NZ706876B2 - A nucleotide sequence and a process thereof - Google Patents
A nucleotide sequence and a process thereof Download PDFInfo
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- NZ706876B2 NZ706876B2 NZ706876A NZ70687612A NZ706876B2 NZ 706876 B2 NZ706876 B2 NZ 706876B2 NZ 706876 A NZ706876 A NZ 706876A NZ 70687612 A NZ70687612 A NZ 70687612A NZ 706876 B2 NZ706876 B2 NZ 706876B2
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- New Zealand
- Prior art keywords
- seq
- expression
- nucleotide sequence
- pastoris
- gene
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- 238000000034 method Methods 0.000 title claims abstract description 24
- 229920001850 Nucleic acid sequence Polymers 0.000 title claims description 15
- 230000014509 gene expression Effects 0.000 claims abstract description 45
- 241000235058 Komagataella pastoris Species 0.000 claims abstract description 32
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 21
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 21
- 238000002955 isolation Methods 0.000 claims abstract description 6
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 24
- 108091006822 Human Serum Albumin Proteins 0.000 claims description 24
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 21
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 16
- 239000000411 inducer Substances 0.000 claims description 15
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 14
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical group OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims description 14
- 239000000600 sorbitol Substances 0.000 claims description 14
- 229920003013 deoxyribonucleic acid Polymers 0.000 claims description 11
- WQZGKKKJIJFFOK-VFUOTHLCSA-N β-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 11
- BJHIKXHVCXFQLS-UYFOZJQFSA-N Fructose Natural products OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 claims description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N D-Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 9
- 239000008103 glucose Substances 0.000 claims description 9
- 238000010367 cloning Methods 0.000 claims description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 6
- 239000004310 lactic acid Substances 0.000 claims description 6
- 235000014655 lactic acid Nutrition 0.000 claims description 6
- FBPFZTCFMRRESA-KAZBKCHUSA-N D-Mannitol Natural products OC[C@@H](O)[C@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KAZBKCHUSA-N 0.000 claims description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N D-sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 5
- 239000005715 Fructose Substances 0.000 claims description 5
- FBPFZTCFMRRESA-KVTDHHQDSA-N Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 5
- CZMRCDWAGMRECN-GDQSFJPYSA-N Sucrose Natural products O([C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1)[C@@]1(CO)[C@H](O)[C@@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-GDQSFJPYSA-N 0.000 claims description 5
- 239000000594 mannitol Substances 0.000 claims description 5
- 235000010355 mannitol Nutrition 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 239000005720 sucrose Substances 0.000 claims description 5
- 230000001131 transforming Effects 0.000 claims description 5
- 229920002472 Starch Polymers 0.000 claims description 4
- 239000008107 starch Substances 0.000 claims description 4
- 235000019698 starch Nutrition 0.000 claims description 4
- 230000003321 amplification Effects 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 102000004142 Trypsin Human genes 0.000 claims 1
- 108090000631 Trypsin Proteins 0.000 claims 1
- 244000195896 dadap Species 0.000 claims 1
- 229960001322 trypsin Drugs 0.000 claims 1
- 239000012588 trypsin Substances 0.000 claims 1
- 108010009384 L-Iditol 2-Dehydrogenase Proteins 0.000 abstract description 24
- 102100013658 SORD Human genes 0.000 abstract description 24
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 32
- 108010043984 Erythrina caffra trypsin inhibitor Proteins 0.000 description 30
- OKTJSMMVPCPJKN-UHFFFAOYSA-N carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 21
- 229910052799 carbon Inorganic materials 0.000 description 21
- 210000004027 cells Anatomy 0.000 description 20
- 229920001184 polypeptide Polymers 0.000 description 19
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 15
- 239000002609 media Substances 0.000 description 9
- 108010033725 Recombinant Proteins Proteins 0.000 description 8
- 102000007312 Recombinant Proteins Human genes 0.000 description 8
- 238000009632 agar plate Methods 0.000 description 8
- 230000001939 inductive effect Effects 0.000 description 7
- 108060000428 AOX Proteins 0.000 description 6
- 102100010989 AOX1 Human genes 0.000 description 6
- 239000000499 gel Substances 0.000 description 6
- 238000010195 expression analysis Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 4
- 102100008175 MGAM Human genes 0.000 description 4
- 229940037201 Oris Drugs 0.000 description 4
- 108090000854 Oxidoreductases Proteins 0.000 description 4
- 102000004316 Oxidoreductases Human genes 0.000 description 4
- 230000002538 fungal Effects 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 210000000349 Chromosomes Anatomy 0.000 description 3
- 241000235648 Pichia Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 241000228245 Aspergillus niger Species 0.000 description 2
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 2
- 229940041514 Candida albicans extract Drugs 0.000 description 2
- 229920001405 Coding region Polymers 0.000 description 2
- 102000002464 Galactosidases Human genes 0.000 description 2
- 108010093031 Galactosidases Proteins 0.000 description 2
- 102000002265 Human Growth Hormone Human genes 0.000 description 2
- 108010000521 Human Growth Hormone Proteins 0.000 description 2
- 239000000854 Human Growth Hormone Substances 0.000 description 2
- 239000006137 Luria-Bertani broth Substances 0.000 description 2
- 108091005771 Peptidases Proteins 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 235000003534 Saccharomyces carlsbergensis Nutrition 0.000 description 2
- 229940081969 Saccharomyces cerevisiae Drugs 0.000 description 2
- 102000003978 Tissue plasminogen activator Human genes 0.000 description 2
- 108090000373 Tissue plasminogen activator Proteins 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 108091006028 chimera Proteins 0.000 description 2
- 239000008121 dextrose Substances 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 230000001105 regulatory Effects 0.000 description 2
- 108091007521 restriction endonucleases Proteins 0.000 description 2
- 230000001225 therapeutic Effects 0.000 description 2
- 229960000187 tissue plasminogen activator Drugs 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- BRZYSWJRSDMWLG-DJWUNRQOSA-N (2R,3R,4R,5R)-2-[(1S,2S,3R,4S,6R)-4,6-diamino-3-[(2S,3R,4R,5S,6R)-3-amino-4,5-dihydroxy-6-[(1R)-1-hydroxyethyl]oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol Chemical compound O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H]([C@@H](C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-DJWUNRQOSA-N 0.000 description 1
- FVFVNNKYKYZTJU-UHFFFAOYSA-N 6-chloro-1,3,5-triazine-2,4-diamine Chemical compound NC1=NC(N)=NC(Cl)=N1 FVFVNNKYKYZTJU-UHFFFAOYSA-N 0.000 description 1
- 229920002287 Amplicon Polymers 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 108091005650 Basic proteases Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 229940080701 Chymosin Drugs 0.000 description 1
- 108090000746 Chymosin Proteins 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 229940110715 ENZYMES FOR TREATMENT OF WOUNDS AND ULCERS Drugs 0.000 description 1
- 102000033147 ERVK-25 Human genes 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 229960000301 Factor VIII Drugs 0.000 description 1
- 102000001690 Factor VIII Human genes 0.000 description 1
- 108010054218 Factor VIII Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 102000006602 Glyceraldehyde-3-Phosphate Dehydrogenases Human genes 0.000 description 1
- 108020004445 Glyceraldehyde-3-Phosphate Dehydrogenases Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 102000005431 Molecular Chaperones Human genes 0.000 description 1
- 108010006519 Molecular Chaperones Proteins 0.000 description 1
- 241000108056 Monas Species 0.000 description 1
- 102000035443 Peptidases Human genes 0.000 description 1
- 241000822135 Ula Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
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- -1 ase Proteins 0.000 description 1
- 230000001580 bacterial Effects 0.000 description 1
- 230000003115 biocidal Effects 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000001413 cellular Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002708 enhancing Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000010359 gene isolation Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 150000004676 glycans Polymers 0.000 description 1
- 239000000383 hazardous chemical Substances 0.000 description 1
- 229940020899 hematological Enzymes Drugs 0.000 description 1
- 230000003301 hydrolyzing Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 230000002934 lysing Effects 0.000 description 1
- 210000004962 mammalian cells Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N oxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 230000036284 oxygen consumption Effects 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
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Classifications
-
- 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
- C07K14/76—Albumins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
-
- 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
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
-
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01014—L-Iditol 2-dehydrogenase (1.1.1.14), i.e. sorbitol-dehydrogenase
Abstract
The present disclosure relates to a process of identification and isolation of sorbitol dehydrogenase promoter from Pichia pastoris. Further, the present disclosure also relates to expression of heterologous proteins under the control of Sorbitol dehydrogenase promoter in Pichia pastoris.
Description
“A NUCLEOTIDE SEQUENCE AND A PROCESS THEREOF”
TECHNICAL FIELD
The present disclosure describes fication and isolation of sorbitol dehydrogenase
promoter from P.pastoris and expression of heterologous protein in P.pastoris under the
influence of said isolated er, specifically expression of Human Albumin (HA) and
Erythrina Trypsin Inhibitor (ETI).
BACKGROUND AND PRIOR ART OF THE DISCLOSURE
Pichia pastoris is a very widely used well-established rial host for producing
therapeutic recombinant proteins. tion of therapeutic recombinant proteins poses
l problems such as clipping at N-terminal (Prabha et.al. 2009) or C-terminal,
introduction of desired glycoforms (Wouter Vervecken et.al. 2004). There is a need for
enhancing the protein productivity and quality to meet the growing demands of the market
and the growing regulatory issues on biosimilarities. This can be achieved by co-expressing
the molecular chaperones (Wei Zhang et.al. 2006), Co-expression of proteases to get the
d end product .In all these cases there is need for additional promoters for carrying out
c manipulation to improve quality and quantity of the recombinant protein.
US Patent No. 8 discloses a DNA segment isolated from a Saccharomyces cerevisiae
sorbitol dehydrogenase gene which is utilized to increase production of a heterologous
polypeptide. US Patent No. 5,139,936 discloses a cloning vector containing a n gene
and the yeast galactosidase (GAL1) regulatory region and promoter in position to increase
expression of the foreign gene. US Patent No. 5,089,398 discloses the use of the promoter
region from the glyceraldehydephosphate dehydrogenase to control expression of a foreign
polypeptide in yeast.
Although several promoters, either constitutive or ble are ble for the Pichia
expression system, AOX1 is employed in most studies and application. AOX1 promoter is a
strong inducible promoter and has tight regulation by carbon sources, expression is highly
repressed when Pichia pastoris is grown in the ce most of other carbon sources other
than Methanol. Methanol is an inducer of AOX1 promoter, methanol is a hazardous
substance due to its high flammability and ty, also the cells growing on methanol have a
very high oxygen consumption which y requires addition of pure oxygen to the culture,
thus increasing the cost of the process and limiting the cultivation capacity at large scale
a Bollok et.al., Recent Patents on biotechnology, 3(2009) 192-201.)
Also host cellular protein (HCP) release from P.pastoris grown on ol is high which is
mainly derived during cell lysis but it occurs to a much lower extent upon growth with
e as a carbon source. As a result cells maintain higher ity and higher purity of the
secreted protein.
Further, the sorbitol dehydrogenase promoter identified in S.cerevisiae is 750bps. Due to
large base pair size, it is not so easy to carry out genetic manipulation as it leads to increase
of the vector size. The instant sure aims at overcoming the issues mentioned above.
Also, there is a need in the art to identify more ers for the expression of heterologous
recombinant proteins in Pichia pastoris which does not require any specific induction for
expression of protein.
SUMMARY OF THE DISCLOSURE
Accordingly, the present disclosure relates to a nucleotide ce set forth as SEQ ID
No.1; a process of isolation of SEQ ID No.1, said process comprising acts of- a) isolating
genomic DNA from P.pastoris, b)amplifying a region comprising SEQ ID No.1 within the
isolated c DNA, and c) subjecting the amplified product to a conventional gel elution
procedure to isolate said SEQ ID No.1; a process of expressing a gene under regulation of
SEQ ID No.1 in a medium comprising non-specific inducer, said process comprising acts ofa
) cloning said gene in an expression construct downstream to the SEQ ID No.1, b)
transforming a host with the expression construct, and c) expressing the gene under the
regulation of said SEQ ID No.1 in a medium comprising non-specific r for production
of hetrologous n; a vector comprising the nucleotide sequence as mentioned above; a
transformed host cell comprising the vector as mentioned above.
BRIEF DESCRIPTION OF ACCOMPANYING FIGURES
Figure 1 shows ction analysis of SDPHA/pMBL208. All the digestions results in the
expected pattern.
Figure 2 shows vector map of SDPHA/pMBL208.
Figure 3 shows screening of colonies for selecting high producer of recombinant protein on
G418 antibiotic. The colony shown in the arrow indicates the higher resistant clone.
Figure 4 shows PCR confirmation of the integrated gene into the P.pastoris genome. All the
clones selected are found to have the gene of interest.
Figure 5 shows expression analysis of HA when grown using sorbitol as a sole carbon
source.
Figure 6 shows expression analysis of HA when grown using methanol as a sole carbon
source.
Figure 7 shows expression analysis of HA when grown using glucose as a sole carbon
source.
Figure 8 shows sion analysis of cell free suprnatant without any ion (cells are
grown in expansion medium).
Figure 9 shows Picture of ETI expression constructs; A) ETI is cloned in the XhoI and
E.coRI restriction sites under the control of sorbitol dehydrogenase promoter. B) ETI with no
promoter upstream.
Figure 10 shows expression analysis of ETI when grown using sorbitol as a sole carbon
source.
Figure 11 shows determination of ETI productivity under different carbon sources.
Figure 12 is a chromatogram showing the sequencing results obtained for sorbitol
dehydrogenase promoter isolated from P.pastoris
DETAILED DESCRIPTION OF DISCLOSURE
The present disclosure s to a nucleotide sequence set forth as SEQ ID No. 1.
In an embodiment of the present, said nucleotide sequence is sorbitol dehydrogenase
promoter.
In another embodiment of the present sure, said tide sequence is identified and
isolated from P.pastoris.
The present sure relates to a process of isolation of SEQ ID No.1, said s
comprising acts of: a) isolating genomic DNA from oris, b) amplifying a region
comprising SEQ ID No.1 within the isolated c DNA, and c) subjecting the amplified
product to a conventional gel elution procedure to isolate said SEQ ID No.1.
In an embodiment of the present disclosure, the amplification of SEQ ID No.1 is carried out
by forward primer set forth as SEQ ID No.2 and e primer set forth as SEQ ID No.3.
The present disclosure relates to a process of expressing a gene under regulation of SEQ ID
No.1 in a medium comprising non-specific inducer, said process comprising acts of: a)
cloning said gene in an expression construct downstream to the SEQ ID No.1, b)
transforming a host with the expression construct, and c) expressing the gene under the
regulation of said SEQ ID No.1 in a medium comprising non-specific inducer for production
of hetrologous protein.
In an embodiment of the present sure, said host is methylotrophic yeast, preferably
oris.
In another embodiment of the present sure, said heterologous protein is selected from a
group comprising human albumin and erythrina trypsin inhibitor.
In yet another embodiment of the present disclosure, said expression is carried out by a nonspecific
inducer.
In still another embodiment of the present disclosure, said non-specific inducer selected from
a group comprising sorbitol, glucose, glycerol, fructose, mannitol, lactic acid, sucrose,
starch, methanol or any combination thereof.
The present sure relates to a vector comprising the nucleotide sequence as mentioned
above.
The t disclosure relates to a transformed host cell sing the vector as mentioned
above.
The present disclosure relates to identification of sorbitol dehydrogenase promoter as a
potential tool to express heterologous recombinant proteins in P.pastoris that does not require
any specific induction for expression.
In an ment of the present disclosure, 210bps promoter is located on the chromosome 1
of oris.
In another ment of the present disclosure, sortibol dehydrogenase promoter is the
st er in P.pastoris.
In an embodiment of the present sure, promoter region available am of Sorbitol
dehydrogenase gene is identified to have CAAT and GC boxes within that region.
In another embodiment of the present disclosure, the promoter is successfully used to s
recombinant proteins such as Human Albumin (HA) and Erythrina Trypsin Inhibitor (ETI).
In another embodiment of the present disclosure, the heterlogous proteins such as human
albumin and erythrina trypsin inhibitor are expressed under the control of sorbitol
dehydrogenase promoter.
In another embodiment of the present disclosure, the sorbitol dehydrogenase promoter
enables the expression of heterlogous proteins under any type of carbon source.
In another embodiment of the present disclosure, the sorbitol dehydrogenase promoter
enables the expression of ogous protein ing sorbitol, e, glycerol, fructose,
mannitol, lactic acid, sucrose, starch and methanol as a carbon source.
In another embodiment of the present disclosure, the sorbitol dehydrogenase gene from
P.pastoris is identified by the ing process: a) ol dehydrogenase gene orthologs is
subjected to NCBI blast which resulted in multiple hits; b) identifying sorbitol dehydrogenase
gene in Pichia pastoris located on the chromosome 1, and; c) analysing the sequence
upstream of the identified ol dehydrogenase gene.
In another embodiment of the present disclosure, expression of a target gene to obtain a
protein of interest under the influence of the chimeric promoter of the instant invention is not
limited by the carbon source being employed. In other words, by making use of the chimeric
promoter of this invention, the protein of interest may be expressed by making use of any
carbon source such as but not limited to sorbitol, e, glycerol, fructose, mannitol, lactic
acid, sucrose,. However, a person d in the art will ize the most potent inducers for
higher expression of a specific type of protein and hence will be able to employ any inducer
having such high potency or likeliness of higher sion than the remaining inducers.
In another embodiment of the present disclosure the term non-specific inducer and carbon
source is used interchangeably in accordance to the description.
In another ment of the present disclosure, recombinant expression system is selected
from prokaryotic and eukaryotic hosts. otic hosts include yeast cells (e.g.,
Saccharomyces cerevisiae or Pichia pastoris), mammalian cells or plant cells. Bacterial and
eukaryotic cells are ble from a number of different sources including commercial
sources to those skilled in the art, e.g., the American Type Culture Collection (ATCC;
Rockville, Md.). Commercial sources of cells used for recombinant protein expression also
provide ctions for usage of the cells. The choice of the expression system depends on
the features desired for the expressed polypeptide.
In another embodiment of the present disclosure, the most preferred host cells are
otrophic yeasts. Strains of a methylotrophic yeast which can be modified using the
present disclosure include, but are not limited to yeast s capable of growing on
methanol, such as yeasts of the genera Pichia, Candida, ula, or Torulopsis. Preferred
methylotrophic yeasts are of the genus Pichia. Methylotrophic yeast strains which can be
modified using the present methods also include those methylotrophic yeast strains which
have been engineered to express one or more heterologous proteins of interest.
In another embodiment of the present disclosure, the term “promoter” is used in the normal
sense of the art, e.g. an RNA polymerase binding site. The term “vector” includes expression
vectors, replicable vectors, transformation vectors and shuttle vectors, including vector
combinations thereof.
In another ment of the present disclosure, the term “expression vector” means a
uct capable of in-vivo or in-vitro expression.
In another embodiment of the present disclosure, preferentially the expression vector is
incorporated in the genome of the organism. The term “incorporated” preferably covers
stable oration into the genome.
In another embodiment of the present disclosure, "expressing polypeptides" is meant as
expression of DNA sequences encoding the polypeptide. "Polypeptides" are polymers of
[alpha]-amino acids which are covalently linked through peptide bonds. ptides e
low molecular weight polymers as well as high molecular weight rs more commonly
referred to as proteins. In addition, a polypeptide can be a phosphopolypeptide,
olypeptide or metallopoly-peptide. Further, one or more polymer chains may be
ed to form a polypeptide. As used herein a "heterologous polypeptide" is a
polypeptide which is not normally expressed and secreted by the ntous fungus used to
express that particular polypeptide. Heterologous polypeptides e polypeptides d
from prokaryotic sources (e.g., [alpha]-amylase from Bacillus species, alkaline protease from
us species, and s hydrolytic enzymes from monas, etc.), polypeptides
derived from eukaryotic sources (e.g., bovine chymosin, human tissue plasminogen activator,
human growth hormone, human interferon, ase, human serum albumin, factor VIII
etc.), and polypeptides, derived from fungal sources other than the expression host (e.g.,
glucoamylase from A.niger and Heterologous polypeptides also include hybrid polypeptides
which comprise a combination of partial or complete polypeptide sequences derived from at
least two different polypeptides each of which may be homologous or heterologous with
regard to the fungal expression host. Examples of such hybrid ptides include: 1) DNA
ces encoding prochymosin fused to DNA sequences encoding the A.niger
glucoamylase signal and pro sequence alone or in conjunction with various amounts of
amino-terminal mature glucoamylase codons, and 2) DNA sequences encoding fungal
glucoamylase or any fungal carboxy protease, human tissue plasminogen activator or human
growth hormone fused to DNA ces encoding a functional signal sequence alone or in
conjunction with various amounts of amino-terminal propeptide condons or mature codons
associated with the functional signal.
The invention is further illustrated by the following examples. The following examples are
provided for rative purposes only and are not intended to limit the scope of the
invention.
EXAMPLES
Example1: Pichia pastoris Sorbitol dehydrogenase Promoter (SDP) Isolation and
Cloning
The coding sequence of sorbitol dehydrogenase gene is identified, which is located on
chromosome no. 1 of oris. The region am to 5` region to the coding sequence of
the identified gene is examined. The examined gene is identified as a promoter region for
Sorbitol dehydrogenase comprising 210bp. The genomic DNA of P.pastoris is isolated using
the conventional gene isolation technique. The isolated gene is ted to polymerase chain
reaction to amplify the examined 210bp region.
The primers used to amplify the examined gene are-
SORFP= 5’ AGA TAT CAA GTT GTA TAT TAT TAA TGG CGG GGC A 3’ (EcoRV at 5’
end)
SORRP = 5’ TCA TAT GTT GGA TAA TAG TGA GTG TAA TGA 3’ (NdeI at 5’ end).
The chromatogram of figure 12 shows the sequencing results obtained for sorbitol
dehydrogenase promoter isolated from P.pastoris. The er region from the region 37 to
246 bp is highlighted within brackets. The amplicon is ted to agarose gel
electrophoresis against a suitable marker. The amplified 210bp region on the gel is eluted
using Qiagen spin columns. The gel purified t is A-tailed under the polymerase chain
reaction. The A-tailed PCR product is ligated to TA vector (pTZ57R) at 16ºC, overnight. The
ligated t is transformed to E coli DH5α by heat shock method and plated on LBA100
and incubated the plates at 37 ˚C overnight.
Example 2: Cloning and restriction analysis of SDPHA/pMBL208
The plasmid pMBL208 having human n gene (HA) with an AOX1 er is
selected. The AOX1 promoter is removed from rHA/pMBL His plasmid by digesting it with
EcoRV and NdeI restriction enzymes. Same restriction enzymes are used to release the
ol dehydrogenase promoter (SDP) from pTZ57R. The released SDP is ligated to the
AOX1 removed vector (fig 2). The ligated product is transformed to E coli DH5α and plated
on LBA100, plates are ted overnight at 37˚C, and the transformed colonies are
screened for t plasmid by colony PCR. The positive clone is isolated and inoculated in
Luria Broth (LB) and grown overnight at 37 ˚C to isolate plasmid, plasmid is isolated from
overnight grown culture using Qiagen mini prep spin s. ed plasmid is confirmed
by restriction digestion (fig.1) with the following restriction enzymes- EcoRV and NdeI,
EcoRV and EcoRI, EcoRI and NdeI and EcoRV.
Example 3: Transformation of P.pastoris with SDPHA/pMBL208.
The plasmid SDPHA/pMBL208 is linearized with EcoRV and transformed to P.pastoris. The
transformed cells are spread on to Yeast nitrogen base (YNB) agar plates without any amino
acids for selection. The plates are incubated at 30°C for two days till the colonies appear.
Example 4: Screening of colonies for higher Geneticin (G418) resistance.
Upon observing growth of the colonies in YNB agar plates, the colonies are picked and
inoculated into Yeast Extract Peptone Dextrose (YPD) in l plate and incubated at
°C, overnight. The es from 96-well plate are d onto YPD agar plates
ning 0.5mg/ml of G418 and incubated the plated at 30°C for 2 days. After 2 days the
colonies showing higher resistance to G418 are selected and streaked onto YPD agar plates as
shown in fig. 3. The colony shown in the arrow indicates the higher resistant clone.
The clones selected based on their resistance to G418 are: 3G11, 4F11, 5F5, 6F10, 4B1,
4F10, 5A1, 7A10. From these selected clones, the genomic DNA is isolated by the Phenol
Chloroform Method. The vector SDPHA/pMBL208 recombination is confirmed by PCR
using the primers SORFP and HSAintRP. This gives amplification of about 750 bps (fig 4).
Figure 4 represents a gel for the PCR confirmation of the integrated gene into the P.pastoris
genome for the clones 3G11, 4F11, 5F5, 6F10, 4B1, 4F10, 5A1, 7A10 and it was observed
that all the clones had proper intergration of gene in to the host genome.
Example 5: Expression Studies in SDPHA/ P.pastoris.
The shake flask induction studies is carried out for 4 selected clones (5F5, 6F10, 5A1 and
7A10) to find out whether the ol promoter is able to produce human albumin (HA). The
cell mass is developed and induction is carried out with three carbon sources such as Glucose,
Methanol and Sorbitol. The shake flask induction samples are analysed using SDS PAGE,
which is depicted in figures 5, 6, 7 and 8. Figure 5 ents the expression of Human
albumin when cells are grown in the medium comprising sorbitol as a carbon source, wherein
lane 1 relates to a n molecular marker, lane 2 relates to Pichia pastoris with the sorbitol
dehydrogenase promoter, lane 3, 4, 5, and 6 relates to HA in clones 5F5, 6F10, 5A1 and
7A10, respectively. Similarly, s 6, 7 relate to the expression of HA protein in Pichia
pastoris when the cells are grown in the medium comprising methanol and glucose,
respectively. Figure 8 relates to expression analysis of cell free supernatant for the clones
5F5, 6F10, 5A1 and 7A10, where cells are grown in expansion medium t any
induction
Results and conclusion:
a) The level of HA expression is good and it is found to be similar with all three carbon
sources; ol, methanol and glucose. The SDS PAGE band intensity obtained in
all cases is equivalent to or more than 2µgs, such band intensity is considered good
expression.
b) Expression is observed during cell mass accumulation.
c) It also shows that the expression levels are increasing in dose dependent manner as it
increases according to the higher G418 ance as clone 5F5 is highest and 7A10 is
the least among the clones selected.
Example 6: Expression of Erythrina Trypsin Inhibitor
In an attempt to find out the robustness of the Sorbitol dehydrogenase promoter for more
number of heterologous protein expressions, Erythrina Trypsin Inhibitor (ETI) is been
chosen. ETI is a small n of ~20 KDa and it is useful for the ty purification of
Retaplase. The vector SDPHA/pMBL208 is digested with XhoI and EcoRI to remove HA
gene from the vector. Synthetic ETI gene is subcloned into pMBL208 in place of HA gene.
The ligated product is transformed to E coli DH5α and plated on LBA100, plates are
incubated overnight at 37˚C and the transformed colonies are ed for correct plasmid by
colony PCR. The positive clone is isolated and inoculated in Luria Broth (LB) and grown
overnight at 37 ˚C to isolate plasmid. The plasmid is isolated from overnight grown culture
using Qiagen mini prep spin columns. Isolated plasmid is confirmed by ction ion
with the following restriction s- EcoRV/NdeI, EcoRV/ EcoRI, NdeI and
EcoRV. ETI gene is cloned into pMBL208 with and without sorbitol dehydrogenase
er (fig.9A and 9B). By doing so it is demonstrated that the production of ETI is due to
the 210 bps sorbitol promoter and not because of any upstream promoter activity.
Example 7: Shake flask expression studies
The plasmid SDPETI/pMBL208 is linearized with EcoRV and transformed to P.pastoris. The
transformed cells are spread on to Yeast nitrogen base (YNB) agar plates without any amino
acids for selection. The plates are incubated at 30°C for two days till the colonies appear.
Upon ing growth of the colonies in YNB agar plates, the colonies are picked and
inoculated into Yeast Extract Peptone Dextrose (YPD) in 96-well plate and ted at
°C, overnight. The cultures from 96-well plate are stamped onto YPD agar plates
containing 0.5mg/ml of G418 and incubated the plated at 30°C for 2 days. After 2 days the
colonies showing higher resistance to G418 are selected and streaked onto YPD agar plates.
The clones selected based on the higher resistant to G418 are clones 1, 2, 3 and 4, these
clones are inoculated into shake flask containing medium, expression of ETI in shake flask is
analysed on 12% SDS PAGE (sodium dodecyl e polyacrylamide gel electrophoresis).
Productivity of sed ETI under different carbon sources is shown in fig 10 and 11, the
arrow mark shows the expressed ETI on the gel.
Results and conclusion:
Sorbitol dehydrogenase promoter is able to es 20kDa ETI when grown in any of the
carbon sources such as sorbitol, glucose, glycerol, fructose, mannitol, sucrose, lactic acid,
starch and methanol. No expression is found without the sorbitol promoter upsetream of ETI.
The productivity of the sed protein is found to be higher when Glycerol is used as a
carbon source. Lactic acid also is been able to produce ETI when compared to other rides
such as monosaccharide, disaccharide and polysaccharides when used as a carbon source.
A ative Expression status for the heterologous protein produced under the influence
of Sorbitol dehydrogenase promoter is shown in table 1 below.
Construct integrated into the Pichia pastoris Inducer/carbon Expression
genome source status
Parent Pichia pastoris host Methanol No
ETI/pMBL208 (ETI CDS without SDP) Methanol No
SDPETI/pMBL208 (ETI CDS under SDP regulation) Methanol Yes
SDPETI/pMBL208 (ETI CDS under SDP tion) Glucose Yes
SDPETI/pMBL208 (ETI CDS under SDP regulation Sorbitol Yes
SDPETI/pMBL208 (ETI CDS under SDP regulation Glycerol Yes
SDPHA/pMBL208 (Human albumin under SDP Methanol Yes
regulation)
SDPHA/pMBL208 (Human albumin under SDP Glucose Yes
tion)
SDPHA/pMBL208 (Human albumin under SDP Sorbitol Yes
regulation)
SDPHA/pMBL208 (Human albumin under SDP ol Yes
regulation)
Claims (13)
1. A nucleotide sequence set forth as SEQ ID No. 1.
2. The nucleotide sequence as claimed in claim 1, wherein said nucleotide sequence is sorbitol ogenase promoter. 5
3. The nucleotide sequence as claimed in claim 1, wherein said nucleotide sequence is identified and isolated from P.pastoris.
4. A s of isolation of SEQ ID No.1, wherein said process comprising acts of: a) isolating genomic DNA from P.pastoris; b) amplifying a region comprising SEQ ID No.1 within the ed genomic 10 DNA; and c) subjecting the amplified product to a conventional gel elution procedure to isolate said SEQ ID No.1.
5. The process as claimed in claim 4, wherein the amplification of SEQ ID No.1 is carried out by forward primer set forth as SEQ ID No.2 and e primer set forth as 15 SEQ ID No.3.
6. A process of expressing a gene under regulation of SEQ ID No.1 in a medium comprising non-specific inducer, wherein said process comprising acts of: a) cloning said gene in an sion construct downstream to the SEQ ID No.1; b) transforming a host with the expression construct; and 20 c) expressing the gene under the regulation of said SEQ ID No.1 in a medium comprising non-specific inducer for production of hetrologous protein.
7. The s as claimed in claim 6, wherein said host is methylotrophic yeast.
8. The process as claimed in claim 6, wherein said logous protein is selected from a group comprising human albumin and erythrina trypsin tor. 25
9. The process as claimed in claim 6, wherein said expression is carried out by a cific inducer.
10. The process as claimed in claim 6, wherein the non-specific inducer selected from a group comprising sorbitol, glucose, glycerol, fructose, mannitol, lactic acid, sucrose, starch, ol or any combination thereof.
11. A vector sing the nucleotide sequence as claimed in claim 1.
12. A transformed host cell comprising the vector as claimed in claim 11; with the proviso that the host cell is not within a human.
13. The process as claimed in claim 6, wherein said host is P.pastoris.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IN4231CH2012 | 2012-10-10 | ||
IN4231/CHE/2012 | 2012-10-10 | ||
PCT/IB2012/056985 WO2014057315A1 (en) | 2012-10-10 | 2012-12-05 | A nucleotide sequence and a process thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ706876A NZ706876A (en) | 2016-03-31 |
NZ706876B2 true NZ706876B2 (en) | 2016-07-01 |
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