WO2019063849A1 - Purification d'un polypeptide d'intérêt - Google Patents

Purification d'un polypeptide d'intérêt Download PDF

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Publication number
WO2019063849A1
WO2019063849A1 PCT/EP2018/085532 EP2018085532W WO2019063849A1 WO 2019063849 A1 WO2019063849 A1 WO 2019063849A1 EP 2018085532 W EP2018085532 W EP 2018085532W WO 2019063849 A1 WO2019063849 A1 WO 2019063849A1
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Prior art keywords
enzyme
anion exchange
nucleic acid
exchange agent
composition
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PCT/EP2018/085532
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English (en)
Inventor
Guilherme Nuno FERREIRA DE PASSOS CORREIA
Robbertus Antonius Damveld
Catarina PEREIRA GALO NEVES
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Dsm Ip Assets B.V.
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Publication of WO2019063849A1 publication Critical patent/WO2019063849A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes

Definitions

  • the present invention relates to the field of microbiology. More specifically, the present invention relates to purification of an enzyme.
  • Downstream processing aims to remove both impurities and contaminants from a certain media.
  • impurities are considered process related and typically include host cell proteins and DNA.
  • impurities can also include culture derived impurities, such as, media components or nutrients, growth hormones or bacterial endotoxins arising from certain nutrients.
  • downstream processing itself can also be a source of impurities, including processing agents (e.g., oxidizing and reducing agents, detergents), inorganic salts (e.g., heavy metals, non-metallic ion), solvents or leachable components.
  • Contaminants should be strictly avoided and must be controlled with appropriate in-process acceptance criteria.
  • Any treatment executed during purification also exerts stress on the proteins, due to possible drastic changes in pH values, protein or salt concentrations, buffers, or solvents, as well as to the shear forces at the liquid stream and surface interfaces.
  • This stress can result in denaturation or aggregation of the proteins with losses in yield and efficacy. Therefore, it is crucial to monitor product quality and functionality during downstream processing with appropriate and fast analytical tools Diibel, S., & Reichert, J. M. (Eds.). (2014). Handbook of therapeutic antibodies (Vol. 1 ). John Wiley & Sons.
  • a conventional sequence of separations starts with the use of techniques which separate components having largest difference in physicochemical properties and ending with separation of molecules with more or less similar properties. Many methods are usually applied in sequence to attain sufficiently high purity levels. The recommended strategy is to use less expensive and simpler methods at early stages, where the volume handled is considerable large, and apply more sophisticated and expensive techniques when the volume remaining is already small.
  • the cells In case the product is intracellular, the cells must be disrupted to release the product, being followed by another separation step.
  • the particle-free liquid is then processed for concentration and purification of the product, by using several unit operations, which are defined not only by the properties of the product, but also by the complexity of separation, scale of operation, economics and desired purity of the product.
  • nucleic acid content especially both DNA and rDNA content must be decreased in the final product.
  • the final product must have a DNA concentration below the detection level of the current analysis methods for these components.
  • the DNA concentration must be reduced by its extraction.
  • Several methods, such as gradient centrifugation, membrane chromatography, precipitation, magnetic separation, usage of filtration aids, flocculation or ion exchange chromatography can be applied.
  • nucleases can be added to the solutions. The advantages and disadvantages of the listed methods are listed in Table 1 here below.
  • Figure 1 depicts the breakthrough curves for pure DNA loading on Sepharose Q XL for ionic strengths of a solution comprising 0 and 500 mM of NaCI, at pH 3, 4, 5 and 7.
  • Figure 2 depicts the results on the purification of a 35.6 kDa Enzyme with a isolectric point of 4.2 (i) before (Buffer A - 0 mM NaCI, pH 5) and (ii) after (Buffer A - 350 mM NaCI, pH 5) optimizing the equilibration.
  • Figure 3 depicts the chromatogram at 280 nm for the anion exchange chromatography of a 10 ml load of phospholipase C feedstock, using Sepharose Q XL a on a 15.7 mL Tricorn 16/200 column packed to a bed height 20 cm.
  • Figure 4 depicts the chromatogram at 280 nm for the anion exchange chromatography of a 10 ml load of phosphatidyl inositol phospholipase feedstock, using Sepharose Q XL a on a 15.7 mL Tricorn 16/200 column packed to a bed height 20 cm.
  • Figure 5 depicts the chromatogram at 280 nm for the anion exchange chromatography of a 10 ml load of Bacillus xylanase feedstock, using Sepharose Q XL a on a 15.7 mL Tricorn 16/200 column packed to a bed height 20 cm.
  • Figure 6 depicts the electrophoresis result after PCR of the samples corresponding to the feedstock and purified material after anion exchange chromatography of a 10 ml load of Bacillus xylanase.
  • the inventors have found that an enzyme can be surprisingly be purified from a nucleic acid by using anion exchange chromatography.
  • the present invention provides for a method for purification of an enzyme in a composition comprising the enzyme and further comprising nucleic acid comprising (i) incubating the composition with an anion exchange agent under conditions where the enzyme does not bind to the anion exchange agent while the nucleic acid does bind to the anion exchange agent, and (ii) separating the composition comprising the enzyme from anion exchange agent comprising the nucleic acid to yield a purified composition comprising the enzyme.
  • the method is herein referred to as a method according to the invention.
  • the composition comprising the enzyme and further comprising nucleic acid may be any type of composition, such as, but not limited to a fermentation broth or a composition that has been purified to a certain extent from a starting composition such as a fermentation broth.
  • the nucleic acid in the composition is regarded as a contaminant that is preferably removed from the composition.
  • the nucleic acid may be any nucleic acid as known to the person skilled in the art and may e.g. be RNA or DNA, either single-stranded or double-stranded.
  • the nucleic acid may be genomic DNA and/or mitochondrial DNA from a host cell that was lysed during fermentation or during downstream processing.
  • the anion exchange agent may be any anion exchange agent known to the person skilled in the art.
  • the person skilled in the art knows anion exchange agents and is able to select a proper anion exchange agent to perform the method according to the invention.
  • a preferred anion exchange agent is a weak or a strong anion exchange agent comprising groups such as DEAE or quaternary ammonium (Q), respectively, or chemically similar groups.
  • the anion exchange agent comprises DEAE or quaternary ammonium (Q) groups.
  • the composition may be a buffered composition and comprising buffering agents known in the art such as acetate, phosphate, Tris, citrate and borate.
  • the composition is incubated with the anion exchange agent, by contacting the composition with the anion exchange agent and keeping the two in contact for a specified period of time.
  • the incubation may be for period of time of seconds to minutes to tens of minutes to hours.
  • the conductivity and/or pH of the composition may be adjusted to the desired values.
  • the person skilled in the art knows how to modulate the pH and the salt concentration of a composition.
  • Conductivity of a composition according to the invention may e.g. be modulated by increasing or decreasing the salt concentration of the composition;
  • the pH of a composition according to the invention may e.g. be modulated by addition of a suitable acid or base; the person skilled in the art will comprehend that when modulating the pH, the conductivity may, as a consequence, be modulated as well.
  • the composition Before contacting the composition with the anion exchange agent, the composition may be diluted or may be concentrated to obtain the desired pH, conductivity, concentration of the enzyme and/or of the nucleic acid.
  • conditions are such that, at least 65% of the nucleic acid binds to the anion exchange agent and/or wherein at least 65% of the enzyme does not bind to the anion exchange agent. More preferably, at least 65, more preferably 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, and most preferably at least 99% of the nucleic acid binds to the anion exchange agent, while at least 65, more preferably 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85,
  • the concentration of nucleic acid in the composition comprising the enzyme and further comprising nucleic acid is at least 1 mg/L (1 ppm), 2 mg/L, 3mg/L, 4mg/L, 5mg/L, 6mg/L, 7mg/L, 8mg/L, 9mg/L, 10mg/L, 15mg/L, 20mg/L, 30mg/L, 40mg/L, 50mg/L, 60mg/L, 70mg/L, 80mg/L, 90mg/L, 100mg/L, 120mg/L, 140mg/L, 160mg/L, 180mg/L, 200mg/L, 220mg/L, 240mg/L, 260mg/L, 280mg/L, 300mg/L, 320mg/L, 340mg/L, 360mg/L, 380mg/L, 400mg/
  • the concentration of nucleic acid in the composition before contacting the composition with the anion exchange agent is at least 10mg/L, 20mg/L, 40mg/L, 60mg/L, 80mg/L, 100mg/L, 200mg/L, 300mg/L, 400mg/L, 500mg/L, 1g/L, 5 g/L, 10g/L, 20g/L, 30g/L. 4g/L, 50g/L, 60g/L, 70g/L, 80g/L, 90g/L, or 100g/L.
  • this is the concentration of enzyme in the composition before contacting the composition with the anion exchange agent.
  • the conditions where the enzyme does not bind to the anion exchange agent while the nucleic acid does bind to the anion exchange agent comprise a pH that is below the iso electric point (IEP) of the enzyme and/or is in the range of 4 to 10.
  • the conditions are such that the pH of the composition is at least 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 0.9 or more preferably at least 1 point lower than the IEP of the enzyme and/or the pH of the composition is in the range of 4 to 10.
  • the pH is the in the range of 4 to 8.
  • the pH is 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 , 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1 , 8.2, 8.3, 8.4, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1 , 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0.
  • the conditions where the enzyme does not bind to the anion exchange agent while the nucleic acid does bind to the anion exchange agent comprise a conductivity in the range of 3 to 150mS/cm.
  • the conductivity is in the range of 10 to 50mS/cm. More preferably, the conductivity is 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150mS/cm.
  • the conditions are typically and preferably such that the protein charge is overall positive such that the protein is electrostatically repelled from the positively charged anion exchange resin.
  • the pH of the composition may be above the IEP of the enzyme, and the protein may overall be negatively charged but the protein may still not bind to the resin.
  • the conductivity of the composition may in such case altered to ensure that the protein will not bind to the resin.
  • the yield of the enzyme in the purified composition is at least 65, more preferably at least 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or even more preferably at least 99% compared to the amount of input enzyme. Most preferably, the yield is 100%.
  • Purified composition is herein defined as the composition after incubation with the anion exchange agent.
  • the amount of nucleic acid in the purified composition is at most 100ppb (100ug/L), preferably at most 90ppb, more preferably at most 80ppb, 70ppb, 60ppb, 50ppb, 40ppb, 30ppb, 20ppb, 10ppb, 9ppb, 8ppb, 7ppb, 6ppb, 5ppb, 4ppb, 3ppb, 2ppb, 1 ppb, 0.9ppb, 0.8ppb, 0.7ppb, 0.6ppb, 0.5ppb, 0.4ppb, 0.3ppb, 0.2ppb, or even more preferably 0.1 ppb.
  • the amount of nucleic acid in the purified composition is at least 2-fold lower, more preferably at least 4-fold lower, even more preferably 6- fold, 10-fold, 20-fold, 40-fold, 60-fold, 80-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600- fold, 700-fold, 800-fold, 900-fold, 1000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, or even more preferably 10.000-fold lower, than in the starting composition.
  • the enzyme may be any enzyme. Such enzyme may be a non-recombinant or a recombinant enzyme.
  • the enzyme is an enzyme selected from the group listed in the section "General Definitions". Such enzyme may be a non-recombinant or a recombinant enzyme.
  • the anion exchange agent may be present in any form suitable for the invention.
  • the anion exchange agent is present as bulk, in a column, on a carrier, or as membrane.
  • the anion exchange agent may be operated in any suitable mode and may be operated in batch-mode or continuous-mode.
  • the enzyme may be non-recombinantly produced in a host cell or may be recombinantly produced in a host cell.
  • the nucleic acid may comprise or may consist of recombinant nucleic acid or of non-recombinant nucleic acid.
  • the nucleic acid comprises recombinant nucleic acid.
  • a method according to the invention preferably further comprises a step selected from the group consisting of: flocculation, precipitation, filtration, cation exchange chromatography, fragmentation, spray drying and lyophilization.
  • the invention provides for a method for the production of an enzyme in a microbial host cell, comprising
  • a method according to the second aspect of the invention further comprises at least one of the steps of:
  • the host cell in the method according to the second aspect of the invention may be any host cell.
  • the host cell may be a prokaryotic- or a eukaryotic-host cell, preferably a bacterium, a yeast, an algae or a filamentous fungus.
  • Preferred host cells are those listed in the section "General Definitions”.
  • the at least one step comprises flocculation, precipitation, filtration, cation exchange chromatography, fragmentation, spray drying or lyophilization.
  • the invention provides for a purified composition comprising the enzyme obtainable by a method according to the first or second aspect of the invention.
  • the features are preferably those of the first or second aspect of the invention.
  • a method for purification of an enzyme in a composition comprising the enzyme and further comprising a nucleic acid comprises (i) incubating the composition with an anion exchange agent under conditions where the enzyme does not bind to the anion exchange agent while the nucleic acid does bind to the anion exchange agent, and (ii) separating the composition comprising the enzyme from anion exchange agent comprising the nucleic acid to yield a purified composition comprising the enzyme.
  • the concentration of enzyme in the composition comprising the enzyme and further comprising nucleic acid is in the range of at least 0.1 to 100gram/L. 5.
  • the conditions where the enzyme does not bind to the anion exchange agent while the nucleic acid does bind to the anion exchange agent comprise a pH that is below the iso electric point (IEP) of the enzyme and/or is in the range of 4 to 10.
  • anion exchange agent is a week or a strong anion exchange agent comprising a group such as DEAE or quaternary ammonium (Q), respectively, or chemically similar groups.
  • a method for the production of an enzyme in a microbial host cell comprising:
  • the host cell is a prokaryotic- or a eukaryotic-host cell, preferably a bacterium, a yeast, an algae or a filamentous fungus.
  • a purified composition comprising the enzyme obtainable by a method according to any one of embodiments 1 - 17.
  • an element may mean one element or more than one element.
  • the word "about” or “approximately” when used in association with a numerical value preferably means that the value may be the given value (of 10) more or less 1 % of the value.
  • nucleic acid and “polynucleotide” are used interchangeably herein and refer to a polymeric form of nucleotides of any length or a defined specific length-range or length, of either deoxyribonucleotides or ribonucleotides, or mixes or analogs thereof.
  • Polynucleotides may be single-stranded or double-stranded and have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, oligonucleotides and primers.
  • a polynucleotide may comprise natural and non-natural nucleotides and may comprise one or more modified nucleotides, such as a methylated nucleotide and a nucleotide analogue or nucleotide equivalent wherein a nucleotide analogue or equivalent is defined as a residue having a modified base, and/or a modified backbone, and/or a non-natural internucleoside linkage, or a combination of these modifications.
  • modifications to the nucleotide structure may be introduced before or after assembly of the polynucleotide.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling compound.
  • sequence identity in the context of the invention of an amino acid- or nucleic acid- sequence is herein defined as a relationship between two or more amino acid (peptide, polypeptide, or protein) sequences or two or more nucleic acid (nucleotide, oligonucleotide, polynucleotide) sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between amino acid or nucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
  • sequence identity with a particular sequence preferably means sequence identity over the entire length of said particular polypeptide or polynucleotide sequence.
  • Similarity between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one peptide or polypeptide to the sequence of a second peptide or polypeptide. In a preferred embodiment, identity or similarity is calculated over the whole sequence (SEQ ID NO:) as identified herein. "Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H.
  • Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1 ): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990).
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990).
  • the well-known Smith Waterman algorithm may also be used to determine identity.
  • Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89: 10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4.
  • a program useful with these parameters is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, Wl. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps).
  • Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine- tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
  • Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place.
  • the amino acid change is conservative.
  • Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; lie to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.
  • a polynucleotide according to the invention is represented by a nucleotide sequence.
  • a polypeptide according to the invention is represented by an amino acid sequence.
  • a nucleic acid construct according to the invention is defined as a polynucleotide which is isolated from a naturally occurring gene or which has been modified to contain segments of polynucleotides which are combined or juxtaposed in a manner which would not otherwise exist in nature.
  • sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases.
  • the skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors.
  • polypeptide is not meant herein to refer to a specific length of the encoded product and, therefore, encompasses peptides, oligopeptides, and proteins.
  • polypeptide refers to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
  • amino acid includes natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
  • Polypeptides further include naturally occurring allelic and engineered variations of the above- mentioned polypeptides and hybrid polypeptides.
  • the polypeptide may be native or may be heterologous to the host cell.
  • the polypeptide may be a collagen or gelatine, or a variant or hybrid thereof.
  • the polypeptide may be an antibody or parts thereof, an antigen, a clotting factor, an enzyme, a hormone or a hormone variant, a receptor or parts thereof, a regulatory protein, a structural protein, a reporter, or a transport protein, protein involved in secretion process, protein involved in folding process, chaperone, peptide amino acid transporter, glycosylation factor, transcription factor, synthetic peptide or oligopeptide, intracellular protein.
  • the intracellular protein may be an enzyme such as, a protease, ceramidases, epoxide hydrolase, aminopeptidase, acylases, aldolase, hydroxylase, aminopeptidase, lipase.
  • the polypeptide may also be an enzyme secreted extracellularly.
  • the present enzyme may belong to the groups of oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, catalase, cellulase, chitinase, cutinase, deoxyribonuclease, dextranase, esterase.
  • the enzyme may be a carbohydrase, e.g.
  • cellulases such as endoglucanases, ⁇ -glucanases, cellobiohydrolases or ⁇ -glucosidases, hemicellulases or pectinolytic enzymes such as xylanases, xylosidases, mannanases, galactanases, galactosidases, pectin methyl esterases, pectin lyases, pectate lyases, endo polygalacturonases, exopolygalacturonases rhamnogalacturonases, arabanases, arabinofuranosidases, arabinoxylan hydrolases, galacturonases, lyases, or amylolytic enzymes; hydrolase, isomerase, or ligase, phosphatases such as phytases, esterases such as lipases, proteolytic enzymes, oxidoreductases such as oxidases, transferases
  • the enzyme may be a phytase.
  • the enzyme may be an aminopeptidase, asparaginase, amylase, a maltogenic amylase, carbohydrase, carboxypeptidase, endo-protease, metallo-protease, serine-protease catalase, chitinase, cutinase, cyclodextrin glycosyltransf erase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, protein deaminase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase, galactolipase, chlorophyl
  • the enzyme can be an enzyme with improved secretion features as described in WO2010/102982.
  • a compound of interest can be a fused or hybrid polypeptide to which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof.
  • a fused polypeptide is produced by fusing a nucleic acid sequence (or a portion thereof) encoding one polypeptide to a nucleic acid sequence (or a portion thereof) encoding another polypeptide.
  • fusion polypeptides include, ligating the coding sequences encoding the polypeptides so that they are in frame and expression of the fused polypeptide is under control of the same promoter(s) and terminator.
  • the hybrid polypeptides may comprise a combination of partial or complete polypeptide sequences obtained from at least two different polypeptides wherein one or more may be heterologous to the host cell.
  • Example of fusion polypeptides and signal sequence fusions are for example as described in WO2010/121933.
  • the cell according to the invention may be a haploid, diploid or polyploid cell.
  • a cell according to the invention is interchangeably herein referred as "a cell”, “a cell according to the invention”, “a host cell”, and as “a host cell according to the invention”; said cell may be any cell, e.g. a prokaryotic, an algae, a microalgae, an algae, a microalgae, marine eukaryote, a Labyrinthulomycetes or a eukaryotic cell.
  • the cell is not a mammalian cell.
  • the prokaryotic host cell is preferably a bacterial host cell.
  • the term "bacterial host cell” includes both Gram-negative and Gram-positive microorganisms.
  • a bacterial host cell according to invention is from a genus selected from the group consisting of Escherichia, Anabaena, Caulobactert, Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus, Propionibacterium, Bacillus, Brevibacterium, Corynebacterium, Rhizobium (Sinorhizobium), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylobacterium, Staphylococcus or Streptomyces.
  • the bacterial host cell is selected from the group consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, B. pumilus, G.
  • a preferred yeast cell is from a genus selected from the group consisting of Candida, Hansenula, Issatchenkia, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, Yarrowia or Zygosaccharomyces; more preferably a yeast host cell is selected from the group consisting of Kluyveromyces lactis, Kluyveromyces lactis NRRL Y-1 140, Kluyveromyces marxianus, Kluyveromyces.
  • thermotolerans Candida krusei, Candida sonorensis, Candida glabrata, Saccharomyces cerevisiae, Saccharomyces cerevisiae CEN.PK1 13-7D, Schizosaccharomyces pombe, Hansenula polymorpha, Issatchenkia orientalis, Yarrowia lipolytica, Yarrowia lipolytica CLIB122, Pichia stipidis and Pichia pastoris.
  • the host cell according to the invention is a filamentous fungal host cell.
  • Filamentous fungi as defined herein include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth ef a/. , In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).
  • the filamentous fungal host cell may be a cell of any filamentous form of the taxon Trichocomaceae (as defined by Houbraken and Samson in Studies in Mycology 70: 1-51. 201 1 ).
  • the filamentous fungal host cell may be a cell of any filamentous form of any of the three families Aspergillaceae, Thermoascaceae and Trichocomaceae, which are accommodated in the taxon Trichocomaceae.
  • the filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligatory aerobic.
  • Filamentous fungal strains include, but are not limited to, strains of Acremonium, Agaricus, Aspergillus, Aureobasidium, Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mortierella, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus, Schizophyllum, Talaromyces, Rasamsonia, Thermoascus, Thielavia, Tolypocladium, and Trichoderma.
  • a preferred filamentous fungal host cell is from a genus selected from the group consisting of Acremonium, Aspergillus, Chrysosporium, Myceliophthora, Penicillium, Talaromyces, Rasamsonia, Thielavia, Fusarium and Trichoderma; more preferably from a species selected from the group consisting of Aspergillus niger, Acremonium alabamense, Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae, Aspergillus fumigatus, Talaromyces emersonii, Rasamsonia emersonii, Rasamsonia emersonii CBS393.64, Aspergillus oryzae, Chrysosporium lucknowense, Fusarium oxysporum, Mortierella alpina, Mortierella alpina ATCC 32222, Myceliophthora thermophila, Trichoderma rees
  • the filamentous fungal host cell according to the invention is an Aspergillus niger.
  • the host cell according to the invention is an Aspergillus niger host cell, the host cell preferably is CBS 513.88, CBS124.903 or a derivative thereof.
  • Preferred strains as host cells according to the present invention are Aspergillus niger CBS 513.88, CBS124.903, Aspergillus oryzae ATCC 20423, IFO 4177, ATCC 1011 , CBS205.89, ATCC 9576, ATCC 14488- 14491 , ATCC 1 1601 , ATCC12892, P. chrysogenum CBS 455.95, P.
  • a host cell according to the invention has a modification, preferably in its genome which results in a reduced or no production of an undesired compound as defined herein if compared to the parent host cell that has not been modified, when analysed under the same conditions.
  • a modification can be introduced by any means known to the person skilled in the art, such as but not limited to classical strain improvement, random mutagenesis followed by selection. Modification can also be introduced by site-directed mutagenesis.
  • Modification may be accomplished by the introduction (insertion), substitution (replacement) or removal (deletion) of one or more nucleotides in a polynucleotide sequence.
  • a full or partial deletion of a polynucleotide coding for an undesired compound such as a polypeptide may be achieved.
  • An undesired compound may be any undesired compound listed elsewhere herein; it may also be a protein and/or enzyme in a biological pathway of the synthesis of an undesired compound such as a metabolite.
  • a polynucleotide coding for said undesired compound may be partially or fully replaced with a polynucleotide sequence which does not code for said undesired compound or that codes for a partially or fully inactive form of said undesired compound.
  • one or more nucleotides can be inserted into the polynucleotide encoding said undesired compound resulting in the disruption of said polynucleotide and consequent partial or full inactivation of said undesired compound encoded by the disrupted polynucleotide.
  • a disruption of a polynucleotide encoding an undesired compound by the insertion of one or more nucleotides in the polynucleotide sequence and consequent partial or full inactivation of said undesired compound by the disrupted polynucleotide.
  • This modification may for example be in a coding sequence or a regulatory element required for the transcription or translation of said undesired compound.
  • nucleotides may be inserted or removed so as to result in the introduction of a stop codon, the removal of a start codon or a change or a frame-shift of the open reading frame of a coding sequence.
  • the modification of a coding sequence or a regulatory element thereof may be accomplished by site-directed or random mutagenesis, DNA shuffling methods, DNA reassembly methods, gene synthesis (see for example Young and Dong, (2004), Nucleic Acids Research 32(7) or Gupta et al. (1968), Proc. Natl. Acad.
  • Preferred methods of modification are based on recombinant genetic manipulation techniques such as partial or complete gene replacement or partial or complete gene deletion.
  • an appropriate DNA sequence may be introduced at the target locus to be replaced.
  • the appropriate DNA sequence is preferably present on a cloning vector.
  • Preferred integrative cloning vectors comprise a DNA fragment, which is homologous to the polynucleotide and / or has homology to the polynucleotides flanking the locus to be replaced for targeting the integration of the cloning vector to this pre-determined locus.
  • the cloning vector is preferably linearized prior to transformation of the cell.
  • linearization is performed such that at least one but preferably either end of the cloning vector is flanked by sequences homologous to the DNA sequence (or flanking sequences) to be replaced.
  • This process is called homologous recombination and this technique may also be used in order to achieve (partial) gene deletion.
  • a polynucleotide corresponding to the endogenous polynucleotide may be replaced by a defective polynucleotide; that is a polynucleotide that fails to produce a (fully functional) polypeptide.
  • the defective polynucleotide replaces the endogenous polynucleotide.
  • the defective polynucleotide also encodes a marker, which may be used for selection of transformants in which the nucleic acid sequence has been modified.
  • a technique based on recombination of cosmids in an E may be replaced by a defective polynucleotide; that is a polynucleotide that fails to produce a (fully functional) polypeptide.
  • the defective polynucleotide replaces the endogenous polynucleotide.
  • the defective polynucleotide also encodes a marker, which may be used for selection of transformants in which the nucleic acid sequence has been modified.
  • coli cell can be used, as described in: A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans (2000) Chaveroche, M-K., Ghico, J-M. and d'Enfert C; Nucleic acids Research, vol 28, no 22.
  • modification wherein said host cell produces less of or no protein such as the polypeptide having amylase activity, preferably a-amylase activity as described herein and encoded by a polynucleotide as described herein, may be performed by established anti-sense techniques using a nucleotide sequence complementary to the nucleic acid sequence of the polynucleotide. More specifically, expression of the polynucleotide by a host cell may be reduced or eliminated by introducing a nucleotide sequence complementary to the nucleic acid sequence of the polynucleotide, which may be transcribed in the cell and is capable of hybridizing to the mRNA produced in the cell.
  • a modification resulting in reduced or no production of undesired compound is preferably due to a reduced production of the mRNA encoding said undesired compound if compared with a parent microbial host cell which has not been modified and when measured under the same conditions.
  • a modification which results in a reduced amount of the mRNA transcribed from the polynucleotide encoding the undesired compound may be obtained via the RNA interference (RNAi) technique (Mouyna et al., 2004).
  • RNAi RNA interference
  • RNA interference techniques described in e.g. WO2008/053019, WO2005/05672A1 and WO2005/026356A1.
  • a modification which results in decreased or no production of an undesired compound can be obtained by different methods, for example by an antibody directed against such undesired compound or a chemical inhibitor or a protein inhibitor or a physical inhibitor (Tour O. et al, (2003) Nat. Biotech: Genetically targeted chromophore-assisted light inactivation. Vol.21 , no. 12: 1505- 1508) or peptide inhibitor or an anti-sense molecule or RNAi molecule (R.S. Kamath_et al, (2003) Nature: Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Vol. 421 , 231-237).
  • the foldase CYPB is a component of the secretory pathway of Aspergillus niger and contains the endoplasmic reticulum retention signal HEEL. Mol. Genet. Genomics. 2001 Dec;266(4):537-545), or by targeting an undesired compound such as a polypeptide to a peroxisome which is capable of fusing with a membrane-structure of the cell involved in the secretory pathway of the cell, leading to secretion outside the cell of the polypeptide (e.g. as described in WO2006/040340).
  • decreased or no production of an undesired compound can also be obtained, e.g. by UV or chemical mutagenesis (Mattern, I.E., van Noort J.M., van den Berg, P., Archer, D. B., Roberts, I.N. and van den Hondel, C. A., Isolation and characterization of mutants of Aspergillus niger deficient in extracellular proteases. Mol Gen Genet. 1992 Aug; 234(2):332-6.) or by the use of inhibitors inhibiting enzymatic activity of an undesired polypeptide as described herein (e.g. nojirimycin, which function as inhibitor for ⁇ -glucosidases
  • the modification in the genome of the host cell according to the invention is a modification in at least one position of a polynucleotide encoding an undesired compound.
  • a deficiency of a cell in the production of a compound, for example of an undesired compound such as an undesired polypeptide and/or enzyme is herein defined as a mutant microbial host cell which has been modified, preferably in its genome, to result in a phenotypic feature wherein the cell: a) produces less of the undesired compound or produces substantially none of the undesired compound and/or b) produces the undesired compound having a decreased activity or decreased specific activity or the undesired compound having no activity or no specific activity and combinations of one or more of these possibilities as compared to the parent host cell that has not been modified, when analysed under the same conditions.
  • a modified host cell according to the invention produces 1 % less of the un-desired compound if compared with the parent host cell which has not been modified and measured under the same conditions, at least 5% less of the un-desired compound, at least 10% less of the undesired compound, at least 20% less of the un-desired compound, at least 30% less of the undesired compound, at least 40% less of the un-desired compound, at least 50% less of the undesired compound, at least 60% less of the un-desired compound, at least 70% less of the undesired compound, at least 80% less of the un-desired compound, at least 90% less of the undesired compound, at least 91 % less of the un-desired compound, at least 92% less of the undesired compound, at least 93% less of the un-desired compound, at least 94% less of the undesired compound, at least 95% less of the un-desired compound, at least 96% less of the undesired
  • sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases.
  • the skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors.
  • Tricorn 10/200 column (GE Healthcare, United Kingdom) was packed with Sepharose Q XL to a total volume of 15.7 mL and 20 cm bed height.
  • the equilibration buffers used for the studies with Sepharose Q XL comprised pH 3, 4, 5 and 7 at 0 and 500 mM.
  • the applied volume of sample was 50 mL (around 2.5 cv).
  • the flow trough was collected during the step of sample loading in 2.0 mL fractions to 2.2 mL wells (Whatman, 96 well plates) placed in cassettes upon fraction collector tray. The fractions were then analyzed to determine the DNA concentration in each well by absorbance at 260 nm.
  • the breakthrough curves for pure DNA loading on Sepharose Q XL are shown in Figure 1 for ionic strengths of a solution comprising 0 and 500 mM of NaCI, at pH 3, 4, 5 and 7. The curves are evidence of better DNA binding at low pH and minimal conductivity.
  • Sepharose resins were purchase from GE Healthcare (UK), DIAION from Residuo (X) and TOYOPEARL from Tosoh (Japan).

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Abstract

La présente invention concerne le domaine de la microbiologie. Plus spécifiquement, la présente invention concerne un procédé de purification d'une enzyme dans une composition comprenant l'enzyme et comprenant en outre un acide nucléique, ledit procédé consistant à (i) incuber la composition avec un agent d'échange d'anions dans des conditions dans lesquelles l'enzyme ne se lie pas à l'agent d'échange d'anions tandis que l'acide nucléique se lie à l'agent d'échange d'anions, et (ii) séparer la composition comprenant l'enzyme d'un agent d'échange d'anions comprenant l'acide nucléique pour produire une composition purifiée comprenant l'enzyme.
PCT/EP2018/085532 2017-12-20 2018-12-18 Purification d'un polypeptide d'intérêt WO2019063849A1 (fr)

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