EP3841210A2 - Tetrahymena thermophila artificial chromosome 1 (ttac1) and its use for the production of recombinant proteins - Google Patents
Tetrahymena thermophila artificial chromosome 1 (ttac1) and its use for the production of recombinant proteinsInfo
- Publication number
- EP3841210A2 EP3841210A2 EP19835861.6A EP19835861A EP3841210A2 EP 3841210 A2 EP3841210 A2 EP 3841210A2 EP 19835861 A EP19835861 A EP 19835861A EP 3841210 A2 EP3841210 A2 EP 3841210A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- seq
- vector
- sequence
- ttacl
- cells
- 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
Links
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Classifications
-
- 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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
Definitions
- the present invention relates to an artificial chromosome designed for homologous and heterologous protein production in Tetrahymena thermophila. More particularly, the invention relates to an artificial chromosome vector which can be used in a circular or linear form for recombinant protein production which is inducible by heat, by virtue of a HSP70.2 promoter.
- Tetrahymena thermophila is a ciliated eukaryotic unicellular organism which is one of the best- characterized unicellular eukaryotes. It bears two nuclei: a transcriptionally silent, diploid germline nucleus (MIC] and a transcriptionally active, polyploid somatic macronucleus (MAC]
- MIC diploid germline nucleus
- MAC transcriptionally active, polyploid somatic macronucleus
- Tetrahymena thermophila as a host for recombinant protein production in a way that is productive and efficient
- various attempts and methods suggested for transforming the organism with the genes of interest comprising microinjection, electroporation and biolistic bombardment
- a cloning vector is 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 obtained from a prokaryotic bacterial cell or virus. Alternatively, they can be constructed using a plasmid, or can be isolated from a cell of a higher organism. They are useful in introducing a nucleic acid fragment into an organism for the purpose of cloning.
- Artificial chromosomes are a sort of cloning vectors. They can carry DNA inserts orders of magnitude larger than is possible with 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. They also ensure the continuity of the vector 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 relates to preparation of plant cell lines that comprise artificial chromosomes. Methods for heterologous nucleic acid insertion into the artificial chromosomes, directing the artificial chromosomes to the selected cells and isolation of the said artificial chromosomes are also provided. Said artificial chromosomes were directed to plant cells.
- Secretory proteins are defined as proteins or polypeptides that are directed to extracellular environment upon production. This direction of proteins can also be used in recombinant protein production. This process usually ensured by signal sequences/peptides. These short peptide sequences conduct the transport of translation complex to the ER surface upon translation. Signal peptides are used in recombinant protein technology in order to obtain proteins of interest as already localized atthe extracellular environment.
- EP 1360306 B1 relates to a nucleic acid coding for phospholipase A1 of ciliates. PLA1 that is defined in this patent application consists of 110 amino acids.
- PLA signal sequence can be used as 36 amino acids long, meaning that the signal peptide would still function when it is comprised of 36 amino acids ( Weide , Herrmann L, Bockau U., NieburN.,Aldag L, Laroy 14/., Contreras R., Tiedtke A., Hartmann M. W. (2006). Secretion of functional human enzymes by Tetrahymena thermophila. BMC Biotechnol. 6:19).
- Antibiotic resistance cassettes are important tools in 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 have been developed specific to the organism. 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] from Tetrahymena thermophila.
- MTT1 Cd-inducible metallothionein gene
- Heat shock proteins are stress response proteins that are found in all eukaryotic organisms studied to date. The genes that translate to 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 explains the use of HSP 90 heat-inducible promoter that belongs to the family of Tetrahymena thermophila heat-shock proteins. Said promoter may have natural nucleotide sequences or promoter-effective fragments thereof. 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 Al.
- Another patent application, CN 101586119 A explains the use of HSP70.2 promoter to express GFP marker gene.
- HSP70.2 that is defined in this patent application consists of 1132 bp sequence. This sequence comprises 1105 bp from HSP70.2 promoter and 5’UTR, 21 bp from HSP70.2 protein coding sequence after ATG start codon and 6 bp BamHI restriction enzyme cloning site.
- Figure 1 shows the elements and sequences that construct TtACl, the vectors that these elements are derived, and circular and linear forms of TtACl.
- Figure 2 shows the comparison of transformation efficiencies of two methods: Electroporation and Biolistic gun method.
- FIG. 3 shows the mRNA analysis of circular and linear TtACl which contains HSP70.2-PLA- TtsfGFP-12xHis cassette. The bands with the expected size (730 bp] was pointed with black arrow.
- FIG. 4 shows the utilisation of linear TtACl vector which contains HSP70.2-PLA-TtsfGFP-12xHis cassette for the production of recombinant TtsfGFP protein.
- A Circular and linear representations of TtACl-sfGFP vector.
- B SDS-PAGE gel analysis, dyed with Coomassie Blue.
- C Immunoprecipitation and Western Blotting Analysis. The TtsfGFP-12xHis protein band with the expected size was pointed by black arrow.
- Figure 5 shows the images taken from light and fluorescence microscopy analysis of (A] circular and (B] linear TtACl-sfGFP bearing and sfGFP expressing cells.
- Figure 6 shows Southern blot analysis of Hindlll-digested genomic DNA of Tetrahymena thermophila transformants containing circular or linear TtACl.
- Said artificial chromosome was designed for unicellular eukaryotic organism Tetrahymena thermophila. The aim is to produce recombinant proteins in this organism, and increase the efficiency of Tetrahymena thermophila as an expression system for recombinant protein production.
- 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 pNeo3 and pNeo4 ( Mochizuki , K. (2008). High efficiency transformation of Tetrahymena using a codon-optimized neomycin resistance gene. Gene , 425(1), 79-83), pH4T2 ( Gaertig ,]., Gu, L, Hai, B., & Gorovsky, M. A. (1994).
- an artificial chromosome for Tetrahymena thermophila comprises a telomere sequence (SEQ ID NO: 1]
- said telomere sequence can be cut from a pPXV-GFP vector and inserted into a backbone vector sequence in order to obtain the artificial chromosome.
- the specific telomere of the instant invention is important and advantageous in that it enables to obtain an artificial chromosome (TtACl] in circular or linear form depending on choice.
- Linear vector presents the advantage of removing the limitation about the size of the insertion. Higher numbers of base pairs can be worked with when linear form of artificial chromosome is used. It is useful when working with relatively large proteins and/or metabolic pathways, which include sets of genes working in coordination.
- the telomere sequence of the present invention can be cut with Sfil enzyme in order to turn the circular vector to a linear one.
- an artificial chromosome for Tetrahymena thermophila comprises a "Tt rDNA C3 ori” origin sequence (SEQ ID NO: 2).
- said origin sequence can be cut from pH4T2 vector and inserted into a backbone vector sequence in order to obtain the artificial chromosome.
- the specific origin sequence of the present invention is favorable in that it provides a replication advantage over other strains for the recombinant protein production in Tetrahymena thermophila [Yu, G.-L. , Hasson, M., and Blackburn, E. H. (1988).
- Tetrahymena thermophila Circular ribosomal DNA plasmids transform Tetrahymena thermophila by homologous recombination with endogenous macronuclear ribosomal DNA. Proc. Natl. Acad. Sci. USA 85, 5151-5155). Tetrahymena thermophila is a well-studied organism in the field of molecular biology. Multiplying to high cell densities with short generation times, being able to be grown in laboratory easily and cost-effectively, ability to introduce produced proteins with post-translational modifications are some of the reasons that this organism is widely used. The latter feature especially applies when proteins from eukaryotic origin are to be produced, because post-translational modifications play an important role in protein function in eukaryotes. Therefore, being a eukaryotic organism itself, Tetrahymena thermophila provides an advantage in recombinant protein production over prokaryotic host organisms.
- 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.
- a modified HSP70.2 promoter of SEQ ID NO: 3 is used in the artificial chromosome disclosed herein.
- 277 bp sequence at the 5’ end along with the 21 bp coding the first 7 amino acids of HSP70.2 protein sequence at the 3’ end and 6 bp BamHl sequence are deleted. Therefore, only the 828 bp long HSP70.2 promoter sequence and the region that comprises 5 'UTR region were used in the present invention.
- 21 bp sequence which is coding the first 7 amino acids of HSP70.2 promoter is a part of a potential mRNA level regulatory element, which causes 7 amino acids to be added to the N terminal of the recombinantly expressed target proteins.
- the promoter is used in this modified form for the first time in the current invention, and it was shown that it functions properly as a strong promoter.
- a pNeo4-ori vector (SEQ ID NO: 4], which is constructed for the first time herein is provided as a vector backbone of the artificial chromosome mentioned hereinabove.
- the inventors have also made use of pNeo3-ori (SEQ ID NO: 9] as a negative control.
- pNeo3-ori and pNeo4-ori vectors are constructed by the addition of C3 replicative origin "Tt rDNA C3 ori” (SEQ ID NO: 2] to the pNeo3 and pNeo4 vectors, respectively.
- the origin sequence was derived from the pH4T2 vector.
- vectors are advantageous over vectors that comprise "Tt rDNA B ori” or Tetrahymena thermophila cells that comprise native rDNA minichromosomes which are bearing "Tt rDNA B ori”, in thattheir nucleotide sequences are known and their transformation efficiencies are high.
- pNeo3-ori and pNeo3 vectors comprise neomycin resistance gene cassettes that were not codon- optimized for Tetrahymena thermophila, whereas neomycin resistance gene cassettes of pNeo-4 and pNeo4-ori vectors were codon-optimized for Tetrahymena thermophila (Mochizuki, K. (2008).
- the methods for transforming the cells with the newly created vector include microinjection, electroporation, and biolistic bombardment
- biolistic gun method and electroporation are preferred.
- Vegetative cells were transformed using biolistic gun method, whereas conjugative cells were transformed using electroporation.
- the vectors were directed to macronucleus of Tetrahymena thermophila in both methods. It is surprisingly noted that biolistic gun method provided two times more efficiency than electroporation in transformation of Tetrahymena thermophila cells.
- the transformation process is followed by selection, which separates the positive transformants from the rest of the cells.
- the most widely used form of selection is antibiotic selection.
- neomycin resistance cassette which was codon-optimized for Tetrahymena thermophila was used. This cassette was inserted in pNeo4 vector and inducible with CdCQ through a MTT1 promoter. With the use of this neomycin resistance cassette, transgenic Tetrahymena strains can be selected with high efficiency.
- PLA Phospholipase A protein
- SEQ ID NO: 5 PLA is attached to the N-terminal of the protein of interest and directs the recombinant protein to Endoplasmic reticulum (ER] in order to be secreted.
- sfGFP-12xHis gene cassette (SEQ ID NO: 6], which was taken from pUC57 vector was inserted into TtACl as a marker gene (TtACl-sfGFP]
- TtACl-sfGFP The location of the insertion on the vector is between the modified HSP70.2 promoter sequence and the sequence that contains BTU2 Beta-tubulin 2 gene’s 3’UTR and transcription termination site.
- sfGFP has a PLA sequence on its N-terminal and 12xHis tag on its C-terminal. The production of this protein proves that the construction of artificial chromosome is successful and TtACl operates within the cell effectively.
- sfGFP protein emits fluorescent light, a trait that makes it easy to track and locate. It is shown in the present invention that sfGFP can get into the ER, be folded to become functional and secreted to extracellular environment sfGFP can also be tagged to other recombinant proteins along with PLA signal sequence, so that the localization of the interested proteins (which are directed to ERwith the help of PLA] can be monitored easily by using the fluorescence microscopy.
- the present invention provides a novel artificial chromosome as a vector for producing recombinant proteins in Tetrahymena thermophila which is constituted by the addition of a telomere (SEQ ID NO: 1] and a "Tt rDNA C3 ori” origin sequence (SEQ ID NO: 2] to a pNeo4-ori (SEQ ID NO: 4] vector which is utilized as a backbone vector.
- Said artificial chromosome is at least 90% homologous to, but preferably identical to SEQ ID NO: 7.
- the novel artificial chromosome according to the present invention comprises a telomere (SEQ ID NO: 1], a "Tt rDNA C3 ori” origin sequence (SEQ ID NO: 2], a modified HSP70.2 promoter (SEQ ID NO: 3], a pNeo4-ori (SEQ ID NO: 4], a PLA signal sequence (SEQ ID NO: 5] and sfGFP marker gene (SEQ ID NO: 6] which results in a nucleotide sequence that is at least 95% homologous to, but preferably identical to SEQ ID NO: 8.
- a method for producing an artificial chromosome as identified above comprising the steps of: providing a backbone vector of an articial chromosome for Tetrahymena thermophila, inserting a telomere sequence (SEQ ID NO: 1] and C3 rDNA origin sequence (SEQ ID NO: 2] to the backbone sequence, and
- the backbone vector according to the present invention can be advantageously selected as pNeo4-ori (SEQ ID NO: 4] so that the artificial chromosome would have a nucleotide sequence that is at least 90% homologous to, but preferably identical to SEQ ID NO: 7.
- the method of the present invention further comprises the steps of inserting a modified HSP70.2 promoter (SEQ ID NO: 3], a PLA signal sequence (SEQ ID NO: 5] and sfGFP marker gene (SEQ ID NO: 6] to the artificial chromosome of the present invention, which results in a nucleotide sequence that is at least 95% homologous to, but preferably identical to SEQ ID NO: 8.
- a modified HSP70.2 promoter SEQ ID NO: 3
- PLA signal sequence SEQ ID NO: 5
- sfGFP marker gene SEQ ID NO: 6
- pNeo4 vector contains a CdCk inducible promoter and transcription terminator sequence in a codon-optimized neomycin resistance cassette.
- pH4T2 vector was used as a template DNA to obtain a "Tetrahymena Artificial Chromosome” by adding C3 replicative origin sequence to pNeo4 vector.
- pH4T2 was subjected to PCR with forward and reverse primers which were constructed respectively as: (5'
- telomere sequence For the completion of the Macronucleus Artificial Chromosome, other critical element that should be added to pNeo4-ori vector is the "telomere sequence”. This sequence provides a restriction site which allows the vector to become linear. It also ensures the stability of copy number of linear artificial chromosome by protecting the ends. In order to obtain the telomere sequence, 2 kbp telomere cassette sequence from pPXV-GFP vector was amplified using PCR.
- the forward and reverse primers used for the process were constructed as follows: forward primer having the sequence of (5' GAAAGGTACCGTAACTGCTGCTGGAATTACACAT 3'] which contains KpnI-HF (NEB, R3142S] restriction site and reverse primer having the sequence of (5' TAAACTCGAGCTGGTGAGTACTCAACCAAGTCATTC 3'] which contains Xhol (NEB, R0146S] restriction site.
- pNeo4-ori vector and 2 kbp DNA that involves telomere cassette were both cut in a way to get sticky ends using Xhol and KpnI-HF enzymes.
- sfGFP Superfolder Green Fluorescent Protein
- NotI-HSP70-PLA 36 /Msd-FseI/12xHis -BTU-Notl expression cassette was cut with the Notl (Thermo, ER0591, 10 U/mI] enzyme from the pUC57 vector.
- pNeo4-ori vector was also treated and cut with the same enzyme. The resulting fragments were cloned with the T4 DNA ligase enzyme. Since the methylation of Mscl enzyme (NEB, R0534L, dcm methylation] interrupts the digestion, transformation was performed on E. coli BL21DE3 expression host (non functional dcm methylase] Resulting recombinant vector was named pNeo4-ori-empty secretion vector.
- a sfGFP sequence which was derived from another pUC57cloning vector was amplified by PCR
- the primers used were: FGFP-Msd (5' GCGTGGCCAATGTCTAAAGGTGAAGAATTATTCACT 3'] and RGFP-Fsel (5' ATAGGCCGGCCATTTGTATAATTCGTCCATACCGTG 3'].
- FGFP-Msd 5' GCGTGGCCAATGTCTAAAGGTGAAGAATTATTCACT 3'
- RGFP-Fsel 5' ATAGGCCGGCCATTTGTATAATTCGTCCATACCGTG 3'.
- PCR product was loaded on 0.7% agarose gel and purified from the gel by the usage of purification kit (Fermentas Genejet Gel Extraction Kit, #K0691, 50 preps]
- DNA fragment which contains sfGFP gene and pNeo4-ori-empty secretion vector were digested by Mscl and Fsel restriction enzymes.
- the digestion products were loaded onto the gel and purified by the purification kit sfGFP sequence and pNeo4-ori-empty secretion vector, both having sticky ends were ligated and transformed to the E. coli XLl-Blue cells.
- Resulting recombinant vector was named pNeo4-ori-sfGFP secretion vector.
- This vector was added a telomere cassette sequence with the methods described above.
- TtACl-sfGFP Artificial Chromosome which can secrete recombinant proteins (in this case, sfGFP] by the induction of heat shock and can be used both in circular or linear forms was produced for the first time ( Figure 4-A]
- the vector can be converted to linear form by the digestion with Sfil enzyme, and can be transformed into the host cell upon purification.
- Tetrahymena CU428-VII and B2086-II strains that are used in transformation process were grown in 50 ml PPY (100 U/ml penicillin, 100 pg/rnl streptomycin] at 30°C up to exponential phase. Then the cells were washed with starving buffer (10 mM Tris HCI, pH 7.5] and left to starve at 30°C for 18-24 hours within a 120 rpm shaker incubator. After incubation, cells were counted and cell densities were adjusted to 3x10 5 cells/ml.
- Conjugation was initiated by mixing 50 ml of each Tetrahymena thermophila CU428 and B2086 strains and incubating at 30°C without shaking. On the 7th hour of the conjugation, nuclei of cells were stained with Hoechst dye and were observed under the fluorescent microscope.
- 1 ml SPP medium (lOOU/ml penicillin, 100 pg/ml streptomycin, 0.025 mg/ml amphotericin B] was added to cuvette and the cells were slowly removed by pipetting from cuvette to sterile 19 ml SPP.
- the cells were diluted to the concentration of 1:100 and 100 pl of samples was distributed to each well of a 96-well microtiter plate. Then the cells were incubated at 30°C for 15 hours without shaking, in order to allow the conjugation to end. After CdCD concentration was increased to the final concentration of 1 pg/ml, cells were incubated for additional ⁇ 1 hour. Then the cells were taken under 100 pg/ml paromomycin stress.
- the number of copies of the experimental vectors was calculated based on neomycin resistance gene which is included in the transformant vectors but not included in the Tetrahymena thermophila genome.
- Brilliant III Ultra-Fast QPCR Master Mix Kit (Katalog #600880 Agilent Technologies] was used in Real-Time PCR.
- the amount of DNA fragments were calculated in term of nanograms and the amount that will be taken from each cell to use at Real-Time PCR was determined as 250 ng.
- TaqMan probe Prob 5' FAM TCTGGTTTCATCGACTGTGG TAMRA 3'] and primers (Fneoreal 5' CTGCTTACCCAATATCATG 3' ve Rneoreal 5' CAAGTTCTTCAGCAATATCA 3'] used in the Real-Time PCR experiments were designed according to neomycin gene by using the Primer3 program (http://bioinfo.utee/primer3-0.4.0/]. The concentrations of probe and the primers were diluted to obtain 500 nM final volume.
- Real-Time PCR was carried out using the Brilliant III Ultra-Fast QPCR Master Mix kit (Katalog #600880 Agilent Technologies]
- the 6 cell sets which constitute the experiment and control groups were collected in a volume of 1,5 ml per week in a period of 2 months. The cell numbers of these samples are not known.
- the primers and probe of GSTmu34 gene (GeneBank Acession no: EAR99884.2], whose number of copies in the macronucleus genome is 45, were used in Real-Time PCR along with the 250 ng sample DNAs of experiment and control groups. The number of GSTmu34 copies obtained from the control Real-Time PCR was divided by 45, this way cell number in 1,5 ml was determined.
- GSTmu34 TaqMan probe having the sequence of (Prob 5' FAM ACGCCAATCCTGAAGAATGGTTCG TAMRA 3'] and GSTmu34 primers having the sequence of (FGSTmu34real 5' TGGCTCAACCTATCCGTTTC 3' and RGSTmu34real 5' TTTGACTTCCCCAACATTCC 3'] were used to construct Real-Time PCR.
- the estimated total copy number of 1,5 ml was divided by the total cell number which was calculated with GSTmu34 gene. Consequently, copy number of each vector in a cell was determined.
- Positive transformant cells that contain TtACl-sfGFP-12xHis vector were grown up to logarithmic growth phase in SPP medium overnight at 30°C. Then the cells were collected by centrifuging at 6500 g for 5 minutes. Thereafter, the medium at the temperature of 38°C was added onto the cells, the induction took place for 1 or 3 hours and then the cells were allowed to recover at 30°C for 24 hours.
- RNA isolation was performed using NucleoSpin RNA kit (Macherey- Nagel CatNo 740955], and treatment with DNase took place.
- cDNA was synthesized using RevertAid First Strand cDNA synthesis kit (Cat No: K1621, Fermentas] ⁇ 2,5 pg mRNA was used in cDNA synthesis reaction. This cDNA was objected to PCR using the primers.
- Figure 3 shows the mRNA analyses of linear TtACl-sfGFP and circular TtACl-sfGFP, respectively.
- a group of positive transformants was induced with heat shock (38°C] for 3 hours, while another set of positive transformants was induced for 3 hours and then incubated at 30°C for 24 hours.
- the first group had higher level of sfGFP mRNA than the second group (The well numbers 4-5 and 12-13 in Figure 3).
- genomic DNAs were purified by standard phenol/chloroform purification method either right after the antibiotic selection or two months after transformation of Tetrahymena thermophila with linear and circular TtACl. Negative control cells were untransformed. All genomic DNA samples (25 pg] were digested overnight with Hindlll restriction enzyme and separated on 0.7% agarose gel.
- Depurination was performed with IX depurination buffer from Southern Breeze Blotting Kit (Sigma, SBRZ-2B] Cut and separated genomic DNA on agarose gel blotted to positively charged 0.45 pm nylon membrane (Sigma, N0144] by the alkaline transfer buffer (0.4M NaOH, 1M NaCl] with GE Healthcare Whatman TurboBlotter Transfer System (GE Healthcare, 10416324] Then, neutralization was performed with IX neutralization buffer from Southern Breeze Blotting Kit. The blotted DNA was cross-linked to the membrane with 0.15 joule/cm 2 at 254 nm UV irradiation by using Strategene UV crosslinker.
- Pre-hybridization was carried out with a buffer (0.5% SDS-6X SSC, 0.1% N-Lauryl sarcosine sodium salt, 0.1% Blocking Solution] from DIG DNA Labeling and Detection Kit (Roche, 11175033910] at 65°C. After pre-hybridization, the membrane was hybridized at 65°C for 16 hours with the DIG labelled neo4 protein coding DNA (795 bp] labelled by DIG DNA Labeling Kit (Roche, 11175033910] in pre-hybridization buffer.
- Membrane was first washed with 2X SSC and % 0.1SDS at room temperature and washed to a final lowest stringency of 0.5X SSC and 0.1% SDS at 65°C. Southern blot membrane was visualized by using the detection procedure of DIG DNA Labeling Kit.
- the genomic DNA isolates used in Southern blot experiments were transformed into E. coli DH5a strains. Colonies were counted and colony PCR reactions were performed from these colonies. Plasmid isolation and restriction fingerprinting were done from positive colonies.
- Southern blot was executed with whole-cell genomic DNA isolates from Tetrahymena thermophila cells transformed with linear and circular TtACl right after the antibiotic selection or at the end of two months period.
- the neo4 gene probe which exclusively found in TtACl vector, is specifically found only on the neo4 carrying arm of the TtACl vector (Ligure 6-C, line 5] Therefore, the controls showed that the probe does not bind to any non-specific genomic DNA bands from negative control Tetrahymena thermophila, and that the probe is working properly (Ligure 6-C, line 4]
- Results of Southern blot analysis showed that genomic DNAs carrying linear TtACl vector digested by Hindlll gave only 5751 bp band as expected.
- TtACl was indeed kept as a linear extrachromosomal replicon in macronucleus (Ligure 6-C, line 3 and line 10].
- undigested genomic DNA carrying linear TtACl vector revealed a DNA band in the size of about 20 kb as a two-fold size in contrary to the expected band of 10.3 kb ( Figure 6-C, line 2 and line 9]
- genomic DNA carrying circular TtACl vector was digested by Hindlll, only 11.1 kb DNA band was observed as expected ( Figure 6-C, line 1 and line 8]
- TtACl was found to be about 22 kb in size as a supercoiled form ( Figure 6-C, line 7]
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Abstract
The present invention relates to a Tetrahymena thermophila artificial chromosome 1 comprising a telomere sequence having SEQ ID NO: 1. In more preferred embodiments, the artificial chromosome of the present invention further comprises a C3 replicative origin "Tt rDNA C3 ori" (SEQ ID NO: 2), a modified HSP70.2 promoter (SEQ ID NO: 3), a PLA signal sequence (SEQ ID NO: 5), and a sfGFP marker gene (SEQ ID NO: 6) to obtain an artificial chromosome in a circular or linear form. In further aspects, the present invention relates to a novel method for obtaining said artificial chromosome, and utilization of it for producing recombinant proteins.
Description
TETRAHYMENA THERMOPHILA ARTIFICIAL CHROMOSOME 1 (TTAC1) AND ITS USE FOR THE PRODUCTION OF RECOMBINANT PROTEINS
Technical Field
The present invention relates to an artificial chromosome designed for homologous and heterologous protein production in Tetrahymena thermophila. More particularly, the invention relates to an artificial chromosome vector which can be used in a circular or linear form for recombinant protein production which is inducible by heat, by virtue of a HSP70.2 promoter.
Background of the Invention
Tetrahymena thermophila is a ciliated eukaryotic unicellular organism which is one of the best- characterized unicellular eukaryotes. It bears two nuclei: a transcriptionally silent, diploid germline nucleus (MIC] and a transcriptionally active, polyploid somatic macronucleus (MAC] Use of Tetrahymena thermophila as an alternative model organism for recombinant protein expression for biological and biotechnological studies has been known for many years. The organism has been on the focus based on its advantages in the field; such as short cell division time (under optimal conditions, the generation time is about 1.5-3 hours], the ability to introduce post-translational modifications to the protein of interest, and easy/cost- effective laboratory handling. Therefore, it is of great interest to use Tetrahymena thermophila as a host for recombinant protein production in a way that is productive and efficient There have been various attempts and methods suggested for transforming the organism with the genes of interest, comprising microinjection, electroporation and biolistic bombardment
A cloning vector is 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 obtained from a prokaryotic bacterial cell or virus. Alternatively, they can be constructed using a plasmid, or can be isolated from a cell of a higher organism. They are useful in introducing a nucleic acid fragment into an organism for the purpose of cloning. Some of the features that 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 nucleic acid, being non-integrative or being designed for site-specific integration, having controlled and stable copy numbers of genes that are being carried, etc.
Artificial chromosomes are a sort of cloning vectors. They can carry DNA inserts orders of magnitude larger than is possible with 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. They also ensure the continuity of the vector 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 relates to preparation of plant cell lines that comprise artificial chromosomes. Methods for heterologous nucleic acid insertion into the artificial chromosomes, directing the artificial chromosomes to the selected cells and isolation of the said artificial chromosomes are also provided. Said artificial chromosomes were directed to plant cells.
Secretory proteins are defined as proteins or polypeptides that are directed to extracellular environment upon production. This direction of proteins can also be used in recombinant protein production. This process usually ensured by signal sequences/peptides. These short peptide sequences conduct the transport of translation complex to the ER surface upon translation. Signal peptides are used in recombinant protein technology in order to obtain proteins of interest as already localized atthe extracellular environment. EP 1360306 B1 relates to a nucleic acid coding for phospholipase A1 of ciliates. PLA1 that is defined in this patent application consists of 110 amino acids. Also, there is a paper which shows the fact that PLA signal sequence can be used as 36 amino acids long, meaning that the signal peptide would still function when it is comprised of 36 amino acids ( Weide , Herrmann L, Bockau U., NieburN.,Aldag L, Laroy 14/., Contreras R., Tiedtke A., Hartmann M. W. (2006). Secretion of functional human enzymes by Tetrahymena thermophila. BMC Biotechnol. 6:19).
Antibiotic resistance cassettes are important tools in 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.
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 have been developed specific to the organism. However, current techniques and tools has its downsides along with the advantages described above. First of all, vectors used are mostly circular. Although being easy to use and be transformed, circular vectors limitthe transformable base pairs of nucleic acid. This in turn, makes it hard to produce large proteins that are coded by large genes by vectors. Also, metabolic pathways require several genes working in cooperation. Therefore, expression systems which can bear larger gene sequences are required. Another problem to be solved about current vectors is that the efficiency of transformation is not at high levels. Efficiency and stability
are important features a vector should have, especially if the protein needs to be produced in large quantities.
Out of the promoters that are studied for recombinant protein production in Tetrahymena thermophila, one of most commonly used promoters is MTT1 promoter. 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] from Tetrahymena thermophila. The activation and deactivation of the promoter are achieved by simply adding or depleting cadmium. Although it’s an efficient promoter to induce production, this promoter is not suitable for good manufacturing practise, since 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 too. A different element with less negative impact on the host and environment would be favourable.
Heat shock proteins (HSPs] are stress response proteins that are found in all eukaryotic organisms studied to date. The genes that translate to 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 explains the use of HSP 90 heat-inducible promoter that belongs to the family of Tetrahymena thermophila heat-shock proteins. Said promoter may have natural nucleotide sequences or promoter-effective fragments thereof. 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 Al. Another patent application, CN 101586119 A, explains the use of HSP70.2 promoter to express GFP marker gene. HSP70.2 that is defined in this patent application consists of 1132 bp sequence. This sequence comprises 1105 bp from HSP70.2 promoter and 5’UTR, 21 bp from HSP70.2 protein coding sequence after ATG start codon and 6 bp BamHI restriction enzyme cloning site.
In consideration of the points mentioned above, an improvement is needed in recombinant expression of proteins in Tetrahymena thermophila.
Brief Description of the Figures
Figure 1 shows the elements and sequences that construct TtACl, the vectors that these elements are derived, and circular and linear forms of TtACl.
Figure 2 shows the comparison of transformation efficiencies of two methods: Electroporation and Biolistic gun method.
Figure 3 shows the mRNA analysis of circular and linear TtACl which contains HSP70.2-PLA- TtsfGFP-12xHis cassette. The bands with the expected size (730 bp] was pointed with black arrow.
Figure 4 shows the utilisation of linear TtACl vector which contains HSP70.2-PLA-TtsfGFP-12xHis cassette for the production of recombinant TtsfGFP protein. A] Circular and linear representations of TtACl-sfGFP vector. B] SDS-PAGE gel analysis, dyed with Coomassie Blue. C] Immunoprecipitation and Western Blotting Analysis. The TtsfGFP-12xHis protein band with the expected size was pointed by black arrow. 1: Protein Ladder (ThermoFisher Scientific PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa Cat No: 26619], 2: Tetrahymena thermophila Cu428 total protein (negative control], 3: Total intracellular soluble protein of cells that were induced for 3 hours, 4: Immunoprecipitation of the total intracellular soluble protein of cells that were induced for 1 hour, 5: Immunoprecipitation of the total intracellular soluble protein of cells that were induced for 3 hours, 6: Immunoprecipitation of the total intracellular soluble protein of cells that were induced for 3 hours and then allowed to recover for 24 hours, 7: Immunoprecipitation of the intracellular soluble protein of Tetrahymena thermophila Cu428 (negative control], 8: Immunopecipitation of the concentrated extracellular medium of the cells that were induced for 3 hours, 9: Immunopecipitation of the concentrated extracellular medium of the cells that were induced for 3 hours and then allowed to recover for 24 hours, 10: Positive control (27 kDa recombinant Tt-sfGFP protein that was isolated from E. coli). During Immunoprecipitation, 6X-His Monoclonal Antibody in the ratio of 1:200 and Protein A (ThermoFisher Scientific, MAI-21315] were used. During Blot analysis, mouse anti-GFP Monoclonal Antibody (Roche, 11814460001] was used as a primary antibody in the ratio of 1:200, and HRP conjugated Rabbit Anti-Mouse IgG Antibody was used as a secondary antibody in the ratio of 1:10000.
Figure 5 shows the images taken from light and fluorescence microscopy analysis of (A] circular and (B] linear TtACl-sfGFP bearing and sfGFP expressing cells.
Figure 6 shows Southern blot analysis of Hindlll-digested genomic DNA of Tetrahymena thermophila transformants containing circular or linear TtACl. A] Localization of neo4 probe and
Hindlll restriction site on linear TtACl vector. B] Ethidium bromide stained agarose gel image of circular and linear TtACl in transformant’s genomic DNAs, either uncut or digested with Hindlll. C] Southern blot membranes with uncut or digested genomic DNAs of Tetrahymena probed with neo4 gene; 1] Tetrahymena thermophila genomic DNA containing TtACl circular vector digested with Hindlll, 2] Tetrahymena thermophila genomic DNA containing linear uncut TtACl vector, 3] Tetrahymena thermophila genomic DNA containing linear TtACl vector digested with Hindlll, 4] Negative control genomic DNA digested with Hindlll from Tetrahymena thermophila untransformed with TtACl vector, 5] TtACl positive control purified from E. coli and digested with Hindlll, 6] Scientific GeneRuler 1 kb DNA Ladder (Thermo #SM031], 7] Tetrahymena thermophila genomic DNA containing circular uncut TtACl vector, 8] Tetrahymena thermophila genomic DNA containing circular TtACl vector digested with Hindlll, 9] Tetrahymena thermophila genomic DNA containing linear uncut TtACl vector, 10] Tetrahymena thermophila genomic DNA containing linear TtACl vector digested with Hindlll. Selection means that cells are in the period after paromomycin selection. Two months indicates that cells are in the period of two months passed after the paromomycin selection. D] and H] Plate of E. coli DH5a transformed with a genomic DNA (2 pg] purified from Tetrahymena thermophila untransformed with TtACl vector as a negative control. There are no colonies formed on the LB-agar plate with ampicillin. E] Plate of E. coli colonies transformed with the circular TtACl vector (60 ng] purified from E. coli as a positive control. There are 907 colonies on plate including ampicillin LB agar. F] E. coli plate transformed with Tetrahymena thermophila genomic DNA containing circular TtACl vector (2 pg] There are 501 colonies on the LB-agar plate with ampicillin. G] E. coli plate transformed with Tetrahymena thermophila genomic DNA containing linear TtACl vector (2 pg]. There are 425 colonies on the LB-agar plate with ampicillin. I] E. coli colonies transformed with linear TtACl vector (60 ng] as a positive control. There are 12 colonies on ampicillin LB agar plate. J] E. coli plate transformed with Tetrahymena thermophila genomic DNA containing circular TtACl vector (2 pg] after two months from paromomycin selection. There are 359 colonies on the LB-agar plate with ampicillin, K] E. coli plate transformed with Tetrahymena thermophila genomic DNA containing linear TtACl vector (2 pg] after two months from paromomycin selection. There are 18 colonies on the LB-agar plate with ampicillin.
Detailed Description of the Invention
In this detailed description, the artificial chromosome of the invention and use thereof for the recombinant protein production is described for better understanding of the subject matter.
Said artificial chromosome was designed for unicellular eukaryotic organism Tetrahymena thermophila. The aim is to produce recombinant proteins in this organism, and increase the
efficiency of Tetrahymena thermophila as an expression system for recombinant protein production.
Artificial chromosomes are constructed by cutting and ligating the necessary elements like origin sequence, transcription termination sequence, telomere sequence, antibiotic resistance cassette for selection, and an effective promoter. This "vector backbone” is then added the gene sequence of protein or enzyme of interest In the present invention, vectors used as source of the backbone elements are preferred as pNeo3 and pNeo4 ( Mochizuki , K. (2008). High efficiency transformation of Tetrahymena using a codon-optimized neomycin resistance gene. Gene , 425(1), 79-83), pH4T2 ( Gaertig ,]., Gu, L, Hai, B., & Gorovsky, M. A. (1994). High frequency vector-mediated transformation and gene replacement in Tetrahymena. Nucleic acids research, 22(24), 5391-5398), and pPXV-GFP [Hauser, K., Haynes, W. 20 ]., Rung, C., Plattner, H., & Kissmehl , R. (2000). Expression of the green fluorescent protein in Paramecium tetraurelia. European journal of cell biology, 79(2), 144-149).
In an aspect of the present invention, an artificial chromosome for Tetrahymena thermophila comprises a telomere sequence (SEQ ID NO: 1] In the context of the present invention, said telomere sequence can be cut from a pPXV-GFP vector and inserted into a backbone vector sequence in order to obtain the artificial chromosome. The specific telomere of the instant invention is important and advantageous in that it enables to obtain an artificial chromosome (TtACl] in circular or linear form depending on choice. Linear vector presents the advantage of removing the limitation about the size of the insertion. Higher numbers of base pairs can be worked with when linear form of artificial chromosome is used. It is useful when working with relatively large proteins and/or metabolic pathways, which include sets of genes working in coordination. The telomere sequence of the present invention can be cut with Sfil enzyme in order to turn the circular vector to a linear one.
In an embodiment of the present invention, an artificial chromosome for Tetrahymena thermophila comprises a "Tt rDNA C3 ori” origin sequence (SEQ ID NO: 2). In the context of the present invention, said origin sequence can be cut from pH4T2 vector and inserted into a backbone vector sequence in order to obtain the artificial chromosome. The specific origin sequence of the present invention is favorable in that it provides a replication advantage over other strains for the recombinant protein production in Tetrahymena thermophila [Yu, G.-L. , Hasson, M., and Blackburn, E. H. (1988). Circular ribosomal DNA plasmids transform Tetrahymena thermophila by homologous recombination with endogenous macronuclear ribosomal DNA. Proc. Natl. Acad. Sci. USA 85, 5151-5155).
Tetrahymena thermophila is a well-studied organism in the field of molecular biology. Multiplying to high cell densities with short generation times, being able to be grown in laboratory easily and cost-effectively, ability to introduce produced proteins with post-translational modifications are some of the reasons that this organism is widely used. The latter feature especially applies when proteins from eukaryotic origin are to be produced, because post-translational modifications play an important role in protein function in eukaryotes. Therefore, being a eukaryotic organism itself, Tetrahymena thermophila provides an advantage in recombinant protein production over prokaryotic host organisms.
Many vectors and respective promoters have been developed for the use in Tetrahymena thermophila. MTT1 promoter which belongs to Cd-inducible metallothionein gene of Tetrahymena thermophila is widely used in recombinant protein production. However, 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.
In a preferred embodiment of the present invention a modified HSP70.2 promoter of SEQ ID NO: 3 is used in the artificial chromosome disclosed herein. Instead of using the whole 1132 bp nucleotide sequence of HSP70.2 promoter as defined in the CN 101586119 A document, 277 bp sequence at the 5’ end along with the 21 bp coding the first 7 amino acids of HSP70.2 protein sequence at the 3’ end and 6 bp BamHl sequence are deleted. Therefore, only the 828 bp long HSP70.2 promoter sequence and the region that comprises 5 'UTR region were used in the present invention. 21 bp sequence which is coding the first 7 amino acids of HSP70.2 promoter is a part of a potential mRNA level regulatory element, which causes 7 amino acids to be added to the N terminal of the recombinantly expressed target proteins. The promoter is used in this modified form for the first time in the current invention, and it was shown that it functions properly as a strong promoter.
In a further embodiment of the present invention, a pNeo4-ori vector (SEQ ID NO: 4], which is constructed for the first time herein is provided as a vector backbone of the artificial chromosome mentioned hereinabove. The inventors have also made use of pNeo3-ori (SEQ ID NO: 9] as a negative control. pNeo3-ori and pNeo4-ori vectors are constructed by the addition of C3 replicative origin "Tt rDNA C3 ori” (SEQ ID NO: 2] to the pNeo3 and pNeo4 vectors, respectively. The origin sequence was derived from the pH4T2 vector. These vectors are advantageous over
vectors that comprise "Tt rDNA B ori” or Tetrahymena thermophila cells that comprise native rDNA minichromosomes which are bearing "Tt rDNA B ori”, in thattheir nucleotide sequences are known and their transformation efficiencies are high. pNeo3-ori and pNeo3 vectors comprise neomycin resistance gene cassettes that were not codon- optimized for Tetrahymena thermophila, whereas neomycin resistance gene cassettes of pNeo-4 and pNeo4-ori vectors were codon-optimized for Tetrahymena thermophila (Mochizuki, K. (2008). High efficiency transformation of Tetrahymena using a codon-optimized neomycin resistance gene. Gene, 425(1), 79-83). As mentioned above, this invention made use of pNeo3-ori vector as a negative control during the experiments. It was noted that the codon optimized pNeo4-ori vector (SEQ ID NO: 4] provides much greater transformation efficiency as compared to pNeo3-ori vector (SEQ ID NO: 9] (Figure 2]
The methods for transforming the cells with the newly created vector include microinjection, electroporation, and biolistic bombardment In the context of the present invention, biolistic gun method and electroporation are preferred. Vegetative cells were transformed using biolistic gun method, whereas conjugative cells were transformed using electroporation. The vectors were directed to macronucleus of Tetrahymena thermophila in both methods. It is surprisingly noted that biolistic gun method provided two times more efficiency than electroporation in transformation of Tetrahymena thermophila cells.
The transformation process is followed by selection, which separates the positive transformants from the rest of the cells. The most widely used form of selection is antibiotic selection. In this study, neomycin resistance cassette which was codon-optimized for Tetrahymena thermophila was used. This cassette was inserted in pNeo4 vector and inducible with CdCQ through a MTT1 promoter. With the use of this neomycin resistance cassette, transgenic Tetrahymena strains can be selected with high efficiency.
Signal sequences are effective tools used for the localization and/or secretion of the recombinant proteins. Phospholipase A protein (PLA] is the signal sequence of choice in the current invention (SEQ ID NO: 5] PLA is attached to the N-terminal of the protein of interest and directs the recombinant protein to Endoplasmic reticulum (ER] in order to be secreted.
To show that the resulting artificial chromosome is capable of directing recombinant protein production in the host organism, sfGFP-12xHis gene cassette (SEQ ID NO: 6], which was taken from pUC57 vector was inserted into TtACl as a marker gene (TtACl-sfGFP] The location of the insertion on the vector is between the modified HSP70.2 promoter sequence and the sequence that contains BTU2 Beta-tubulin 2 gene’s 3’UTR and transcription termination site. sfGFP has a
PLA sequence on its N-terminal and 12xHis tag on its C-terminal. The production of this protein proves that the construction of artificial chromosome is successful and TtACl operates within the cell effectively. sfGFP protein emits fluorescent light, a trait that makes it easy to track and locate. It is shown in the present invention that sfGFP can get into the ER, be folded to become functional and secreted to extracellular environment sfGFP can also be tagged to other recombinant proteins along with PLA signal sequence, so that the localization of the interested proteins (which are directed to ERwith the help of PLA] can be monitored easily by using the fluorescence microscopy.
To this end, in preferred embodiments, the present invention provides a novel artificial chromosome as a vector for producing recombinant proteins in Tetrahymena thermophila which is constituted by the addition of a telomere (SEQ ID NO: 1] and a "Tt rDNA C3 ori” origin sequence (SEQ ID NO: 2] to a pNeo4-ori (SEQ ID NO: 4] vector which is utilized as a backbone vector. Said artificial chromosome is at least 90% homologous to, but preferably identical to SEQ ID NO: 7.
In more preferred embodiments, the novel artificial chromosome according to the present invention comprises a telomere (SEQ ID NO: 1], a "Tt rDNA C3 ori” origin sequence (SEQ ID NO: 2], a modified HSP70.2 promoter (SEQ ID NO: 3], a pNeo4-ori (SEQ ID NO: 4], a PLA signal sequence (SEQ ID NO: 5] and sfGFP marker gene (SEQ ID NO: 6] which results in a nucleotide sequence that is at least 95% homologous to, but preferably identical to SEQ ID NO: 8.
In a further aspect of the present invention, there is provided a method for producing an artificial chromosome as identified above, comprising the steps of: providing a backbone vector of an articial chromosome for Tetrahymena thermophila, inserting a telomere sequence (SEQ ID NO: 1] and C3 rDNA origin sequence (SEQ ID NO: 2] to the backbone sequence, and
obtaining the articial chromosome.
As noted above, the backbone vector according to the present invention can be advantageously selected as pNeo4-ori (SEQ ID NO: 4] so that the artificial chromosome would have a nucleotide sequence that is at least 90% homologous to, but preferably identical to SEQ ID NO: 7.
Preferably, the method of the present invention further comprises the steps of inserting a modified HSP70.2 promoter (SEQ ID NO: 3], a PLA signal sequence (SEQ ID NO: 5] and sfGFP marker gene (SEQ ID NO: 6] to the artificial chromosome of the present invention, which results in a nucleotide sequence that is at least 95% homologous to, but preferably identical to SEQ ID NO: 8.
In another aspect of the present invention, there is provided a novel use of the artifical chromosome as identified above for recombinant protein production in Tetrahymena thermophila.
A) Construction of Tetrahymena Artificial Chromosome
In order to construct TtACl artificial chromosome in E. coli by cloning method, ampicillin resistance gene cassette which provides antibiotic selection in this bacterial system and pNeo4 vector (NCBI Accession No: EU606202] which comprises E. coli origin sequence that enables the replicative amplification of the cloning vector have been used. Since this vector was designed for deletion of macronuclear genes of Tetrahymena thermophila (= knockout], even though it contains a neomycin (paromomycin] antibiotic resistance cassette providing selection, it does not provide the vector with an origin sequence that will provide 8,000-10,000 copies of the vector. However, pNeo4 vector contains a CdCk inducible promoter and transcription terminator sequence in a codon-optimized neomycin resistance cassette. pH4T2 vector was used as a template DNA to obtain a "Tetrahymena Artificial Chromosome” by adding C3 replicative origin sequence to pNeo4 vector. pH4T2 was subjected to PCR with forward and reverse primers which were constructed respectively as: (5'
TGATAAGCTTTAGGTTTTAGGAATCGCGGC 3'] and (5' TATGCCTGCAGGTCGACTCTAGAGGATCC 3']. By this way, 4 kbp DNA fragment which contains C3 replicative origin that enables 10.000 copies was amplified. The ends of the origin DNA fragment was cut using Hindlll (Thermo, # ER0645] and Sail (Thermo, # ER0501] restriction enzymes. In this way, the ends were turned into sticky ends. pNeo4 vector containing the neomycin resistance gene was also cut with Hindlll and Sail restriction enzymes and sticky ends were obtained. These cut pNeo4 vector and 4 kbp DNA fragment which contains C3 replicative origin were visualized on 0.7% agarose gel, cut from the gel under the UV cabinet of 365 nm wavelength and purified using the purification kit (Thermo, Genejet Gel Extraction Kit, # K0691] Sticky end C3 origin and pNeo4 vector were treated with T4 DNA ligase enzyme (NEB, M0202S], and transformed to NEB 10-beta competent E coli cells using the heat shock method. The recombinant vector which was purified from NEB 10-beta E. coli cells was named pNeo4-ori (pNeo4 with C3 origin sequence added]. The fact that the vector was produced successfully was confirmed by fingerprint analysis using double digestion with Hindlll- Sall restriction enzymes, and single digestion with Xbal (Takara, 1093 A] enzyme.
For the completion of the Macronucleus Artificial Chromosome, other critical element that should be added to pNeo4-ori vector is the "telomere sequence”. This sequence provides a restriction site which allows the vector to become linear. It also ensures the stability of copy number of linear artificial chromosome by protecting the ends. In order to obtain the telomere sequence, 2 kbp
telomere cassette sequence from pPXV-GFP vector was amplified using PCR. The forward and reverse primers used for the process were constructed as follows: forward primer having the sequence of (5' GAAAGGTACCGTAACTGCTGCTGGAATTACACAT 3'] which contains KpnI-HF (NEB, R3142S] restriction site and reverse primer having the sequence of (5' TAAACTCGAGCTGGTGAGTACTCAACCAAGTCATTC 3'] which contains Xhol (NEB, R0146S] restriction site. pNeo4-ori vector and 2 kbp DNA that involves telomere cassette, were both cut in a way to get sticky ends using Xhol and KpnI-HF enzymes. These cut pNeo4-ori vector and 2 kbp DNA fragment which contains telomere cassette were visualized on 0.7% agarose gel, cut under the UV cabinet of 365 nm wavelength and purified using the purification kit (Thermo, Genejet Gel Extraction Kit, # K0691] Sticky end pNeo4-ori vector and telomere cassette were ligated with T4 DNA ligase enzyme (NEB, M0202S], and transformed to E. coli XLl-Blue cells using the heat shock method. Single colonies were selected from LB-agar-amp plate and purified. This recombinant vector was named as pNeo4-ori-tel (TtACl] vector. The fact that the vector was produced successfully was confirmed by fingerprint analysis using double digestion with Xhol and KpnI-HF restriction enzymes, and single digestion with Sfil (Thermo, ER1821] enzyme. Using the cloning of the DNA fragment which contains telomere cassette, it is ensured that the spacer DNA fragment located between the two telomeric sequences can be cut on Sfil restriction site and the vector can be converted to its linear form. This way, the object of this invention, "Tetrahymena thermophila Macronucleus Artificial Chromosome 1” (TtACl] was acquired (Figure 1]
To confirm the use of this vector in recombinant technologies in order to produce proteins of interest in the medical-pharmaceutical fields, an expression cassette which contains a marker gene was placed into the vector. However, in this vector version, since the Notl enzyme in the telomere cassette was to be used in the cloning of the marker gene cassette, the insertion of the marker gene cassette to the pNeo4-ori vector was performed prior to the insertion of the telomere cassette. As a result, pTtACl-sfGFP vector was obtained.
This way, by the usage of Mscl-Fsel cloning sites which reside between the HSP70.2 (Heat Shock Promoter] promoter and BTU2 transcription termination sequence, codon-optimized sfGFP (Superfolder Green Fluorescent Protein] cassette was synthetically produced. sfGFP protein which was coded by sfGFP gene of pUC57 vector, was introduced 36 amino acids long Tetrahymena Phospholipase A enzyme’s secretion signal sequence (PLA36] on its N-terminal, and 12xHis affinity tag coding sequence on its C-terminal. The resulting cassette was named as Tt- sfGFP secretion gene cassette, since it contains PLA36 tag (Figure 4]
Synthetically produced NotI-HSP70-PLA36/Msd-FseI/12xHis -BTU-Notl expression cassette was cut with the Notl (Thermo, ER0591, 10 U/mI] enzyme from the pUC57 vector. pNeo4-ori vector
was also treated and cut with the same enzyme. The resulting fragments were cloned with the T4 DNA ligase enzyme. Since the methylation of Mscl enzyme (NEB, R0534L, dcm methylation] interrupts the digestion, transformation was performed on E. coli BL21DE3 expression host (non functional dcm methylase] Resulting recombinant vector was named pNeo4-ori-empty secretion vector.
A sfGFP sequence which was derived from another pUC57cloning vector was amplified by PCR The primers used were: FGFP-Msd (5' GCGTGGCCAATGTCTAAAGGTGAAGAATTATTCACT 3'] and RGFP-Fsel (5' ATAGGCCGGCCATTTGTATAATTCGTCCATACCGTG 3']. As a result sfGFP sequence was added Mscl on its 5' end and Fsel on its 3' end. PCR product was loaded on 0.7% agarose gel and purified from the gel by the usage of purification kit (Fermentas Genejet Gel Extraction Kit, #K0691, 50 preps] DNA fragment which contains sfGFP gene and pNeo4-ori-empty secretion vector were digested by Mscl and Fsel restriction enzymes. The digestion products were loaded onto the gel and purified by the purification kit sfGFP sequence and pNeo4-ori-empty secretion vector, both having sticky ends were ligated and transformed to the E. coli XLl-Blue cells. Resulting recombinant vector was named pNeo4-ori-sfGFP secretion vector. This vector was added a telomere cassette sequence with the methods described above. By the end of this last process, TtACl-sfGFP Artificial Chromosome which can secrete recombinant proteins (in this case, sfGFP] by the induction of heat shock and can be used both in circular or linear forms was produced for the first time (Figure 4-A] The vector can be converted to linear form by the digestion with Sfil enzyme, and can be transformed into the host cell upon purification.
B) Transformation of Resulting Tetrahymena thermophila Vectors to Vegetative Cells by Biolistic Gun Method and to Conjugative Cells by Electroporation
Transformation of Vegetative Cells
By the utilisation of biolistic gun method, gold particles coated with vector DNA were sprayed on the cell under certain pressure. While passing through the cell, some of the vector DNAs were left in the nucleus. After transformation, the cells were transferred to IX SPP medium, and kept at 30°C without shaking in order to allow the cells to recover. Since antibiotic resistance is to be gained by the induction of cadmium-inducible MTT1 promoter, 0,5 pg/ml CdCD was added to the medium. After the recovery period, paromomycin stress was established up to 100 pg/ml concentration and the amount of CdCD was increased up to 1 pg/ml. Then the cells were diluted and transferred to 96-well microplate in a way that each well contains 100 mΐ of dilution. The transformation yield was evaluated using the microplate. The cells were monitored for 6 days: on the 3rd day they were transferred to 300 pg/ml antibiotic, and on the 5th day they were transferred to 500 pg/ml antibiotic. This process was followed by the counting of the positive
transformant wells. Poisson Distribution formula was used in order to calculate the total number of transformant cells. These experiments were conducted three times, independent from each other [Bruns, P. J., & Cassidy-Hanley, D. (1999). Biolistic transformation of macro-and micronuclei. In Methods in cell biology (Vol. 62, pp. 501-512). Academic Press).
Transformation of Conjugative Cells
With the help of BioRad Gene Pulser device, desalted vector DNAs were elecroporated into the conjugative cells. While lipids in the membranes of electroporated cells were opened under electrical currents, some of the vector DNAs were entrapped in the macronucleus of the cells. E. coli NEB 10-beta cells that bear pNeo4-ori, pNeo3-ori, circular TtACl and linear TtACl were grown in 100 ml LB overnight at 37°C. Vectors were isolated with standard phenol-chloroform method and dissolved in 100 mΐ elution buffer (EB; Tris pH 8.0, Thermo, #K0691] [Kado, C. A., & Liu, S. (1981). Rapid procedure for detection and isolation of large and small plasmids. Journal of bacteriology, 145(3), 1365-1373). Tetrahymena CU428-VII and B2086-II strains that are used in transformation process were grown in 50 ml PPY (100 U/ml penicillin, 100 pg/rnl streptomycin] at 30°C up to exponential phase. Then the cells were washed with starving buffer (10 mM Tris HCI, pH 7.5] and left to starve at 30°C for 18-24 hours within a 120 rpm shaker incubator. After incubation, cells were counted and cell densities were adjusted to 3x105 cells/ml. Conjugation was initiated by mixing 50 ml of each Tetrahymena thermophila CU428 and B2086 strains and incubating at 30°C without shaking. On the 7th hour of the conjugation, nuclei of cells were stained with Hoechst dye and were observed under the fluorescent microscope. On the 9th hour of the conjugation, 100 ml of conjugant cells were centrifuged at 6.500 g for 7 minutes, pellet was washed with 10 mM HEPES buffer (pH 7.5] and then suspended with 1 ml of sterile 10 mM HEPES buffer (pH 7.5] Vectors (15 pg DNA in 125 mΐ HEPES buffer] and 125 mΐ suspended cells that are in the process of conjugation were mixed and immediately transferred to 0.4 cm electroporation cuvette (BioRad] BioRad Gene Pulser device was adjusted to the values of 200 W, 25 pF capacitor and 0.44 kV and cells were shocked ( Gaertig , ]., & Gorovsky, M. A. (1992). Efficient mass transformation of Tetrahymena thermophila by electroporation of conjugants. Proceedings of the National Academy of Sciences, 89(19), 9196-9200; Barchetta, S., La Terza,A., Ballarini, P., Pucciarelli, S., & Miceli, C. (2008). Combination of two regulatory elements in the Tetrahymena thermophila HSP70-1 gene controls heat shock activation. Eukaryotic cell, 7(2), 379-386). Then, 1 ml SPP medium (lOOU/ml penicillin, 100 pg/ml streptomycin, 0.025 mg/ml amphotericin B] was added to cuvette and the cells were slowly removed by pipetting from cuvette to sterile 19 ml SPP. The cells were diluted to the concentration of 1:100 and 100 pl of samples was distributed to each well of a 96-well microtiter plate. Then the cells were incubated at 30°C for 15 hours without shaking, in order to allow the conjugation to end. After CdCD concentration was increased to the final
concentration of 1 pg/ml, cells were incubated for additional ~ 1 hour. Then the cells were taken under 100 pg/ml paromomycin stress. The cells were let for the selection at 30°C, while paromomycin concentration was increased up to 300 pg/ml at 3th day and to 600 pg/ml at 6th day. All wells of 96-microtiter plate were counted for positive transformants every day of the experiments. Total transformant cell numbers were estimated with the help of the Poisson distribution formula. Estimated number of total transformants formula is S = 200 x A x (-loge(96-
B]/96], where A is the dilution factor, and B is the number of wells containing paromomycin resistant cells.
C) Determination of TtACl Vector Copy Number by Real-Time PCR Method
The number of copies of the experimental vectors was calculated based on neomycin resistance gene which is included in the transformant vectors but not included in the Tetrahymena thermophila genome. For this purpose, Brilliant III Ultra-Fast QPCR Master Mix Kit (Katalog #600880 Agilent Technologies] was used in Real-Time PCR.
In order to constitute a "DNA Copy Number Standard Curve” which will be used as a base in Real- Time PCR copy number calculations, pNeo4-ori vector whose base content and number are known was used. To generate a standard curve, dilutions from 214.3 ng/mΐ pNeo4-ori vector were obtained (since the minimum expected copy number of genes except rDNA genes is 45 per cell in this project, the copy number was diluted below this limit]. Vector copy numbers in each dilution was calculated. (The designated standard copy numbers are: Standard 1=2.170.000, Standard 2=217.000, Standard 3=21.700, Standard 4=2170, Standard 5=217, Standard 6=21.7] Real-Time PCR reaction was carried out using the Brilliant III Ultra-Fast QPCR Master Mix Kit (Katalog #600880 Agilent Technologies] The conditions were as follows: 3 minutes long initial denaturation at 95°C, followed by 40 cycles of 15 seconds at 95°C and 30 seconds at 60°C.
For the experimental groups on the other hand, transformed cells which completed the antibiotic selection period (7 days under paromomycin stress] were selected and tranferred to glass tubes. The cells were kept under paromomycin stress (100 pg/ml] for 2 months, the samples were collected weekly during this 2 months period, and total genomic and/or vector DNA was purified. This process was followed by RNase treatment: 10 mg/ml RNase solution was added to DNA dilution, then kept overnight at 55°C and centrifuged at 31.000 g for 45 minutes the next day. As a result of the centrifuge, DNA was precipitated and dissolved in the water. These isolated genomic/vector DNAs were used as templates in Real-Time PCR, and copy number data was obtained. In order to understand the level of stability of artificial chromosome vectors, comparative Real-Time analysis for two groups (Par+ experimental group and Par- control group]
were conducted for 2 months. The cell groups according to the vectors they contain could be listed as:
Paromomycin stress / Par+ Experimental Group;
1. The cells containing pNeo4-ori
2. The cells containing circular TtACl
3. The cells containing linear TtACl
No Paromomycin stress / Par- Control Group;
1. The cells containing pNeo4-ori
2. The cells containing circular TtACl
3. The cells containing linear TtACl
The amount of DNA fragments were calculated in term of nanograms and the amount that will be taken from each cell to use at Real-Time PCR was determined as 250 ng. TaqMan probe (Prob 5' FAM TCTGGTTTCATCGACTGTGG TAMRA 3'] and primers (Fneoreal 5' CTGCTTACCCAATATCATG 3' ve Rneoreal 5' CAAGTTCTTCAGCAATATCA 3'] used in the Real-Time PCR experiments were designed according to neomycin gene by using the Primer3 program (http://bioinfo.utee/primer3-0.4.0/]. The concentrations of probe and the primers were diluted to obtain 500 nM final volume. Real-Time PCR was carried out using the Brilliant III Ultra-Fast QPCR Master Mix kit (Katalog #600880 Agilent Technologies]
The 6 cell sets which constitute the experiment and control groups were collected in a volume of 1,5 ml per week in a period of 2 months. The cell numbers of these samples are not known. The primers and probe of GSTmu34 gene (GeneBank Acession no: EAR99884.2], whose number of copies in the macronucleus genome is 45, were used in Real-Time PCR along with the 250 ng sample DNAs of experiment and control groups. The number of GSTmu34 copies obtained from the control Real-Time PCR was divided by 45, this way cell number in 1,5 ml was determined. In addition to the neomycin probe and the primers of experimental and control groups, GSTmu34 TaqMan probe having the sequence of (Prob 5' FAM ACGCCAATCCTGAAGAATGGTTCG TAMRA 3'] and GSTmu34 primers having the sequence of (FGSTmu34real 5' TGGCTCAACCTATCCGTTTC 3' and RGSTmu34real 5' TTTGACTTCCCCAACATTCC 3'] were used to construct Real-Time PCR. The estimated total copy number of 1,5 ml was divided by the total cell number which was calculated with GSTmu34 gene. Consequently, copy number of each vector in a cell was determined.
D) The Usability of TtACl Linear Vector as Protein Expression Vector in Tetrahymena thermophila and the Production of Recombinant sfGFP-12xHis Protein
In order to confirm the usability of TtACl-sfGFP linear vector in protein expression studies, designed vector was transformed into the cells by using the biolistic gun. Antibiotic selection was performed as described above.
Positive transformant cells that contain TtACl-sfGFP-12xHis vector were grown up to logarithmic growth phase in SPP medium overnight at 30°C. Then the cells were collected by centrifuging at 6500 g for 5 minutes. Thereafter, the medium at the temperature of 38°C was added onto the cells, the induction took place for 1 or 3 hours and then the cells were allowed to recover at 30°C for 24 hours.
The mRNA production of recombinant sfGFP-12xHis upon induction was controlled by cDNA analysis. For this purpose, RNA isolation was performed using NucleoSpin RNA kit (Macherey- Nagel CatNo 740955], and treatment with DNase took place. cDNA was synthesized using RevertAid First Strand cDNA synthesis kit (Cat No: K1621, Fermentas] ~2,5 pg mRNA was used in cDNA synthesis reaction. This cDNA was objected to PCR using the primers.
In an attempt to confirm the protein expression of recombinant sfGFP-12xHis from the transformant cells, soluble protein isolation performed using the Triton base lysis buffer. The extracellular environement was concentrated using Stirred Cell Ultrafiltration device (Millipore, Model 8050] for further analysis. Additional affinity purifications were performed with Immunoprecipitation using anti-12xHis Monoclonal antibody (1:200] and protein A (1:200] (Pierce™ Protein A 25 Agarose CatNo: 20333] Isolated and concentrated proteins were run on Glycine-SDS PAGE gel (5-12%] and dyed with Coomassie blue (Figure 4] The light and fluorescence microscopy analysis of sfGFP producing cells were carried out using Olympus Bx50 fluorescence microscope and Olympus DP72 camera (Figure 5]
In the light of these experiments, it has been shown that the transformation that has been carried out using biolistic gun is 2 times more efficient than the transformation with electroporation. The transformation efficiency of pNeo4-ori vector with telomere (TtACl] is lower than that of circular pNeo4-ori without telomere. However, the number of transformant cells was adequate for the single cell clone selection (Figure 2]
Under the paromomycin stress, the copy number of TtACl linear vector in the cell has been calculated weekly. The results were 1.328 for first week and 4.500 for the last week. For another set of cells, the results were 1.710 and 7.462 respectively. When the paromomycin stress was
removed, the number of copies was reduced by 3, 3-4, 3 fold (Table 1] This situation has been observed in other vectors too. Nevertheless, these findings prove that conservation of linear TtACl vector for 2 months in the cell under paromomycin stress is possible. Although the circular TtACl form is more effective, it is proved for the first time with this invention that linear form of TtACl is also expressed with stability in Tetrahymena thermophila.
Table 1
Figure 3 shows the mRNA analyses of linear TtACl-sfGFP and circular TtACl-sfGFP, respectively. A group of positive transformants was induced with heat shock (38°C] for 3 hours, while another set of positive transformants was induced for 3 hours and then incubated at 30°C for 24 hours. In the outcome of this experiment, it has been shown that the first group had higher level of sfGFP mRNA than the second group (The well numbers 4-5 and 12-13 in Figure 3).
Since phospholipase A gene (PLA36] was inserted to sfGFP-12xHis gene’s N-terminal as a signal sequence, the protein was expected to be secreted to the extracellular environment The findings of SDS-PAGE gel and Western Blotting analyses showed that both group of cells (the cells that were induced for 3 hours and the cells that were induced for 3 hours and then incubated for 24 hours] secreted the 27 kDa sfGFP-12xHis. Western Blotting analysis showed that total intracellular soluble protein contains sfGFP- 12xHis both with uncut PLA (smaller protein band] and without PLA (larger protein band] (Figure 4] In addition, the production of recombinant sfGFP- 12xHis was also proved with Fluorescence Microscopy. In this analysis, the TtACl in circular form showed more fluorescence emission than linear TtACl did, in accordance with the copy numbers (Figure 5]
E) Southern Blot Analysis with the neo4 Probe
The genomic DNAs were purified by standard phenol/chloroform purification method either right after the antibiotic selection or two months after transformation of Tetrahymena thermophila with linear and circular TtACl. Negative control cells were untransformed. All genomic DNA samples (25 pg] were digested overnight with Hindlll restriction enzyme and separated on 0.7% agarose gel. Depurination was performed with IX depurination buffer from Southern Breeze Blotting Kit (Sigma, SBRZ-2B] Cut and separated genomic DNA on agarose gel blotted to positively charged 0.45 pm nylon membrane (Sigma, N0144] by the alkaline transfer buffer (0.4M NaOH, 1M NaCl] with GE Healthcare Whatman TurboBlotter Transfer System (GE Healthcare, 10416324] Then, neutralization was performed with IX neutralization buffer from Southern Breeze Blotting Kit. The blotted DNA was cross-linked to the membrane with 0.15 joule/cm2 at 254 nm UV irradiation by using Strategene UV crosslinker. Pre-hybridization was carried out with a buffer (0.5% SDS-6X SSC, 0.1% N-Lauryl sarcosine sodium salt, 0.1% Blocking Solution] from DIG DNA Labeling and Detection Kit (Roche, 11175033910] at 65°C. After pre-hybridization, the membrane was hybridized at 65°C for 16 hours with the DIG labelled neo4 protein coding DNA (795 bp] labelled by DIG DNA Labeling Kit (Roche, 11175033910] in pre-hybridization buffer. Membrane was first washed with 2X SSC and % 0.1SDS at room temperature and washed to a final lowest stringency of 0.5X SSC and 0.1% SDS at 65°C. Southern blot membrane was visualized by using the detection procedure of DIG DNA Labeling Kit.
F) Retransformation of E coli with Genomic DNA Isolates from Tetrahymena thermophila Transformed with TtACl
The genomic DNA isolates used in Southern blot experiments were transformed into E. coli DH5a strains. Colonies were counted and colony PCR reactions were performed from these colonies. Plasmid isolation and restriction fingerprinting were done from positive colonies.
Southern blot was executed with whole-cell genomic DNA isolates from Tetrahymena thermophila cells transformed with linear and circular TtACl right after the antibiotic selection or at the end of two months period. The neo4 gene probe, which exclusively found in TtACl vector, is specifically found only on the neo4 carrying arm of the TtACl vector (Ligure 6-C, line 5] Therefore, the controls showed that the probe does not bind to any non-specific genomic DNA bands from negative control Tetrahymena thermophila, and that the probe is working properly (Ligure 6-C, line 4] Results of Southern blot analysis showed that genomic DNAs carrying linear TtACl vector digested by Hindlll gave only 5751 bp band as expected. Thus, TtACl was indeed kept as a linear extrachromosomal replicon in macronucleus (Ligure 6-C, line 3 and line 10]. However, undigested genomic DNA carrying linear TtACl vector revealed a DNA band in the size
of about 20 kb as a two-fold size in contrary to the expected band of 10.3 kb (Figure 6-C, line 2 and line 9] On the other hand, after genomic DNA carrying circular TtACl vector was digested by Hindlll, only 11.1 kb DNA band was observed as expected (Figure 6-C, line 1 and line 8] In genomic DNA carrying undigested circular TtACl vector line, TtACl was found to be about 22 kb in size as a supercoiled form (Figure 6-C, line 7]
Maintainability of TtACl as an extrachromosomal circular or linear replicon in the macronucleus of Tetrahymena thermophila was also analyzed by transforming E. coli DH5a with these isolated genomic DNAs on LB-agar-ampicillin plate (Figures 6-D to 6-K] The negative control of 2 pg genomic DNA from untransformed Tetrahymena thermophila produced no colonies as expected (Figure 6-D and Figure 6-H], whereas 60 ng of circular TtACl purified from E. coli as a positive control template gave 907 colonies (Figure 5-E] In the experimental group, E. coli transformant plate with genomic DNA isolates from Tetrahymena thermophila transformed with circular TtACl vector (2 pg] had 501 colonies (Figure 6-F] Genomic DNA isolate (2 pg] from Tetrahymena thermophila transformed with linear TtACl vector gave unexpectedly 425 colonies (Figure 6-G] However, positive control of 60 ng linear TtACl purified from E. coli DH5a gave only 12 colonies (Figure 6-1] Colony PCR based screening of these E. coli colonies resulted with very low positive colonies; none out of 35 colonies for the linear TtACl, 4 positives in 21 colonies for the circular TtACl and 5 positives in 27 colonies for the pNeo4-ori (Data not given]. Additionally, after the transformant selection under antibiotic presence, some of the transformant cells were grown in paromomycin free media until the end of second month. Genomic DNAs were isolated from these Tetrahymena thermophila cells and the genomic DNAs were transformed into E. coli DH5a strains. After the transformation of genomic DNA carrying circular TtACl vector (2 pg], E. coli transformation plate had 359 colonies (Figure 6-J] whereas genomic DNA isolate (2 pg] containing linear TtACl vector gave only 18 colonies (Figure 6-K]
When the results of two E. coli retransformation experiments were compared, colony numbers were sharply dropping in the transformant cells kept in paromomycin free media. Data from Southern blot analysis, copy number studies with the real-time PCR and E. coli retransformation experiments cooperatively proved that TtACl vector is maintained as an extrachromosomal linear vector but also as a circular vector in the macronucleus of Tetrahymena thermophila during a period of two months (Figure 6]
As a result, with the present invention it has been demonstrated that both circular and linear forms of TtACl effectively function in different copy numbers by the virtue of shortened PLA sequence and modified inducible heat shock promoter HSP70.2. It has been also proven that
Tetrahymena thermophila artificial chromosome, which was produced for the first time, maintains its activity in the cell as inserted to macronucleus in both Par+ and Par- conditions for 2 months.
Claims
1. An artifical chromosome for Tetrahymena thermophila comprising a telomere having SEQ ID NO: 1.
2. An artifical chromosome according to claim 1, further comprising a C3 rDNA origin sequence having SEQ ID NO: 2.
3. An artifical chromosome according to claim 1, further comprising a modified HSP70.2 promoter having SEQ ID NO: 3.
4. An artifical chromosome according to claims 1 to 3, further comprising a vector backbone pNeo4-ori having SEQ ID NO: 4.
5. An artifical chromosome according to claim 1, further comprising a PLA signaling sequence having SEQ ID NO: 5.
6. An artifical chromosome according to claim 1, further comprising a marker gene casette having SEQ ID NO: 6.
7. An artifical chromosome according to any of the preceding claims having a nucleotide sequence which is at least 95% homologous to SEQ ID NO: 7.
8. An artifical chromosome according to any of the preceding claims having a nucleotide sequence which is at least 90% homologous to SEQ ID NO: 8.
9. A method for producing an artificial chromosome as defined in claim 1 comprising the steps of: providing a backbone vector of an articial chromosome for Tetrahymena thermophila, inserting a telomere sequence (SEQ ID NO: 1] and C3 rDNA origin sequence (SEQ ID NO: 2] to the vector backbone, and
obtaining the articial chromosome.
10. A method according to claim 9 wherein the backbone vector is pNeo4-ori (SEQ ID NO: 4] so that the artificial chromosome would have a nucleotide sequence that is at least 90% homologous to SEQ ID NO: 7.
11. A method according to claim 9 wherein the method further comprises the steps of inserting a modified HSP70.2 promoter (SEQ ID NO: 3], a PLA signal sequence (SEQ ID NO: 5] and
sfGFP marker gene (SEQ ID NO: 6] which results in a nucleotide sequence that is at least 95% homologous to SEQ ID NO: 8.
12. Use of the artifical chromosome according to claim 1 for recombinant protein production in Tetrahymena thermophila.
13. A nucleotide sequence pNeo4-ori having SEQ ID NO: 4.
14. A modified HSP70.2 promoter having SEQ ID NO: 3.
15. A nucleotide sequence pNeo3-ori having SEQ ID NO: 9.
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| TR201813880A TR201813880A2 (en) | 2018-09-25 | 2018-09-25 | ARTIFICIAL CHROMOSOME OF TETRAHYMENA THERMOPHILA (TtAC1) AND ITS USE FOR RECOMBINANT PROTEIN PRODUCTION |
| PCT/TR2019/050498 WO2020068014A2 (en) | 2018-09-25 | 2019-06-24 | Tetrahymena thermophila artificial chromosome 1 (ttac1) and its use for the production of recombinant proteins |
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| US20030033617A1 (en) | 1996-04-10 | 2003-02-13 | Gyula Hadlaczky | Artificial chromosomes, uses thereof and methods for preparing artificial chromosomes |
| US6670154B1 (en) * | 1998-07-27 | 2003-12-30 | Ronald A. Hitzeman | Automatic eukaryotic artificial chromosome vector |
| DE10105152A1 (en) | 2001-01-22 | 2002-07-25 | Hartmann Marcus | New nucleic acid encoding ciliate phospholipase A1, useful e.g. for causing secretion of proteins from Tetrahymena |
| DE602006020140D1 (en) | 2005-07-13 | 2011-03-31 | Cilian Ag | HEAT-INDUSTRIAL TETRAHYMENA PROMOTERS AND THEIR USE |
| CN101586119B (en) | 2009-05-15 | 2011-04-06 | 中国科学院水生生物研究所 | Tetrahymena transgenic carrier containing HSP70 promoter and GFP and preparation method and use thereof |
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