WO2014128135A1 - Use of an amino acid auxotrophy cured prokaryotic strain for the recombinant production of a polypeptide - Google Patents
Use of an amino acid auxotrophy cured prokaryotic strain for the recombinant production of a polypeptide Download PDFInfo
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- WO2014128135A1 WO2014128135A1 PCT/EP2014/053171 EP2014053171W WO2014128135A1 WO 2014128135 A1 WO2014128135 A1 WO 2014128135A1 EP 2014053171 W EP2014053171 W EP 2014053171W WO 2014128135 A1 WO2014128135 A1 WO 2014128135A1
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- 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
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- 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 current invention is in the field of recombinant polypeptide production. More precisely herein is reported a method for the recombinant production of a non- glycosylated polypeptide using an auxotrophy cured prokaryotic strain in a chemically defined minimal growth medium.
- therapeutic polypeptides In recent years the production of therapeutic polypeptides has steadily increased and it is likely that therapeutic polypeptides will become the biggest group of therapeutics available for the treatment of various diseases in the near future. The impact of therapeutic polypeptides emerges from their specificity, such as specific target recognition and/or binding function.
- Cell cultures are used in fermentative processes to produce substances and in particular polypeptides.
- processes in which the cell cultures are genetically unmodified and form their own metabolic products and processes in which the organisms are genetically modified in such a manner that they either produce a larger amount of their own substances such as polypeptides or produce foreign substances.
- the organisms producing the substances are supplied with a nutrient medium which guarantees the survival of the organisms and enables the production of the desired target compound. Numerous culture media are known for these purposes which enable an optimal cultivation of the specific host.
- the use of a chemically defined minimal growth medium in the cultivation of a recombinant cell for the recombinant production of therapeutic polypeptides is advantageous. It enables easy development of downstream processing and purification of the produced therapeutic polypeptide, provides for a robust productions process due to minimized raw material differences and reduces costs of goods.
- a chemically defined minimal growth medium does not comprise free amino acids and it is required to use prototrophic cell lines which have intact metabolic pathways to produce the required amino acids from the available components of the chemically defined minimal growth medium.
- E.coli is used as host cell line
- generally wild-type strains such as MG1655, W3110 or BL21, are employed. These strains show good growth characteristics but inferior product titer.
- Mutant prokaryotic strains which have been obtained by non-directed mutagenesis and selection, show profound differences in their genomic DNA when compared to wild-type strains.
- the mutant strains have been selected based on the maximum product titer that can be obtained. As the mutant strains harbor a number of auxotrophies they cannot be used for cultivation in a chemically defined minimal growth medium. The mutant strains required the feeding of amino acids to complement their auxotrophies resulting in increased cultivation costs.
- the auxotrophy cured strain can grow on a chemically defined minimal growth medium to which the substance corresponding to the auxotrophy that has been cured no further needs to be added. In case of expensive substances a reduction of the costs of goods can be achieved and a recombinant polypeptide can be produced more economically.
- the obtainable product titer can be maintained or even increased compared to the non-cured strain. It has furthermore been found that the obtainable product titer using an auxotrophy cured prokaryotic strain is higher than the product titer that can be obtained with a corresponding prototrophic wild-type strain.
- an amino acid auxotrophy cured prokaryotic strain as reported herein has a different metabolism and metabolic fluxes when compared to a corresponding prototrophic wild-type strain in which such a deletion and cure has not been performed.
- the inactivation and re-activation of certain enzymes within a prokaryotic strain has a detectable/pronounced effect on the entire metabolism of the strain, which can be seen, e.g. in the increased productivity of such a strain compared to a corresponding prototrophic wild-type strain.
- One aspect as reported herein is an amino acid auxotrophy cured prokaryotic strain, characterized in that - at least one deficiency in an essential amino acid metabolic pathway has been cured, and the strain can grow in chemically defined minimal growth medium that is free of the amino acid corresponding to the auxotrophy that has been cured in the auxotrophy cured prokaryotic strain.
- the auxotrophy cured prokaryotic strain is an auxotrophy cured
- One aspect as reported herein is an amino acid auxotrophy cured prokaryotic cell, characterized in that at least one deficiency in an essential amino acid metabolic pathway has been cured, and the cell can grow in chemically defined minimal growth medium that is free of the amino acid corresponding to the auxotrophy that has been cured in the auxotrophy cured prokaryotic cell.
- the auxotrophy cured prokaryotic cell is an auxotrophy cured E.coli cell.
- auxotrophy cured prokaryotic strain/cell as reported herein can be used in a method for the recombinant production of a therapeutic polypeptide wherein compared to the non-auxotrophy cured strain/cell the supplementation of at least one amino acid residue is not required.
- One aspect as reported herein is a method for producing a polypeptide in a
- (recombinant) prokaryotic cell comprising the following step: cultivating a (recombinant) prokaryotic cell, which has been obtained by introducing into the genome of a parent prokaryotic cell a nucleic acid curing an amino acid auxotrophy of the parent prokaryotic cell, comprising one or more nucleic acids encoding the polypeptide in a chemically defined minimal growth medium and recovering the polypeptide from the prokaryotic cell or the periplasm of the prokaryotic cell or from the medium.
- One aspect as reported herein is a method for producing a polypeptide in a (recombinant) prokaryotic cell, comprising the following steps: curing an prokaryotic cell that is auxotrophic for at least one amino acid from at least one amino acid auxotrophy, cultivating the amino acid auxotrophy cured (recombinant) prokaryotic cell comprising one or more nucleic acids encoding the polypeptide in a chemically defined minimal growth medium and recovering the polypeptide from the prokaryotic cell or the periplasm of the prokaryotic cell or from the medium.
- One aspect as reported herein is a method for producing a polypeptide in a (recombinant) prokaryotic cell, comprising the following step: - cultivating a (recombinant) prokaryotic cell comprising one or more nucleic acids encoding the polypeptide in a chemically defined minimal growth medium and recovering the polypeptide from the prokaryotic cell or the periplasm of the prokaryotic cell or from the medium, wherein the (recombinant) prokaryotic cell is an amino acid auxotrophy cured prokaryotic cell.
- the auxotrophy cured prokaryotic cell has at least one further (non-cured) amino acid auxotrophy.
- This auxotrophy can be used for the selection of recombinants/transformants after the introduction of one or more nucleic acids encoding a polypeptide of interest (the polypeptide).
- the growth of the amino acid auxotrophy cured prokaryotic cell compared to the non-cured prokaryotic cell (parent cell) under the same cultivation conditions and in the same growth medium requires the supplementation of fewer amino acids to the growth medium.
- the chemically defined minimal growth medium is free of the amino acid corresponding to the auxotrophy that has been cured in the auxotrophy cured prokaryotic cell.
- the cultivation of the auxotrophy cured prokaryotic cell requires supplementation of one or two or three or four amino acids less to the growth medium than required for the cultivation of the non-auxotrophy cured prokaryotic cell (parent/parent cell).
- the amino acid corresponding to the auxotrophy that has been cured in the auxotrophy cured prokaryotic cell is not added during the cultivation.
- the cultivation is in the absence of the amino acid corresponding to the auxotrophy that has been cured in the auxotrophy cured prokaryotic cell.
- the prokaryotic cell is an E.coli cell.
- the polypeptide is a full length antibody chain, a single chain antibody, a single domain antibody, a scFv, a scFab, or a conjugate of one of the before with a non-antibody polypeptide.
- the polypeptide is a conjugate of a scFv or scFab with a cytotoxic agent.
- One aspect as reported herein is the use of an amino acid auxotrophy cured prokaryotic strain for the (recombinant) production of a polypeptide.
- the amino acid auxotrophy cured prokaryotic strain at least one deficiency in an essential amino acid metabolic pathway has been cured, and the amino acid auxotrophy cured strain can grow in chemically defined minimal growth medium that is free of the amino acid corresponding to the auxotrophy that has been cured in the auxotrophy cured prokaryotic strain.
- the auxotrophy cured prokaryotic strain is an auxotrophy cured E.coli strain.
- polypeptide is a full length antibody chain, a single chain antibody, a single domain antibody, a scFv, a scFab, or a conjugate of one of the before with a non-antibody polypeptide.
- polypeptide is a conjugate of a scFv or scFab with a cytotoxic agent.
- the auxotrophy cured prokaryotic strain is a non- lysogenic strain.
- a lysogenic strain is infected with a temporal bacteriophage, i.e. a non-lysogenic strain is free of bacteriophages.
- the cure is a directed cure targeting only the amino acid auxotrophy.
- the cure is the introduction of one or more enzymes required for the synthesis of the auxotrophic amino acid.
- the prokaryotic strain has the genotype thi-1, AompT, ApyrF.
- One aim in the development of large scale recombinant polypeptide production processes is the reduction of the required amount, i.e. the consumption, of medium components during the cultivation. Especially the reduction of the consumption of expensive medium components, such as amino acids, is advantageous in view of costs of goods.
- This aim has been achieved by curing an amino acid auxotrophic strain from at least one, some, or all of its amino acids auxotrophies in order to avoid feeding of expensive amino acids and therewith to reduce the costs of the recombinant production of a polypeptide of interest. Additionally a chemical defined medium can be used which results in an improved process stability and facilitates downstream processing (DSP). Thus, herein is reported a method for the recombinant production of a non- glycosylated polypeptide using an auxotrophy cured prokaryotic strain in a chemically defined minimal growth medium.
- auxotrophy cured strain can grow on a chemically defined minimal growth medium to which the substance corresponding to the auxotrophy that has been cured no further needs to be added.
- expensive substances such as e.g. amino acids
- a reduction of the costs of goods can be achieved and a recombinant polypeptide can be produced more economically.
- the obtainable product titer can be maintained or even increased. It has furthermore been found that the obtainable product titer using an auxotrophy cured prokaryotic strain is higher than the product titer that can be obtained with a corresponding prototrophic wild-type strain.
- One aspect as reported herein is a method for producing a polypeptide in a prokaryotic cell, comprising the following step: cultivating a prokaryotic cell, which has been obtained by introducing into the genome of a parent prokaryotic cell a nucleic acid curing an amino acid auxotrophy of the parent prokaryotic cell, comprising one or more nucleic acids encoding the polypeptide in a chemically defined minimal growth medium and recovering the polypeptide from the prokaryotic cell or the periplasm of the prokaryotic cell or from the medium.
- One aspect as reported herein is a method for producing a polypeptide in a prokaryotic cell, comprising the following steps: curing an prokaryotic cell that is auxotrophic for at least one amino acid from at least one amino acid auxotrophy, - cultivating the amino acid auxotrophy cured prokaryotic cell comprising one or more nucleic acids encoding the polypeptide in a chemically defined minimal growth medium and recovering the polypeptide from the prokaryotic cell or the periplasm of the prokaryotic cell or from the medium.
- One aspect as reported herein is a method for producing a polypeptide in a (recombinant) prokaryotic cell, comprising the following step: - cultivating a (recombinant) prokaryotic cell comprising one or more nucleic acids encoding the polypeptide in a chemically defined minimal growth medium and recovering the polypeptide from the prokaryotic cell or the periplasm of the prokaryotic cell or from the medium, wherein the (recombinant) prokaryotic cell is an amino acid auxotrophy cured prokaryotic cell, wherein growth of the amino acid auxotrophy cured prokaryotic cell compared to the non-cured prokaryotic cell (parent cell) under the same cultivation conditions and in the same growth medium requires the supplementation of fewer amino acids to the growth medium, and wherein the chemically defined minimal growth medium is free of the amino acid corresponding to the auxotrophy that has been cured in the auxotrophy cured prokaryotic cell.
- amino acid auxotrophic prokaryotic cell is a prokaryotic cell that cannot synthesize an essential amino acid e.g. due to a mutation or deletion within a gene locus comprising the structural gene encoding the proteins of the corresponding biosynthetic pathway. Without the addition of the respective amino acid to the cultivation medium the cell cannot proliferate.
- the auxotrophy can be for any amino acid.
- the prokaryotic cell can also be auxotrophic for more than one amino acid.
- the amino acid auxotrophic prokaryotic cell is auxotrophic for at least two amino acids.
- the amino acid auxotrophic prokaryotic cell is auxotrophic for at least two, at least three, at least four, at least five amino acids.
- the amino acid auxotrophic prokaryotic cell is auxotrophic for at least 2 and up to 5, or up to 10, or up to 15 amino acids.
- the amino acid auxotrophic prokaryotic cell is auxotrophic for two to five amino acids, or two to three amino acids, or for two amino acids, or for three amino acids, or for four amino acids.
- the (amino acid) auxotrophy cured cell has at least one amino acid auxotrophy, or at least two amino acid auxotrophies, or at least three amino acid auxotrophies, or from one to three amino acid auxotrophies, or two amino acid auxotrophies.
- the amino acid auxotrophic prokaryotic cell is in one embodiment a bacterial cell.
- the cell is an Escherichia cell, or a Bacillus cell, or a Lactobacillus cell, or a Corynebacterium cell, or a Yeast cell (Saccharomyces,
- the cell is an Escherichia coli cell, or a Bacillus subtilis cell, or a Lactobacillus acidophilus cell, or a Corynebacterium glutamicum cell, or a Pichia pastoris yeast cell.
- Prokaryotic cells that can be used in the method as reported herein can comprise one or more amino acid auxotrophies.
- Leucine biosynthetic pathway can be selected from the LeuB6 deficient cells 13-6, ⁇ 148, ⁇ 156, ⁇ 2224, ⁇ 462, ⁇ 463, ⁇ 474, ⁇ 478, ⁇ 515, ⁇ 65, ⁇ 697, ⁇ 760, 2000k MSE248, 342-167, 342MG, 679-680, ⁇ 586, ⁇ 592, ⁇ 593, AA100, ⁇ 7852, ⁇ 787, AB1102, AB1111, AB1115, AB1122, AB1129, AB113, AB1132, AB1133, AB114, AB1157, AB1157-D, AB1314, AB1330, AB1331, AB1881,
- PA20SR, PA200 SR, PA201 SR, PA214SRT6R, PA265 SR, PA309, PDE70, PA340, PA340/T6, PA360, PA414, PAM161, PAM162, PAM163, PAM164, PAM660, PAT84, PB349, PB69, PCI, PC2, PC3, PC5, PC6, PC8, PJ1, PJ2, PJ3, PJ4, PJ5, PJ C600 ( CRSR), W208 SR AzR, W2660, LAM-, W945his, WA2127, WA2379, WA2548, WA2552, WA2574, WA2899, WA921, WA946, WA960,
- the prokaryotic cell is an E.coli K12 cell or an E.coli B cell.
- the term "growth” refers to the change in viable cell density in a cultivation. This change can be a change over time.
- the viable cell density can be determined by determining the optical density at 578 nm.
- the growth is the increase of viable cell density during a time period.
- the time period is the time period of 0 to 30 hours of the cultivation after inoculation.
- the term “same” denotes that a second value is within +/- 20 % of a first value.
- the term “same” refers to cultivation time, cultivation volume, cultivation temperature, seeding cell density, aeration rate, pH value, stirrer speed, feed rate (if applicable), etc.
- the same cultivation conditions are the identical cultivation conditions.
- the same cultivation medium is the identical cultivation medium.
- the cultivating can be with any method.
- the cultivating is a batch cultivating, a fed-batch cultivating, a perfusion cultivating, a semi-continuous cultivating, or a cultivating with full or partial cell retention.
- the cultivating is a high cell density cultivating.
- high cell density cultivating denotes a cultivating method wherein the dry cell weight of the cultivated prokaryotic cell is at one point in the cultivating at least 10 g/L. In one embodiment the dry cell weight is at one point in the cultivating at least 20 g/L, or at least 50 g/L, or at least 100 g/L, or more than 100 g/L. In order to reach such a high cell density state the volume of feed and/or adjustment solutions added during the cultivating has to be as small as possible. Methods for the determination of dry cell weight are reported e.g. in Riesenberg, D., et al., Appl. Microbiol. Biotechnol.
- an amino acid auxotrophy cured prokaryotic strain characterized in that - at least one deficiency in an essential amino acid metabolic pathway has been cured, the strain can grow in chemically defined minimal growth medium that is free of the amino acid corresponding to the auxotrophy that has been cured in the auxotrophy cured prokaryotic strain, and - the productivity of the auxotrophy cured strain is improved compared to the productivity of the parental non-auxotrophy cured strain as well as compared to corresponding wild-type strains (corresponding genotype).
- the auxotrophy cured prokaryotic strain is an auxotrophy cured E.coli strain.
- One aspect as reported herein is an amino acid auxotrophy cured prokaryotic cell, characterized in that at least one deficiency in an essential amino acid metabolic pathway has been cured, and the cell can grow in chemically defined minimal growth medium that is free of the amino acid corresponding to the auxotrophy that has been cured in the auxotrophy cured prokaryotic cell.
- the auxotrophy cured prokaryotic cell is an auxotrophy cured E.coli cell.
- auxotrophy cured prokaryotic strain/cell as reported herein can be used in a method for the recombinant production of a therapeutic polypeptide wherein compared to the non-auxotrophy cured strain/cell the supplementation of at least one amino acid residue is not required.
- One aspect as reported herein is a method for producing a polypeptide in a
- (recombinant) prokaryotic cell comprising the following step: cultivating a (recombinant) prokaryotic cell comprising one or more nucleic acids encoding the polypeptide in a chemically defined minimal growth medium and recovering the polypeptide from the prokaryotic cell or the periplasm of the prokaryotic cell or from the medium, wherein the (recombinant) prokaryotic cell is an amino acid auxotrophy cured prokaryotic cell.
- the growth of the amino acid auxotrophy cured prokaryotic cell compared to the non-cured prokaryotic cell (parent cell) under the same cultivation conditions and in the same growth medium requires the supplementation of fewer amino acids to the growth medium.
- the chemically defined minimal growth medium is free of the amino acid corresponding to the auxotrophy that has been cured in the auxotrophy cured prokaryotic cell.
- the cultivation of the auxotrophy cured prokaryotic cell requires supplementation of one or two or three or four amino acids less to the growth medium than required for the cultivation of the non-auxotrophy cured prokaryotic cell (parent cell).
- the auxotrophy cured prokaryotic cell has at least one further (non-cured) amino acid auxotrophy.
- the amino acid corresponding to the auxotrophy that has been cured in the auxotrophy cured prokaryotic cell is not added during the cultivation.
- the cultivation is in the absence of the amino acid corresponding to the auxotrophy that has been cured in the auxotrophy cured prokaryotic cell.
- the prokaryotic cell is an E.coli cell.
- polypeptide is a full length antibody chain, a single chain antibody, a single domain antibody, a scFv, a scFab, or a conjugate of one of the before with a non-antibody polypeptide.
- polypeptide is a conjugate of a scFv or scFab with a cytotoxic agent.
- the viable cell density at 30 hours after inoculation of the cultivation is higher in the cultivation of the auxotrophy cured strain than in the cultivation of the non-cured prokaryotic cell.
- One aspect as reported herein is the use of an amino acid auxotrophy cured prokaryotic strain for the (recombinant) production of a polypeptide.
- the amino acid auxotrophy cured prokaryotic strain at least one deficiency in an essential amino acid metabolic pathway has been cured, and the amino acid auxotrophy cured strain can grow in chemically defined minimal growth medium that is free of the amino acid corresponding to the auxotrophy that has been cured in the auxotrophy cured prokaryotic strain.
- the auxotrophy cured prokaryotic strain is an auxotrophy cured
- polypeptide is a full length antibody chain, a single chain antibody, a single domain antibody, a scFv, a scFab, or a conjugate of one of the before with a non-antibody polypeptide.
- polypeptide is a conjugate of a scFv or scFab with a cytotoxic agent.
- the method for producing a polypeptide in a prokaryotic cell comprises the following step: cultivating a prokaryotic cell comprising one or more nucleic acids encoding the polypeptide in a chemically defined minimal growth medium and recovering the polypeptide from the prokaryotic cell or the periplasm of the prokaryotic cell or from the medium, wherein the prokaryotic cell is an amino acid auxotrophy cured prokaryotic cell, wherein growth of the amino acid auxotrophy cured prokaryotic cell compared to the non-cured prokaryotic cell under the same cultivation conditions and in the same growth medium requires the supplementation of fewer amino acids to the growth medium, and wherein the chemically defined minimal growth medium is free of the amino acid corresponding to the auxotrophy that has been cured in the auxotrophy cured prokaryotic cell.
- An amino acid auxotrophy cured prokaryotic strain characterized in that at least one deficiency in an essential amino acid metabolic pathway has been cured, and the strain can grow in chemically defined minimal growth medium that is free of the amino acid corresponding to the auxotrophy that has been cured in the auxotrophy cured prokaryotic strain.
- the strain according to item 1 characterized in that the auxotrophy cured prokaryotic strain is an auxotrophy cured E.coli strain.
- An amino acid auxotrophy cured prokaryotic cell characterized in that at least one deficiency in an essential amino acid metabolic pathway has been cured, and the cell can grow in chemically defined minimal growth medium that is free of the amino acid corresponding to the auxotrophy that has been cured in the auxotrophy cured prokaryotic cell.
- the strain according to item 3 characterized in that the auxotrophy cured prokaryotic cell is an auxotrophy cured E.coli cell.
- a method for producing a polypeptide in a (recombinant) prokaryotic cell comprising the following step: cultivating a (recombinant) prokaryotic cell, which has been obtained by introducing into the genome of a parent prokaryotic cell a nucleic acid curing an amino acid auxotrophy of the parent prokaryotic cell, comprising one or more nucleic acids encoding the polypeptide in a chemically defined minimal growth medium and recovering the polypeptide from the prokaryotic cell or the periplasm of the prokaryotic cell or from the medium.
- a method for producing a polypeptide in a (recombinant) prokaryotic cell comprising the following steps: curing an prokaryotic cell that is auxotrophic for at least one amino acid from at least one amino acid auxotrophy, cultivating the amino acid auxotrophy cured (recombinant) prokaryotic cell comprising one or more nucleic acids encoding the polypeptide in a chemically defined minimal growth medium and recovering the polypeptide from the prokaryotic cell or the periplasm of the prokaryotic cell or from the medium.
- a method for producing a polypeptide in a (recombinant) prokaryotic cell comprising the following step: cultivating a (recombinant) prokaryotic cell comprising one or more nucleic acids encoding the polypeptide in a chemically defined minimal growth medium and recovering the polypeptide from the prokaryotic cell or the periplasm of the prokaryotic cell or from the medium, wherein the (recombinant) prokaryotic cell is an amino acid auxotrophy cured prokaryotic cell.
- the method according to any one of items 5 to 7, characterized in that the auxotrophy cured prokaryotic cell has at least one further (non-cured) amino acid auxotrophy.
- the method according to any one of items 5 to 10 characterized in that the cultivation of the auxotrophy cured prokaryotic cell requires supplementation of one or two or three or four amino acids less to the growth medium than required for the cultivation of the non-auxotrophy cured prokaryotic cell (parent/parent cell).
- the method according to any one of items 5 to 11 characterized in that the amino acid corresponding to the auxotrophy that has been cured in the auxotrophy cured prokaryotic cell is not added during the cultivation.
- the method according to any one of items 5 to 12 characterized in that the cultivation is in the absence of the amino acid corresponding to the auxotrophy that has been cured in the auxotrophy cured prokaryotic cell.
- SEQ ID NO: 01 Human interferon derived sequence.
- SEQ ID NO: 02 Hexa-histidine affinity tag.
- SEQ ID NO: 03 IgA protease cleavage site.
- SEQ ID NO: 04 Tetranectin apolipoprotein A-I fusion polypeptide amino acid sequence.
- SEQ ID NO: 05 N-terminal extended Tetranectin apolipoprotein A-I fusion polypeptide amino acid sequence obtained after IgA protease cleavage.
- SEQ ID NO: 06 Tetranectin apolipoprotein A-I fusion polypeptide amino acid sequence without affinity tag and IgA protease cleavage (N-terminal shortened fusion polypeptide).
- FIG. 1 Trend plot of the L-leucine concentration (CSPZ-2: diamonds;
- wild-type strain 66C5 squares, normal temperature
- wild-type strain 66C5 at elevated temperature triangles
- wild-type strain 66C5 squares, normal temperature
- wild-type strain 66C5 at elevated temperature triangles
- E.coli K12 strain CSPZ-2 (leuB, proC, trpE, thi-1, ApyrF) was cured from its genetic defects in the genes leuB, proC, proBA and trpE. This was done by using a method for directed genetic engineering of E.coli chromosomal genes (Gene Bridges, Heidelberg, Germany; http://www.genebridges.com; see e.g. WO 99/29837, WO 01/04288, US 6,355,412). In detail the following stepwise changes were made :
- the E.coli CSPZ-2 has a TCG to TTG point mutation in the locus leuB which results in a Serine to Leucine amino acid exchange at Position 286 in the corresponding 3-isopropylmalate dehydrogenase making this enzyme defective.
- the strain is therefore not able to grow on cultivation medium not supplemented with L-Leucine.
- With an oligonucleotide encoding the wild-type Ser286 with flanking sequences the genomic mutation in strain CSPZ-2 was exchanged to generate the cured strain CSPZ-3 (CSPZ-2 leuB + ) which can grow on M9 medium plates supplemented with L-Proline but does not require L-Leucine. Repair of proC (b0386) and proBA (b0242 and b0243) loci:
- the strain CSPZ-3 still requires the supplementation of Proline to grow on M9 minimal medium.
- a PCR-amplification of the proC locus resulted in an approx. 2.4 kb fragment.
- E.coli K12 wild-type strain
- a 1,024 bp fragment was expected (Blattner, F.R., et al, Science 277 (1997) 1453-1474).
- Sequencing of the PCR product revealed the disruption of the proC locus by IS 186 (1,335 bp) transposition into the genome.
- the proBA locus was characterized by PCR, but the amplification failed indicating the absence of the proBA operon.
- a PCR product amplified from E.coli strain MG1655 genomic DNA was used for Red/ET recombination to cure the deficiency in the proC locus.
- the proBA operon was inserted via Red/ET recombination into the infA-serW intergenic region of the genome to restore prototrophic phenotype in CSPZ-4.
- the integration was verified by PCR amplifying and sequencing of the integration site and by plating on M9 minimal medium agar plates.
- the Proline prototrophic, auxotrophy cured strain was named CSPZ-5. Repair of the trpE (M264) locus:
- DNA sequencing of the trpE locus of the parental strain CSPZ-2 revealed the deletion of 9 nucleotides resulting in the loss of Glul39, Glu 140 and Argl41. This mutation does not lead to a Tryptophan auxotrophic phenotype but may affect enzyme activity and lead to reduced growth. Therefor the mutated trpE locus was exchanged by the wild-type locus via Red/ET recombination. Successful restoration of the trpE gene was confirmed by sequencing and the resulting strain was named CSPZ-6.
- the tetranectin-apolipoprotein A-I fusion polypeptide was prepared by recombinant means.
- the amino acid sequence of the expressed fusion polypeptide in N- to C-terminal direction is as follows:
- CDLPQTHSL SEQ ID NO: 01
- HHHHHH SEQ ID NO: 02
- the tetranectin-apolipoprotein A-I fusion polypeptides as described above are precursor polypeptides from which the tetranectin-apolipoprotein A-I fusion polypeptides was released by enzymatic cleavage in vitro using IgA protease.
- the precursor polypeptide encoding fusion gene was assembled with known recombinant methods and techniques by connection of appropriate nucleic acid segments. Nucleic acid sequences made by chemical synthesis were verified by DNA sequencing. The expression plasmid for the production of tetranectin-apolipoprotein A-I was prepared as follows.
- Plasmid 4980 (4980-pBRori-URA3-LACI-SAC) is an expression plasmid for the expression of core-streptavidin in E.coli. It was generated by ligation of the 3142 bp long EcoRI/Celll-vector fragment derived from plasmid 1966 (1966- pBRori-URA3-LACI-T-repeat; reported in EP-B 1 422 237) with a 435 bp long core-streptavidin encoding EcoRI/Celll-fragment.
- the core-streptavidin E.coli expression plasmid comprises the following elements: the origin of replication from vector pBR322 for replication in E.coli (corresponding to bp position 2517-3160 according to Sutcliffe, G., et al,
- the core-streptavidin expression cassette comprising
- T5 hybrid promoter T5-PN25/03/04 hybrid promoter according to Bujard, H., et al, Methods Enzymol. 155 (1987) 416-433 and Stueber, D., et al, Immunol. Methods IV (1990) 121-152
- T5 hybrid promoter T5-PN25/03/04 hybrid promoter according to Bujard, H., et al, Methods Enzymol. 155 (1987) 416-433 and Stueber, D., et al, Immunol. Methods IV (1990) 121-152
- the core-streptavidin gene two bacteriophage-derived transcription terminators
- the ⁇ - ⁇ terminator Rosarz, E., et al, Nature 272 (1978) 410-41
- the fd- terminator Beck E. and Zink, B. Gene 1-3 (1981) 35-58
- the lacl repressor gene from E.coli Ferabaugh P.J. Nature 274 (1978) 7
- the final expression plasmid 5816 for the expression of the tetranectin- apolipoprotein A-I precursor polypeptide was prepared by excising the core- streptavidin structural gene from plasmid 4980 using the singular flanking EcoRI and Celll restriction endonuclease cleavage site and inserting the EcoRI/Celll restriction site flanked nucleic acid encoding the precursor polypeptide into the 3142 bp long EcoRI/CelII-4980 vector fragment.
- Plasmid 5830 is identical to plasmid 5816 except that the codons encoding the tripeptide QKK are changed from caa aaa aag (plasmid 5816) to cag aag aag (plasmid 5830).
- Plasmid 5836 is identical to plasmid 5816 except that the codons encoding the tripeptide QKK are changed from caa aaa aag (plasmid 5816) to cag aag aag (plasmid 5830).
- the encoding fusion gene for expression of a shortened tetranectin-apolipoprotein A-I fusion protein is assembled with known recombinant methods and techniques by connection of appropriate nucleic acid segments. Nucleic acid sequences made by chemical synthesis are verified by DNA sequencing.
- the expression plasmid for the production of the fusion protein of SEQ ID NO: 06 can be prepared as follows:
- Plasmid 4980 (4980-pBRori-URA3-LACI-SAC) is an expression plasmid for the expression of core-streptavidin in E.coli. It was generated by ligation of the 3142 bp long EcoRI/Celll -vector fragment derived from plasmid 1966 (1966-pBRori- URA3-LACI-T-repeat; reported in EP-B 1 422 237) with a 435 bp long core- streptavidin encoding EcoRI/Celll-fragment.
- the core-streptavidin E.coli expression plasmid comprises the following elements: the origin of replication from vector pBR322 for replication in E.coli (corresponding to bp position 2517-3160 according to Sutcliffe, G., et al, Quant. Biol. 43 (1979) 77-90),
- the core-streptavidin expression cassette comprising
- T5 hybrid promoter T5-PN25/03/04 hybrid promoter according to Bujard, H., et al, Methods. Enzymol. 155 (1987) 416-433 and Stueber,
- the final expression plasmid 5836 for the expression of the shortened tetranectin- apolipoprotein A-I fusion protein can be prepared by excising the core-streptavidin structural gene from plasmid 4980 using the singular flanking EcoRI and Celll restriction endonuclease cleavage site and inserting the EcoRI/Celll restriction site flanked nucleic acid encoding the fusion protein into the 3142 bp long EcoRI/CelII-1 plasmid fragment. d) Plasmid 3036
- Plasmid 3036 for recombinant production of IgA-Protease is based on the vector OripBR-URA3 -EK-IFN (see e.g. US 6,291,245). Plasmid 3036 differs from OripBR-URA3 -EK-IFN by the presence of a lacl repressor gene and the target gene encoding the IgA protease protein.
- the lacl repressor gene was derived from plasmid pUHAl (Stueber, D., et al, In: Immunological Methods IV
- the IgA-Protease expression plasmid 3036 comprises the following elements: the origin of replication from vector pBR322 for replication in E.coli (corresponding to bp position 2517-3160 according to Sutcliffe, G., et al, Quant. Biol. 43 (1979) 77-90), the URA3 gene of Saccharomyces cerevisiae coding for orotidine 5'- phosphate decarboxylase (Rose, M., et al, Gene 29 (1984) 113-124) which allows plasmid selection by complementation of E.coli ApyrF mutant strains (uracil auxotrophy),
- T5 hybrid promoter T5-PN25/03/04 hybrid promoter according to Bujard, H., et al, Methods. Enzymol. 155 (1987) 416-433 and Stueber, D., et al, Immunol. Methods IV (1990) 121-152
- T5 hybrid promoter T5-PN25/03/04 hybrid promoter according to Bujard, H., et al, Methods. Enzymol. 155 (1987) 416-433 and Stueber, D., et al, Immunol. Methods IV (1990) 121-152
- a synthetic ribosomal binding site according to Stueber, D., et al. (see before), - Open-reading-frame of IgA Protease (Neisseria gonorrhoeae, GenBank
- ApyrF ApyrF
- the transformed E.coli cells were first grown at 37 °C on agar plates. A colony picked from this plate was transferred to a 3 mL roller culture and grown at 37 °C to an optical density of 1-2 (measured at 578 nm). Then 1000 culture were mixed with 1000 sterile 86%-glycerol and immediately frozen at -80 °C for long time storage.
- CDM chemical defined medium
- the trace elements solution contains MnS04*H20 1.28 g/1, ZnS04*7H20 1.70 g/L, H3B03 0.30 g/L, (NH4)6Mo7024*4H20 0.18 g/L, CoC12*6H20 0.25 g/L, CuS04*5H20 0.22 g/L, EDTA 0.75 g/L.
- the trace elements solution contains FeS04*7H20 10 g/L, ZnS04*7H20 2.25 g/L, MnS04*H20 2.13 g/L, CuS04*5H20 1.0 g/L, CoC12*6H20 0.42 g/L, (NH4)6Mo7024*4H20 0.3 g/L, H3B03 0.50 g/L, thiamine-HCL 2.0 g/L solubilized in IN HC1 solution.
- the feed 1 solution contained 700 g/L glucose*H20, KH2P04 8.43 g/L and K2HP04*3H20 22.38 g/L.
- Feed 2 comprises 585 g/L L-proline and 150.3 g/L MgS04*7H20.
- the alkaline solution for pH regulation was an aqueous 12.5 % (w/v) NH3 solution supplemented with 11.25 g/L L-methionine, 51.5 g/L L-proline and 56.25 g/L L- leucine (see WO 2012/028522).
- the rate of feed 2 is kept constant, while the rate of feed 1 is increased stepwise with a predefined feeding profile from 50 to finally 160 g/h within 8.5 hours.
- carbon dioxide off gas concentration leveled above 2% the aeration rate was constantly increased from 10 to 20 L/min within 5 hours.
- the expression of recombinant tetranectin-apolipoprotein A-l fusion protein was induced by the addition of 2.4 g IPTG at an optical density of 150.
- the electrophoresis was run for 60 min. at 200 V and thereafter the gel was transferred to a GelDOC EZ Imager (Bio-Rad) and processed for 5 min. with UV radiation. Gel images were analyzed using Image Lab analysis software (Bio-Rad).
- the activity of the purified and lipidated TN-ApoAl was measured in an eight day radioactive efflux assay.
- Cells (THP-1) were differentiated by PMA (phorbol myristate acetate) to macrophages. These cells were loaded with acetylated LDL and 3 H-labeled cholesterol. The supernatant was discarded and cells were incubated for 5 hours with equilibration medium to remove non-specifically bound cholesterol.
- the lipidated TN-ApoAl was added which enabled the export of the 3 H-labeled cholesterol out of the cells during the following 18 hours. Radioactivity was measured in the supernatant and in the cell lysate.
- the above mentioned fermentation process was used to express tetranectin- apolipoprotein A-l in the parental strain CSPZ-2 which is auxotrophic against L-leucine and L-proline and in the auxotrophy cured strain CSPZ-6. Despite the latter strain does not need the feeding of the amino acids L-leucine and L-proline any more, the performance of this auxotrophy cured strain was tested with the supplemented fermentation medium for direct performance comparison to strain CSPZ-2.
- the auxotrophy cured strain is growing faster than the parental strain despite the same medium and fermentation process was used (see Figure 1).
- the auxotrophy cured strain is faster expressing more product than the parental strain despite the same medium and fermentation process was used (see Figure 2).
- the auxotrophy cured strain can now metabolize L-leucine and L-proline due to intact metabolic pathways. Therefore the growth of the auxotrophy cured strain is faster under glucose limited cultivation conditions of the applied fed-batch process.
- the auxotrophy cured strain had a significant better performance than the parental strain on the same chemical defined fermentation medium supplemented with the amino acids L-leucine and L-proline. The growth is accelerated due to the possibility to metabolize the contained amino acids and this may also improve product formation. The conclusion from this is to use a prototroph E.coli production strain.
- the E.coli K12 strains CSPZ-2 (leuB, proC, trpE, thi-1, ApyrF) and CSPZ-6 (thi-1, ApyrF) were transformed by electroporation with the expression plasmid 3036.
- the transformed E.coli cells were first grown at 37 °C on agar plates. A colony picked from this plate was transferred to a 3 mL roller culture and grown at 37 °C to an optical density of 1-2 (measured at 578 nm). Then 1000 culture were mixed with 1000 sterile 86%-glycerol and immediately frozen at -80 °C for long time storage. The correct product expression of this clone was first verified in small scale shake flask experiments and analyzed with SDS-Page prior to the transfer to the 10 L fermenter. Pre-cultivation:
- CDM chemical defined medium
- the trace elements solution contains FeS04*7H20 10.0 g/L, ZnS04 * 7H20 2.25 g/L, MnS04 * H20 2.13 g/L, H3B03 0.50 g/L, (NH4)6Mo7024 * 4H20 0.3 g/L, CoC12*6H20 0.42 g/L, CuS04 * 5H20 1.0 g/L dissolved in 0.5M HC1.
- the electrophoresis was run for 60 min. at 200 V and thereafter the gel was transferred the GelDOC EZ Imager (Bio-Rad) and processed for 5 min. with UV radiation. Gel images were analyzed using Image Lab analysis software (Bio-Rad).
- auxotrophy cured strain has significant advantages over a wild-type E.coli strain which is also prototrophic growth and product formation of the K12 strain MG1655 (with deleted pyrF gene to fit to the antibiotic free selection system) within the same fermentation process was explored.
- the E.coli K12 strains CSPZ-6 (thi-1, ApyrF) and MG1655 derivative (ApyrF, AompT) were transformed by electroporation with the expression plasmid 5816
- the transformed E.coli cells were first grown at 37 °C on agar plates. A colony picked from this plate was transferred to a 3 mL roller culture and grown at 37 °C to an optical density of 1-2 (measured at 578nm). Then 1000 culture were mixed with 1000 sterile 86%-glycerol and immediately frozen at - 80 °C for long time storage.
- CDM chemical defined medium
- the trace elements solution contains MnS04*H20 1.28 g/L, ZnS04*7H20 1.70 g/L, H3B03 0.30 g/L, (NH4)6Mo7024*4H20 0.18 g/L, CoC12*6H20 0.25 g/L, CuS04*5H20 0.22 g/L, EDTA 0.75 g/L.
- the trace elements solution contains FeS04*7H20 10 g/L, ZnS04*7H20 2.25 g/L, MnS04*H20 2.13 g/L, CuS04*5H20 1.0 g/L, CoC12*6H20 0.42 g/L, (NH4)6Mo7024*4H20 0.3 g/L, H3B03 0.50 g/L, thiamine-HCl 2.0 g/L solubilized in IN HC1 solution.
- the feed 1 solution contained 700 g/L glucose*H20, KH2P04 8.43 g/L and K2HP04*3H20 22.38 g/L.
- Feed 2 comprises only 150.3 g/L MgS04*7H20.
- the alkaline solution for pH regulation was an aqueous 12.5 % (w/v) NH3 solution supplemented with 11.25 g/L L-methionine.
- the rate of feed 2 is kept constant, while the rate of feed 1 is increased stepwise with a predefined feeding profile from 50 to finally 160 g/h within 8.5 hours.
- carbon dioxide off gas concentration leveled above 2% the aeration rate was constantly increased from 10 to 20 L/min within 5 hours.
- the expression of recombinant tetranectin-apolipoprotein A-l fusion protein was induced by the addition of 2.4 g IPTG at an optical density of 150.
- the within the cytoplasm soluble expressed tetranectin-apolipoprotein A-I is transferred to insoluble protein aggregates, the so called inclusion bodies, with a heat step where the whole culture broth in the fermenter is heated to 50 °C for one hour before harvest (see e.g. EP-B 1 486 571). Thereafter, the content of the fermenter was centrifuged with a flow-through centrifuge (13,000 rpm, 13 1/h) and the harvested biomass was stored at -20 °C until further processing.
- the synthesized tetranectin-apolipoprotein A-I fusion proteins were found exclusively in the insoluble cell debris fraction in the form of insoluble protein aggregates, so-called inclusion bodies (IBs).
- the activity of the purified and lipidated TN-ApoAl was measured in an eight day radioactive efflux assay.
- Cells (THP-1) were differentiated by PMA (phorbol myristate acetate) to macrophages. These cells were loaded with acetylated LDL and 3 H-labeled cholesterol. The supernatant was discarded and cells were incubated for 5 hours with equilibration medium to remove non-specifically bound cholesterol. The lipidated TN-ApoAl was added which enabled the export of the 3 H-labeled cholesterol out of the cells during the following 18 hours. Radioactivity was measured in the supernatant and in the cell lysate. Results:
- the above mentioned fermentation process was used to express tetranectin- apolipoprotein A-I in the auxotrophy cured strain CSPZ-6 and the prototrophic MG1655 derivative strain representing the K12 wild-type strain MG1655. Both strains do not need the feeding of the amino acids L-leucine and L-proline which where consequently excluded from the medium and feeds. This distinctly reduces cost of goods for the process.
- Both, the auxotrophy cured and the wild-type strain are growing faster on medium without the amino acids L-leucine and L-proline due to the lack of growth inhibitory effects of higher concentrations of L-leucine (see Example 3).
- Final optical density of the auxotrophy cured strain is well improved in this medium lacking the amino acids, while the growth of the wild-type strain is significant reduced after induction of product formation and in the late phase of fermentation (see Figure 4).
- the prototroph wild-type E.coli MG1655 derivative strain has significant deficiencies in yields of growth and product.
- the auxotrophy cured strain showed a better performance than a prototroph wild-type E.coli strain on the same chemical defined fermentation medium even if the amino acids L-leucine and L-proline are not supplemented. Therefore it is advantageous to cure high producing E.coli strains from their amino acid auxotrophies in order to allow a cultivation on chemically defined minimal growth medium and profit from the advantages of using such a chemically defined minimal growth medium instead of using a prototrophic wild-type E.coli strain like MG1655 or the closely related W3110 (Vijayendran, C, et al, J. Biotechnol. 128 (2007) 747-761) as can further be seen in Example 6.
- the auxotrophy cured strain CSPZ-6 has significant advantages over the prototrophic wild-type E.coli strain W3110 (with deleted pyrF gene to fit to the antibiotic free selection system) growth and product formation within the same fermentation process was explored.
- the transformed E.coli cells were first grown at 37 °C on agar plates. A colony picked from this plate was transferred to a 3 mL roller culture and grown at 37 °C to an optical density of 1-2 (measured at 578nm). Then 1000 culture were mixed with 1000 mL sterile 86%-glycerol and immediately frozen at -80 °C for long time storage.
- CDM chemical defined medium
- the trace elements solution contains FeS04*7H20 10.0 g/L, ZnS04*7H20 2.25 g/L, MnS04*H20 2.13 g/L, H3B03 0.50 g/L, (NH4)6Mo7024*4H20 0.3 g/L, CoC 12*6H20 0.42 g/L, CuS04*5H20 1.0 g/L dissolved in 0.5 M HC1.
- the trace elements solution contains FeS04*7H20 10 g/L, ZnS04*7H20 2.25 g/L, MnS04*H20 2.13 g/L, CuS04*5H20 1.0 g/L, CoC12*6H20 0.42 g/L,
- Feed 2 comprisesKH2P04 52.7 g/L, K2HP04*3H20 139.9 g/L and (NH4)2HP04 132.1 g/L. All components were dissolved in deionized water.
- the alkaline solution for pH regulation was an aqueous 12.5 % (w/v) NH3 solution supplemented with 11.25 g/L L-methionine.
- the rate of feed 2 is kept constant, while the rate of feed 1 is increased stepwise with a predefined feeding profile from 60 to finally 160 g/h within 7 hours.
- carbon dioxide off gas concentration leveled above 2 % the aeration rate was constantly increased from 10 to 20 L/min within 5 hours.
- the expression of recombinant tetranectin-apolipoprotein A-I fusion protein was induced by the addition of 2.4 g IPTG at an optical density of 120.
- the within the cytoplasm soluble expressed tetranectin-apolipoprotein A-I is transferred to insoluble protein aggregates, the so called inclusion bodies, with a heat step where the whole culture broth in the fermenter is heated to 50 °C for 1 hour before harvest (see e.g. EP-B 1 486 571). Thereafter, the content of the fermenter was centrifuged with a flow-through centrifuge (13,000 rpm, 13 L/h) and the harvested biomass was stored at -20 °C until further processing.
- the synthesized tetranectin- apolipoprotein A-I fusion proteins were found exclusively in the insoluble cell debris fraction in the form of insoluble protein aggregates, so-called inclusion bodies (IBs).
- the electrophoresis was run for 60 min. at 200 V and thereafter the gel was transferred the GelDOC EZ Imager (Bio-Rad) and processed for 5 min. with UV radiation. Gel images were analyzed using Image Lab analysis software (Bio-Rad). With the three standards a linear regression curve was calculated with a coefficient of >0.99 and thereof the concentrations of target protein in the original sample was calculated.
- the activity of the purified and lipidated TN-ApoAl was measured in an eight day radioactive efflux assay.
- Cells (THP-1) were differentiated by PMA (phorbol myristate acetate) to macrophages. These cells were loaded with acetylated LDL and 3 H-labeled cholesterol. The supernatant was discarded and cells were incubated for 5 hours with equilibration medium to remove non-specifically bound cholesterol.
- the lipidated TN-ApoAl was added which enabled the export of the 3 H-labeled cholesterol out of the cells during the following 18 hours. Radioactivity was measured in the supernatant and in the cell lysate.
- the fermentation with the wild-type strain 66C5 was repeated with elevated temperatures for batch phase (37°C) and fed-batch phase (30°C), but product yield could only be improved to 6.9 g/L (see Figure 9).
- the prototroph wild-type E.coli strain has significant deficiencies in direct comparison with the auxotrophy cured strain.
- the auxotrophy cured strain CSPZ-6 had a much better performance than a prototroph wild-type E.coli strain W3110 derivate on the same chemical defined fermentation medium without the supplementation of other amino acids than methionine. Therefore it is advantageous to cure E.coli strains from their amino acids auxotrophies in order to use a chemically defined minimal medium for cultivation and to profit from the advantages of using such a medium instead of using a prototrophic wild-type E.coli strain, such as for example MG1655 or W3110.
- auxotrophy cured strain CSPZ-6 has significant advantages over the prototrophic wild-type E.coli B strain BL21 (with deleted pyrF gene to fit to the antibiotic free selection system, named CSPZ-14) the growth and product formation within the same fermentation process has been explored.
- the transformed E.coli cells were first grown at 37 °C on agar plates. A colony picked from this plate was transferred to a 3 mL roller culture and grown at 37 °C to an optical density of 1-2 (measured at 578 nm). Then 1000 culture were mixed with 1000 sterile 86%-glycerol and immediately frozen at - 80 °C for long time storage. The correct product expression of this clone was first verified in small scale shake flask experiments and analyzed with SDS-Page prior to the transfer to the 10 L fermenter.
- CDM chemical defined medium
- CDM chemical defined medium
- the trace elements solution contains FeS04*7H20 10.0 g/L, ZnS04 * 7H20 2.25 g/L, MnS04 * H20 2.13 g/L, H3B03 0.50 g/L, (NH4)6Mo7024 * 4H20 0.3 g/L, CoC12*6H20 0.42 g/L, CuS04 * 5H20 1.0 g/L dissolved in 0.5M HC1.
- KH 2 PO 4 1.58 g/L, (NH 4 ) 2 HP0 4 7.47 g/L, K2HP04*3H20 13.32 g/L, citrate 2.07 g/L, L-methionine 1.22 g/L, NaHC0 3 0.82 g/L, trace elements solution 7.3 mL/L, MgS0 4 *7H 2 0 0.99 g/L, thiamine*HCl 20.9 mg/L, glucose*H 2 0 29.3 g/L, biotin
- the trace elements solution contains FeS04*7H20 10 g/L, ZnS04*7H20 2.25 g/L, MnS04*H20 2.13 g/L, CuS04*5H20 1.0 g/L, CoC12*6H20 0.42 g/L, (NH4)6Mo7024*4H20 0.3 g/L, H3B03 0.50 g/L solubilized in 0.5M HC1 solution.
- the feed 1 solution contained 700 g/L glucose*H20, 7.4 g/L MgS0 4 *7H 2 0 and 0.1 g/L FeS04*7H20.
- Feed 2 comprises KH 2 P0 4 52.7 g/L, K2HP04*3H20 139.9 g/L and (NH4)2HP04 66.0 g/L. All components were dissolved in deionized water.
- the alkaline solution for pH regulation was an aqueous 12.5 % (w/v) NH 3 solution supplemented with 11.25 g/L L-methionine.
- the rate of feed 2 is kept constant, while the rate of feed 1 is increased stepwise with a predefined feeding profile from 60 to finally 160 g/h within 7 hours.
- carbon dioxide off gas concentration leveled above 2 % the aeration rate was constantly increased from 10 to 20 L/min within 5 hours.
- the expression of recombinant tetranectin-apolipoprotein A-I fusion protein was induced by the addition of 2.4 g IPTG at an optical density of approx. 150.
- the within the cytoplasm soluble expressed tetranectin-apolipoprotein A-I is transferred to insoluble protein aggregates, the so called inclusion bodies, with a heat step where the whole culture broth in the fermenter is heated to 50 °C for 1 hour before harvest (see e.g. EP-B 1 486 571). Thereafter, the content of the fermenter was centrifuged with a flow-through centrifuge (13,000 rpm, 13 L/h) and the harvested biomass was stored at -20 °C until further processing.
- the synthesized tetranectin- apolipoprotein A-I fusion proteins were found exclusively in the insoluble cell debris fraction in the form of insoluble protein aggregates, so-called inclusion bodies (IBs). Analysis of product formation:
- the electrophoresis was run for 60 min. at 200 V and thereafter the gel was transferred the GelDOC EZ Imager (Bio-Rad) and processed for 5 min. with UV radiation. Gel images were analyzed using Image Lab analysis software (Bio-Rad). With the three standards a linear regression curve was calculated with a coefficient of >0.99 and thereof the concentrations of target protein in the original sample was calculated.
- the activity of the purified and lipidated shortened TN-ApoAl was measured in an eight day radioactive efflux assay.
- Cells (THP-1) were differentiated by PMA (phorbol myristate acetate) to macrophages. These cells were loaded with acetylated LDL and 3 H-labeled cholesterol. The supernatant was discarded and cells were incubated for 5 hours with equilibration medium to remove non- specifically bound cholesterol.
- the lipidated shortened TN-ApoAl was added which enabled the export of the 3 H-labeled cholesterol out of the cells during the following 18 hours. Radioactivity was measured in the supernatant and in the cell lysate.
- the above mentioned fermentation process was used to express a shortened tetranectin-apolipoprotein A-I fusion protein in the auxotrophy cured strain CSPZ-6 and in the prototrophic strain CSPZ-14 representing the E.coli B wild-type strain BL21. Both strains do not need the feeding of the amino acids L-leucine and L-proline which where consequently excluded from the medium and feeds. This reduces cost of goods for the process.
- the wild-type strain and BL21 derivate CSPZ-14 despite being prototrophic and the fermentation was inoculated with an equal amount of cells, grew significantly slower from the start when compared to the auxotrophy cured strain CSPZ-6 on the same chemically defined medium and under the same cultivation conditions (see Figure 10).
- the glucose feeding started 1.2 hours later, but the increase of the feed rate followed the same profile as in the comparative fermentation.
- the final optical densities differ greatly between both strains. While the auxotrophy cured strain reached an optical density of 266 before the final heat step the optical density of the wild-type strain was decreasing form a maximum at hour 33 to only 143 at the end of the fermentation (hour 47).
- the auxotrophy cured strain CSPZ-6 had a much better performance than a prototroph wild-type E.coli strain CSPZ-14 (BL21 ApyrF derivate) on the same chemical defined fermentation medium without the supplementation of other amino acids than methionine. Therefore it is advantageous to cure E.coli strains from their amino acids auxotrophies in order to culture them on chemically defined minimal medium and profit from the advantages of using such a medium instead of using a prototrophic wild-type E.coli strain, such as for example MG1655, W3110 or BL21.
- CSPZ-6 has significant advantages over the prototrophic wild-type E.coli K12 strain MG1655 (with deleted pyrF gene to fit to the antibiotic free selection system, named CSPZ-9) growth and IgA- protease product formation within the same fermentation process between the two strains has been explored.
- the transformed E.coli cells were first grown at 37 °C on agar plates. A colony picked from this plate was transferred to a 3 mL roller culture and grown at 37 °C to an optical density of 1-2 (measured at 578 nm). Then 1000 culture were mixed with 1000 sterile 86%-glycerol and immediately frozen at -80 °C for long time storage. The correct product expression of this clone was first verified in small scale shake flask experiments and analyzed with SDS-Page prior to the transfer to the 10 L fermenter. Pre-cultivation:
- CDM chemical defined medium
- the trace elements solution contains FeS04*7H20 10.0 g/L, ZnS04 * 7H20 2.25 g/L, MnS04 * H20 2.13 g/L, H3B03 0.50 g/L, (NH4)6Mo7024 * 4H20 0.3 g/L, CoC12*6H20 0.42 g/L, CuS04 * 5H20 1.0 g/L dissolved in 0.5M HC1.
- the trace elements solution contains FeS04*7H20 10 g/L, ZnS04*7H20 2.25 g/L, MnS04*H20 2.13 g/L, CuS04*5H20 1.0 g/L, CoC12*6H20 0.42 g/L,
- Feed 2 comprises KH 2 P0 4 52.7 g/L, K2HP04*3H20 139.9 g/L and (NH4)2HP04 66.0 g/L. All components were dissolved in deionized water.
- the alkaline solution for pH regulation was an aqueous 12.5 % (w/v) NH 3 solution supplemented with 11.25 g/L L-methionine.
- the expression of recombinant IgA-protease was induced by the addition of 2.4 g IPTG at an optical density of approx. 150.
- the fermentation is conducted up to 48 hours despite there is no significant decrease in optical density.
- the content of the fermenter was centrifuged the next day with a flow-through centrifuge (13,000 rpm, 13 L/h) and the harvested biomass was stored at -20 °C until further processing.
- the synthesized IgA-protease is found exclusively in the insoluble cell debris fraction in the form of insoluble protein aggregates the so-called inclusion bodies (IBs).
- the electrophoresis was run for 60 min. at 200 V and thereafter the gel was transferred the GelDOC EZ Imager (Bio-Rad) and processed for 5 min. with UV radiation. Gel images were analyzed using Image Lab analysis software (Bio-Rad). With the three standards a linear regression curve was calculated with a coefficient of >0.99 and thereof the concentrations of target protein in the original sample was calculated.
- the activity of the refolded and purified IgA-protease was tested with the molecule tetranectin-apolipoprotein A-I fusion polypeptides as described above which is a precursor polypeptide from which the tetranectin-apolipoprotein A-I fusion polypeptides was released by enzymatic cleavage in vitro using this IgA protease. Cleavage activity was in the expected range.
- strain CSPZ-9 had significantly slowed down after 30 hours of cultivation and as a consequence of the continuous feeding of feed 1 the glucose concentration in the medium increased to 16 g/L. Therefore the fermentation was terminated after 37 hours of cultivation.
- Metabolite analysis revealed a significant increase in concentrations of ammonia, glutamate, iron, magnesium and acetate in the medium due to the significant decrease in growth rate.
- the final optical densities differ greatly between both strains. While the cured strain reached 362 the optical density of the wild-type strain was decreasing form a maximum at hour 24 of 256 to only 177 at the end of the fermentation (hour 37) (see Figure 12).
- the auxotrophy cured strain CSPZ-6 showed improved growth characteristics compared to the prototroph wild-type E.coli strain CSPZ-9 (MG1655 ApyrF derivate) on the same chemical defined fermentation medium without the supplementation of other amino acids. Therefore it is useful to cure highly productive E.coli strains from its amino acids auxotrophies in order to be able to culture them on chemically defined minimal medium and profit from the advantages of using such a medium and instead of using a prototrophic wild-type
- E.coli strain derivate from MG1655, W3110 or BL21 E.coli strain derivate from MG1655, W3110 or BL21.
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Priority Applications (9)
Application Number | Priority Date | Filing Date | Title |
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EP14706005.7A EP2959006B1 (en) | 2013-02-22 | 2014-02-19 | Use of an amino acid auxotrophy cured prokaryotic strain for the recombinant production of a polypeptide |
CN201480009903.4A CN105026575A (en) | 2013-02-22 | 2014-02-19 | Use of an amino acid auxotrophy cured prokaryotic strain for the recombinant production of a polypeptide |
US14/769,594 US10155970B2 (en) | 2013-02-22 | 2014-02-19 | Use of an amino acid auxotrophy cured prokaryotic strain for the recombinant production of a polypeptide |
PL14706005T PL2959006T3 (en) | 2013-02-22 | 2014-02-19 | Use of an amino acid auxotrophy cured prokaryotic strain for the recombinant production of a polypeptide |
SG11201505916XA SG11201505916XA (en) | 2013-02-22 | 2014-02-19 | Use of an amino acid auxotrophy cured prokaryotic strain for the recombinant production of a polypeptide |
JP2015558428A JP6412509B2 (en) | 2013-02-22 | 2014-02-19 | Use of prokaryotic strains that eliminate amino acid auxotrophy for recombinant production of polypeptides |
ES14706005.7T ES2688044T3 (en) | 2013-02-22 | 2014-02-19 | Use of a prokaryotic strain with suppression of auxotrophies for amino acids for recombinant production of a polypeptide |
SI201430869T SI2959006T1 (en) | 2013-02-22 | 2014-02-19 | Use of an amino acid auxotrophy cured prokaryotic strain for the recombinant production of a polypeptide |
HRP20181414TT HRP20181414T1 (en) | 2013-02-22 | 2018-09-03 | Use of an amino acid auxotrophy cured prokaryotic strain for the recombinant production of a polypeptide |
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US10155970B2 (en) | 2018-12-18 |
CN110541018A (en) | 2019-12-06 |
HRP20181414T1 (en) | 2018-10-19 |
SG11201505916XA (en) | 2015-09-29 |
JP6412509B2 (en) | 2018-10-24 |
EP2959006B1 (en) | 2018-07-18 |
TWI628281B (en) | 2018-07-01 |
CN105026575A (en) | 2015-11-04 |
SI2959006T1 (en) | 2018-10-30 |
TW201444975A (en) | 2014-12-01 |
AR094716A1 (en) | 2015-08-19 |
US20150376674A1 (en) | 2015-12-31 |
JP2016507243A (en) | 2016-03-10 |
PL2959006T3 (en) | 2018-11-30 |
EP2959006A1 (en) | 2015-12-30 |
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