 promoter is used in the GFP gene cassette, which can be physically triggered by heat shock and is suitable for good manufacturing practices.
  • the transcription and translation terminating BTU2 termination sequence is also included in this cassette.
  • HSP70.2 promoter Heat Shock Promoter
  • BTU2 termination sequence was synthetically manufactured to Shinegene Company.
  • HSP70.2 promoter and BTU2 sequences which were synthetically generated and cloned into the pUC57 vector were amplified by PCR reaction. With this reaction; The Ascl restriction sequence was added to the 3' end of the HSP70.2 promoter sequence and the Pmel restriction sequence was added before the BTU2 termination sequence.
  • the TtsfGFP gene cassette was amplified with primers containing the Ascl and Pmel restriction sequence ( Yilmaz ; G., & Arslanyolu, M. 2015.
  • TtsfGFP-6xHis cassette which was prepared with the vector pUC19-3'NTS-Neo4 (TtAC2.2) which was purified by restriction with BamHI (Thermo, #ER0052) and Cfr9I (Thermo, #ER0171) enzymes was ligated and transformed into E. coli.
  • the plasmid isolated from the positive clones detected by colony PCR procedures was taken to double enzyme restriction with BamHI and Cfr9I enzymes and single enzyme restriction with EcoRI and Hindlll enzymes, and the presence of TtsfGFP-6xHis gene cassette was confirmed.
  • This newly created vector was named pUC19-3'NTS- Neo4-TtsfGFP (TtAC2.3) ( Figure 1) 4. Insertion of Telomere Sequence
  • telomeres In order to protect the ends of the chromosome in linear form, telomeres must also be added to the ends of the chromosome in accordance with biomimetics. Telomere sequences generally assume roles of maintaining the stability of linear chromosomal ends, completing DNA replication at the ends, and performing nuclear transport during meiosis and karyokinesis (Kirk, K. E., Blackburn, E. H. 1995. An unusual sequence arrangement in the telomeres of the germ-line micronucleus in Tetrahymena thermophila. Genes & development , 9(1), 59-71).
  • the purified vector pUC19- 3'NTS-Neo4-TtsfGFP (TtAC2.3) and the telomere cassette sequence were cut with restriction enzymes Apal (Thermo, #ER1411) and Xhol (Thermo, #ER0691) to make the sticky ends and ligated. The structural positioning is confirmed by Xhol and Apal double restriction enzyme digestion.
  • Wild type 21 kbp long Tetrahymena thermophila macronuclear rDNA chromosome contains an origin region (5'NTS) (Reischmann, KP, Zhang, Z., & Kapler, GM 1999. Long range cooperative interactions regulate the initiation of the Tetrahymena thermophila rDNA minichromosome. Nucleic acids research, 27 (15), 3079-3089), the locus containing the rDNA genes and the 3'NTS sequence as 2 copies in the palindromic orientation and is protected by telomere at the ends.
  • 5'NTS origin region
  • the 5'NTS alleles containing the Tetrahymena thermophila rDNA origin sequence are differentiated and the B type allele is recessive compared to the C3 type allele due to a 42 base deletion.
  • the rDNA replication superiority or dominance order of alleles is as C3>B>C3 rmml or C3 rmm4 (Yaeger, PC, Orias, E., Shaiu, WL, Larson, DD, & Blackburn, EH 1989.
  • the replication advantage of a free linear rRNA gene is restored by somatic recombination in Tetrahymena thermophila , Molecular and cellular biology, 9 (2), 452-460).
  • Transformant cells obtained from circular vectors formed by the presence of only one copy of this rDNA locus (5'NTS-rDNA genes-3'NTS), which are found in two palindromic copies, have been shown to maintain stable copies of this vector within the macronucleus genome (Yu, G. L, & Blackburn, E. H. 1989. Transformation of Tetrahymena thermophila with a mutated circular ribosomal DNA plasmid vector. Proceedings of the National Academy of Sciences, 86(21 ), 8487-8491 ). Furthermore, the vector pMND-1 containing the entire palindromic rDNA gene locus without deletion reached 9,000 copies ( Blomberg , P., Randolph, C., Yao, C.
  • PCR reaction was performed using Herculase Fusion DNA Polymerase II enzyme (Agilent, #600675) and the genomic DNA purified from wild-type Tetrahymena thermophila C3368.1 strain as the source DNA.
  • the steps of the PCR reaction were optimized as follows: initial denaturation at 92 °C for 2 minutes, 10 cycles of 92 °C denaturation for 10 cycles, hybridization for 20 seconds at 56 °C, elongation at 68 °C for 7 minutes, 20 cycles of denaturation at 20 °C for 10 seconds, hybridization at 56 °C for 20 seconds, 68 °C elongation for 7 minutes (so as to increase 20 seconds in each cycle) and one-time 68 °C final elongation.
  • the first stage product of the two-stage PCR reaction was produced. This PCR product was used as the source of DNA in the second reaction, and the amount was increased.
  • the PCR product containing 10.3 kbp sequence of "5'NTS containing C3 origin-rDNA genes-3'NTS” was purified from the gel, and was integrated into the pJET1.2/ blunt-ended cloning vector with Clonejet PCR Cloning Kit (Thermo, #K1231) The size of the cloned region was confirmed by PCR and the DNA sequence was verified by DNA sequence analysis.
  • deletion of the BamHI restriction enzyme site (which is found between the Neo4 and TtsfGFP cassettes seen in the TtAC2.3 vector construct) was initiated by Xma-Sall enzyme restriction of the TtsfGFP and Neo4 cassettes.
  • Some changes to the HSP70.2, TtsfGFP gene and BTU2 sequence in the TtsfGFP gene cassette were also performed at this stage.
  • the first is the convertion of the G (guanine) base of the ATG start codon of the region encoding the artificial 11 amino acid HSP70 protein sequence, which is involuntarily added to the N-terminus of a produced protein and could function as a possible IRES element sequence residing at the 3' end of the HSP70.2 promoter, to the T (thymine) base by mutation.
  • This was carried out by PCR using a specific primer set.
  • an A (adenine) base was added prior to the Pmel enzyme restriction sequence at the 3' end of the HSP70.2 promoter sequence.
  • Pmel sequence was inserted on the forward primer of TtsfGFP and Ascl sequence was inserted on the reverse primer.
  • the SfaAI restriction enzyme sequence was also inserted on the reverse primer of the BTU2 sequence.
  • the TtsfGFP gene cassette was regenerated by ligating the modified HSP70.2 promoter, TtsfGFP gene and BTU2 sequences with overlapping PCR. No changes were performed on the Neo4 cassette sequence, but the SfaAI restriction enzyme recognition sequence instead of BamHI was added to the forward primer.
  • TtsfGFP and Neo4 cassettes were ligated with overlapping PCR and integrated into the vector with Xmal-Sall enzymes. These steps yielded the artificial chromosome TtAC2 (SEQ ID NO: 4) Sequential accuracy of the TtAC2 artificial chromosome was confirmed by DNA sequence analysis ( Figure 1)
  • the gold particles are coated with designed chromosome DNAs and sent into the cell with the help of a biolistic gun under certain pressure and the artificial chromosome (TtAC2) vector DNAs are transferred to the nucleus while passing through the cell.
  • TtAC2 artificial chromosome
  • the gold particles must be coated with DNA.
  • 1 mL of linearized or circular vector DNA 10 mL 2.5 M CaCl 2 and 4 mL of spermidine were rapidly added. After each addition, it was vortexed for about 3 seconds and all components were shaken for 10 minutes at 4 °C. At the end of shaking, the samples were centrifuged at 4 °C and 10,000 g for 6 seconds to collect DNA-coated gold particles. The supernatant was gently removed with a micropipette and the washing steps proceeded.
  • Chromosome DNA and DNA-bound gold particles were washed and centrifuged with 100 mL of 70% ethanol. After the supernatant was removed by pipetting again, 100 mL of 100% ethanol was added to the pellet and the same process was repeated. Subsequently, 10 mL of 100% ethanol was added and dissolved on gold particles bound to the vector DNA and this mixture was transferred to the middle part of the macrocarrier chamber of the biolistic gun. The macrocarrier was stored in the desiccator to remove alcohol.
  • Tetrahymena thermophila CU428 cells of B origin were grown in SPP medium, and when they reached a density of 2x10 5 cells/ml, they were fasted in 50 ml fasting buffer (10 mM Tris-HCl pH 7.4) On the day of transformation, starving cells at a density of 2x10 5 cells/ml and a volume of 50 ml were collected by centrifugation at 1100 g for 3 minutes in a conical bottom tube to obtain 10 million cells for each firing.
  • the firing step was started with the biolistic gun.
  • a 900 psi rupture disc wetted with isopropanol was placed in the holding apparatus.
  • the holding apparatus is mounted by rotating it onto the threaded helium port in the particle bombardment apparatus.
  • the petri dish containing the cells was placed on the lowest shelf of the device to provide the necessary conditions for firing. Firing was performed when the conditions were ready. 10 ml of SPP was added to the transformed cells at 30 °C and allowed to incubate at 30 °C until further firing was complete. All biolistic gun transformation was performed in a sterile cabinet
  • the antibiotic amount was increased to 1000 mg/ml and the selection was continued and positive transformant cells showing dynamic viability were transferred to glass tubes.
  • the paromomycin suppression applied to the "antibiotic experimental group (Par+)” was reduced by 100 mg/ml and the transformant cells were maintained at this suppression for months.
  • "non-antibiotic experiment group (Par-)” was also tubed, but antibiotic addition to the cells was not performed during monthly passaging.
  • paromomycin and cadmium were added to the antibiotic-suppression tubes every 3 days. Once a week Pen/Strep and Amphotericin were added to the cells in all tubes and these cells were transferred to NEFF medium and passagings were performed. After the first 2 months, the cells in the tube were monitored for a long term by monthly passaging.
  • Untransformed Tetrahymena thermophila CU428 cells were used as negative control of the production assay of recombinant TtsfGFP.
  • Modified HSP70.2 promoters of transformant cell lines were induced by heat shock from weekly or monthly samples, production of TtsfGFP-6xHis recombinant protein was monitored by microscopic methods and SDS-PAGE and western blot analyzes.
  • the cells to be monitored were grown in shaker incubator in 15 ml or 25 ml NEFF medium until the logarithmic growth phase is reached. After incubation, the cells were pelleted by centrifugation at 1100 g for 5 minutes in a swing rotor centrifuge.
  • the medium previously added Pen/Strep and amphotericin, was heated at 38 °C, and a volume sufficient to dissolve the pellet was added very slowly onto the cell pellets to dissolve the cells.
  • the dissolved cells were transferred to larger surface flasks containing sterile 15 ml or 25 ml medium.
  • the cells were subjected to heat shock for 3 hours in a 38 °C water bath. Light and fluorescence microscope analyzes of the cells were performed after this 3 hour TtsfGFP generation period.
  • the Olympus Ix53 microscope with fluorescent apparatus was used with a GFP filter and a Black- and-white 10x-IR camera (for imaging at the end of 24 months) and the Olympus Bx50 microscope with the GFP filter and the DP72 camera (for imaging at the end of the 5th month) (40X magnification).
  • In-vivo microscopic cell analyzes showed that the cells had strong GFP fluorescence at 5 months (Figure 2-D) and at 24 months ( Figure 2-E)
  • the neomycin resistance gene which is not present in the Tetrahymena thermophila genome but is found in the artificial chromosomes of the invention, was used as marker gene to determine the number of copies of TtAC2 vectors.
  • Taqman probe and primers which are suitable for the neomycin resistance-marker gene were designed.
  • Taqman probes and primers were also designed for the catalase gene (GenBank: XP_001026590.2), which would help to detect the number of cells per nanogram of DNA due to its presence in the Tetrahymena genome as a single copy.
  • DNA isolation from 1 week, 2 month and 7 month cell pellets for Par+ and Par- experimental groups was performed using DNA isolation kit (Promega, Wizard Genomic DNA Purification Kit, A1120) to determine the number of copies of artificial chromosome vectors. In this way, the quality and concentration of the Real-Time PCR experiments were investigated and the nanograms of the DNAs were determined. Purified DNAs were run on agarose gel to confirm their usability in Real-Time PCR.
  • Taqman Probe and primers designed according to neomycin resistance gene using Primer3 program are as follows:
  • Taqman Probe 5' FAM TCTGGTTTCATCGACTGTGG TAMRA 3'
  • Reverse primer 5' CAAGTTCTTCAGCAATATCA 3'
  • the robe and primers designed for the catalase gene are as follows:
  • Taqman Probe 5' FAM AATCCCTGAAAGAGTCGTCCACGC TAMRA 3'
  • Reverse primer 5' CCCCAAGCACCAGCACCTTTG 3'
  • the probe and primer concentrations described above were diluted to the final concentration of 10,000 nM. For the reaction they were used at a concentration of 500 nm.
  • TtAC2 vector whose base content and number are known was used.
  • three independent dilutions from 267 ng/mL TtAC2 vector were performed. The R 2 value of the plotted standard curve is 0.989 and the productivity is found to be 101.7%.
  • the real-time PCR reaction was established with the Brilliant III Ultra-Fast QPCR Master Mix (Catalog #600880, Agilent Technologies) kit.
  • the real-time PCR reaction was carried out with a program of 40 cycles of 15 sec at 95 °C and 30 sec at 60 °C after the 3 minutes of pre-denaturation at 95 °C.
  • Genomic DNAs that were purified from cells containing antibiotic-suppressed Par+ circular TtAC2 and Par+ linear TtAC2 and Par- circular TtAC2 and Par-linear TtAC2 were used along with their non-template reaction controls for real-time PCR reaction.
  • Genomic DNAs of the experimental group were diluted to 50 ng/mL and the template genomic DNA was used as 100 ng/mL in the reaction.
  • TtAC2 artificial chromosome vector is maintained in the macronucleus genome in circular and linear form of the Par+ and Par- experimental groups (Table 1)
  • Table 1 The circular and linear forms of TtAC2 differ in the number of copies between Par+ and Par- experimental groups. While the experimental groups without antibiotic suppression showed a rapid increase in the number of copies in the first weeks, it was observed that the number of copies decreased in time. While the experimental groups under antibiotic suppression had a slow increase in the number of copies in the first weeks, the increase in the number of copies gained momentum in time. This suggests that instead of applying antibiotic suppression immediately after transformation, the cells should be suppressed after the first week based on the natural predominance of the C3 allele on the B allele.
  • TtAC2 has an artificial chromosome properties and can be maintained by the cell for a long time without paromomycin suppression, which is of great importance for industrial use.

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Abstract

La présente invention concerne un chromosome artificiel de tétrahyména thermophila (TtAC2) ayant une séquence nucléotidique qui est homologue d'au moins 80 % à SEQ ID No : 4 et comprenant un promoteur HSP70.2 modifié ayant la SEQ ID No : 1, une séquence 3´NTS ayant la SEQ ID No : 2, "5´NTS contenant une origine C3, les gènes ADNr et la séquence 3´NTS" ayant la SEQ ID No : 3. Le chromosome artificiel de tétrahyména thermophila, qui est formé par biomimétisme du minichromosome de tétrahymena thermophila macronucléus, comprend en outre une séquence de télomère, une cassette de Neo4 et une cassette de gène marqueur TtsfGFP. Dans le cadre de la présente invention, l'invention concerne également un procédé d'obtention dudit chromosome artificiel de tétrahyména thermophila. Dans un autre aspect, la présente invention concerne un procédé de production par recombinaison d'ADN génomique homologue et/ou hétérologue, d'ARN et/ou de protéines et/ou de dérivés de ceux-ci à l'aide d'un chromosome artificiel de tétrahyména thermophila en tant que système/plateforme de production. Dans un autre aspect, la présente invention concerne l'utilisation du chromosome artificiel de tétrahymena thermophila en tant que système/plateforme de production pour produire par recombinaison de l'ADN génomique homologue et/ou hétérologue, de l'ARN et/ou des protéines et/ou des dérivés de ceux-ci.
PCT/TR2019/051165 2019-03-22 2019-12-20 Chromosome artificiel 2 de tétrahyména thermophila (ttac2) et son utilisation dans la production de protéines recombinantes Ceased WO2020197518A1 (fr)

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