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WO2000066751A1 - Cassette d'expression servant a la production efficace d'une proteine - Google Patents

Cassette d'expression servant a la production efficace d'une proteine Download PDF

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Publication number
WO2000066751A1
WO2000066751A1 PCT/NL2000/000261 NL0000261W WO0066751A1 WO 2000066751 A1 WO2000066751 A1 WO 2000066751A1 NL 0000261 W NL0000261 W NL 0000261W WO 0066751 A1 WO0066751 A1 WO 0066751A1
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Prior art keywords
ala
protein
gly
leu
thr
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PCT/NL2000/000261
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English (en)
Inventor
Pieter De Geus
Adriana Marina Riemens
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Dsm N.V.
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Priority to AU44369/00A priority Critical patent/AU4436900A/en
Publication of WO2000066751A1 publication Critical patent/WO2000066751A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C12N9/84—Penicillin amidase (3.5.1.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • 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
    • C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
    • C12P35/02—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin by desacylation of the substituent in the 7 position
    • 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
    • C12P37/00—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin
    • C12P37/06—Preparation of compounds having a 4-thia-1-azabicyclo [3.2.0] heptane ring system, e.g. penicillin by desacylation of the substituent in the 6 position
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention relates to a new expression cassette for the efficient production of a protein.
  • secretion pathway is slowly being unravelled, starting at the molecular level with the recognition that secreted proteins are translated as a precursor polypeptide carrying an N-terminal extension, the secretion signal peptide, which upon translocation across the membrane is cleaved off by an enzyme, the signal peptidase, to yield the mature protein (J.M. Gennity, M.Inouye. 1991. Curr.Opin.Biotechnol . 2: 661-667).
  • Penicillin G acylase (E.coli strains), transcription of the acylase gene needs to be induced by the addition of aryl fatty acids (e.g. phenylacetic acid) and is sensitive to catabolite repression (S.Scherrer ibid.).
  • Translation of Penicillin G acylase appears to be inhibited at temperatures above 30°C, resulting in the unfavourable situation that the temperature by which both host cell growth is optimal and efficient synthesis of the desired periplasmic protein occurs, do not coincide (Keilman, C, Wanner, G., Boeck, A., 1993, Biol.Chem. Hoppe Seyler 374, 983-992). Folding of penicillin G acylase occurs properly at 22 °C, whereas at 26°C only 50% of the enzyme is still active, and at 30°C most of the enzyme occurs in an inactive form.
  • Km(R) kanamycin resistance gene
  • Tc (R) tetracyclin resistance gene
  • Cm(R) chloramphenicol resistance gene
  • ori327 origin of replication from plasmid pBR327
  • trpP modified trp promoter
  • ECpga E. coli PenG acylase .
  • Km(R) kanamycin resistance gene
  • Tc (R) tetracyclin resistance gene
  • Cm(R) chloramphenicol resistance gene
  • ori327 origin of replication from plasmid pBR327
  • trpP modified trp promoter
  • ECpga E. coli PenG acylase.
  • AFss A. faecalis secretion signal sequence
  • aroP aro promoter.
  • the present invention provides an expression cassette comprising a promoter sequence, a secretion signal and a DNA sequence encoding a protein, wherein said secretion signal is heterologous.
  • This secretion signal originates from Alcaligenes faecalis .
  • the expression cassette comprises a DNA sequence encoding the mature part of a naturally secreted protein, which DNA sequence is heterologous with respect to Alcaligenes faecalis, i.e. the DNA sequence does not originate form Alcaligenes faecalis.
  • the DNA sequence encoding a protein preferably is a ⁇ -lactam acylase, e.g. Penicillin G acylase of E . coli origin, or originating from Escherichia coli, Kluyvera citrophila, Providencia rettgeri, Arthrobacter viscosus or Bacillus megaterium.
  • the expression cassette comprises a promoter sequence preferably selected from the group consisting of aro, lac, trp and tac promoter.
  • the expression cassette may be integrated in a host chromosome, or may be present on a plasmid which is capable of propagation in a suitable host.
  • Various hosts or host cell strains may be used, for example E. coli or Pseudomonas species are suitable hosts.
  • an E . coli host cell strain is used which is capable of production and secretion of the protein.
  • a process for the preparation of the protein encoded by the expression cassette characterised by growing the host cell strain in a suitable nutrient medium allowing initiation of expression of said protein in the host cell strain, whereby that host cell strain produces said protein, followed by harvesting said protein from that medium or host cell.
  • the host cell strain is preferably grown at a temperature of 20-30°C, more preferably 22-28°C.
  • a process for the preparation of an amino ⁇ -lactam compound is provided, by the application of the protein of present invention. This process is optionally followed by a process for the preparation of a semisynthetic ⁇ -lactam antibiotic, wherein the corresponding amino ⁇ -lactam compound, is subsequently reacted with a suitable side chain derivative by applying a suitable ⁇ -amino acid hydrolase.
  • said semisynthetic ⁇ - lactam antibiotic is preferably selected from the group consisting of Amoxicillin, Ampicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Cefotaxim, Cephalexin, Cefadroxil, Cephradine, Cefetamet, Cefroxadine, Cefatrizine, Cefoperazon, Cefprozil, Cefaclor, Loracarbef, Cefazolin, Cefotiam and Cefamandole.
  • E. coli Penicillin G acylase is a heterodimeric protein located in the periplasmic space and thus has to be translocated across the cytoplasmic membrane.
  • the single peptide chain (preproprotein) resulting from translation of the messenger RNA not only loses the signal peptide yielding the proprotein, but subsequently requires proteolytic modification by removal of a central portion of 54 amino acids resulting in the mature form of two closely associated subunits, i.e. a small ⁇ -subunit and a large ⁇ - subunit (G. Schumacher et al . ibid.) .
  • Hydrolases and specifically acylases are involved in the enzymatic preparation of ⁇ -lactams.
  • ⁇ - Lactam acylases such as penicillin G acylases (E.C. 3.5.1.11, benzylpenicillin amidohydrolase or penicillin amidase) , glutaryl acylases, adipyl acylases and dicarboxylate acylases can be obtained from various microorganisms and can be used in enzymatic steps for semi-synthetic preparation of ⁇ -lactam antibiotics such as penicillins, cephalosporins and their derivatives.
  • Penicillin acylases are efficiently able to cleave the benzyl side chain off penicillin G and penicillin V yielding 6-APA.
  • Penicillin G acylase is a member of a family of evolutionary related Penicillin G acylases from different Gram positive (e.g. Arthrobacter viscosus and Bacillus megaterium) and Gram negative bacteria (e.g. E . coli, Kluyvera citrophila and Providencia rettgeri) .
  • Gram positive e.g. Arthrobacter viscosus and Bacillus megaterium
  • Gram negative bacteria e.g. E . coli, Kluyvera citrophila and Providencia rettgeri
  • other enzymes have been identified displaying different properties like e.g. altered substrate specificities or different stability under application conditions.
  • Alcaligenes faecalis Penicillin G acylase R.M.D.
  • Glutaryl, adipyl or benzyl acylases are capable of specifically hydrolyzing the glutaryl, adipyl or benzyl side chain of the respective Cephalosporin C derivative, yielding 7-ACA and 7-ADCA.
  • 6-APA, 7-ACA and 7-ADCA are important precursors for semi-synthetic ⁇ -lactam antibiotics such as Amoxicillin, Ampicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Cefotaxim, Cephalexin, Cefadroxil, Cephradine, Cefetamet, Cefroxadine, Cefatrizine, Cefoperazon, Cefprozil, Cefaclor, Loracarbef, Cefazolin, Cefotiam and Cefamandole .
  • antibiotics such as Amoxicillin, Ampicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Cefotaxim, Cephalexin, Cefadroxil, Cephradine, Cefetamet, Cefroxadine, Cefatrizine, Cefoperazon, Cefprozil, Cefaclor, Loracarbef, Cefazolin, Cefotiam and
  • ⁇ -amino acid ester hydrolases there are ⁇ -amino acid ester hydrolases. It has been shown that these hydrolysing enzymes can be used for the reverse reaction too, i.e. are capable of attachment of a desired side chain to a ⁇ -lactam structure: such as the condensation of activated side chain derivatives such as D- (-) -phenylglycine (PG) , D- (-) -2 , 5-dihydrophenylglycine, D- (-) -4-hydroxyphenylglycine (HPG) , lH-tetrazoleacetic acid, (2-aminothiazol-4-yl) acetic acid and D- (- ) -mandelic acid and amides or methyl esters thereof, with amino ⁇ - lactams such as 6-amino-penicillanic acid (6-APA) , 7- aminocephalosporanic acid (7-ACA) , and 7- aminodesacet
  • acylases are involved in many enzymatic reactions in pharmaceutical processes.
  • efficient synthesis of the desired acylase proteins in the periplasmic compartment remains a major problem since the temperatures at which both host cell growth and protein synthesis is optimal do not always coincide.
  • expression of Penicillin G acylase needs to be tightly controlled by the cell, because both the secretion pathway and the folding process are very sensitive to high level expression of the protein.
  • the latter phenomenon is not uncommon in recent biotechnological research involving E. coli as a production organism: several (classes of) proteins of interest have been observed to be blocked in either efficient secretion, resulting in accumulation of intracellular inactive complexes, or after secretion are incorrectly folded in the periplasm. Examples can be found e.g. in the expression and secretion of single-chain antibody fragments (J.E. Somerville Jr, S.C. Goshorn, H.P Fell,
  • the basic element to realise protein expression is called the expression cassette.
  • This cassette comprises: a promoter with accessory regulatory regions for induction or repression involved in directing the transcription of the downstream DNA into messenger RNA followed by a region, which in the messenger RNA is responsible for the recognition and correct positioning of the starting point of translation, the actual protein assembly mechanism. From this starting point onwards the nucleotide sequence of the messenger RNA determines the amino acid sequence of the encoded protein. Embedded in this amino acid sequence, a secretion signal is present targeting the protein to a cellular compartment, e.g. the cell membrane, periplasmic space (gram-negative bacteria only) , peptidoglycan layer, the outer membrane (gram- negative bacteria only), or completely out of the cell.
  • a cellular compartment e.g. the cell membrane, periplasmic space (gram-negative bacteria only) , peptidoglycan layer, the outer membrane (gram- negative bacteria only), or completely out of the cell.
  • secretion is defined as at least one membrane crossing effectuated by the secretion signal.
  • the expression cassette may contain signals mediating efficient transcription termination, translation termination, as well as sequences increasing messenger RNA stability. In the so-called expression cassettes, a strong promoter ensuring high level messenger RNA availability must be present.
  • the promotor used in the expression cassette according to the invention may be selected from the well-known set of inducible promoters for highly expressed operons/genes like the lactose operon ( lac, lacUV5) , the arabinose operon ( ara) , the tryptophan operon ( trp) , and the operon encoding enzymes common to the biosynthesis of all aromatic amino acids (aro) , or functional hybrids of these, e.g. the tac promoter, which is a fusion of the trp and the lac promoter (E.Amann, J.Brosius, M.Ptashne. 1983. Gene 25: 161-178).
  • constitutive promoters can be used providing for a constant supply of messenger RNA throughout the cell's life. Similar strategies can be used to improve translation of the messenger RNA pool by introducing, via recDNA methodology, 5' untranslated leader regions from efficiently translated messenger RNA's like those obtainable from the tuf gene encoding the highly expressed Elongation Factor Tu protein, or modified or synthetic variants of the tryptophan operon. Transcription terminators, possibly also contributing to messenger RNA stability can be selected either from the native gene to be expressed or from sources like rRNA genes or viral operons, e.g. the ribosomal RNA terminator, or the fd terminator (J. Sambrook, E.F. Fritsch, T. Maniatis. 1989.
  • the extrachromosomal elements in which the expression cassettes can be inserted may be plasmid or virus derived, and preferably but not necessarily comprise a marker enabling selection of cells harbouring the element, as well as DNA sequences responsible for autonomous propagation and/or equal distribution of the element within the host cell and its daughter cells.
  • chromosomal integration can be envisaged.
  • the expression cassette comprises a DNA fragment encoding the mature part of a protein, preferably a secreted protein (crossing at least one membrane) , more preferably an acylase, most preferably one or both subunits of a ⁇ - lactam acylase, in combination with a DNA fragment encoding a secretion signal peptide of a heterologous protein i.e. originating from a different species, i.e. A. faecalis, which is subsequently introduced into a production host and which yield high expression levels of said proteins.
  • the embodiment of the expression cassette last mentioned is additionally modified by replacement of the original promoter by preferably the trp promoter or the aro promoter.
  • messenger RNA translation and plasmid stability may be applied to the recDNA constructs used to create the actual production strain e.g. addition of the Transcription terminator of phage fd, or the introduction of the partitioning function par from plasmid pSClOl (G. Churchward, P. Linder, L. Caro, 1983. Nucleic Acid Res. 11:5645-5659) .
  • extrachromosomal elements for example plasmids ColEl, ColD, R1162, RK2 or derivatives which may be present in either predetermined low copy numbers or, often dynamic, high copy numbers and which are capable of propagation or autonomous replication in e.g. E . coli strains HB101, B7, RV308, DH1, HMS174, W3110, BL21.
  • the enzyme obtained in the present invention may be used as isolated free enzymes, or preferably immobilised on various types of water- insoluble carrier materials known in the art (WO 97/04086 and WO99/01566) .
  • plasmid vectors should be free of ⁇ - lactamase activity (which would destroy the substrate for the acylase) .
  • plasmid pMC5 was used (P. Stanssens, H.-J. Fritz. 1989. Nucleic Acids Res. 17: 4441-4454) .
  • trp promoter For directing transcription and translation in the expression cassette, a modified trp promoter was designed which lacked the attenuator region, contained an adapted ribosome binding site, and within the translation start codon incorporated a -Vdel restriction site.
  • a DNA fragment according to this design was assembled synthetically using an Applied Biosystems DNA synthesizer. The complete nucleotide sequence of this fragment is in SEQ ID nr 1.
  • the native gene encoding E.coli Penicillin G acylase (G.Schumacher et al . ibid.) was obtained in two parts : a. the N-terminal coding region was obtained by PCR using the following two oligonucleotides: 5'-
  • Clones harbouring the correct Sphl-Smal fragment were identified by colony hybridization techniques using synthetic oligonucleotides designed on the published sequence.
  • Plasmid pBR327 was digested with Oral and Aatll, treated with T4 DNA polymerase to obtain blunt ends on both sides of the fragment, and treated with T4 DNA ligase in the presence of a purified Haell fragment from pBGS18+.
  • the ligation mixture was transformed to E. coli strain DHl and cells resistant to tetracyclin and kanamycin were analysed for the presence of recombinant plasmid. Plasmid was identified by restriction enzyme mapping and a new plasmid named pBRK ( Figure 1) was found as being the expected combination of fragments.
  • Plasmid pBRK was digested with Styl, treated with T4 DNA polymerase to create a blunt end, then digested with EcoRI , ligated to the BcoRI-Smal fragment containing the expression cassette for the Penicillin G acylase from pMCtrpEC, and transformed to E . coli strain HB101 (ATCC33694) to yield plasmid pKECtrp ( Figure 1) .
  • the Alcaligenes faecalis secretion signal coding region was linked with the coding region for the mature E.coli Penicillin G acylase coding region employing the fusion PCR technique.
  • Two oligonucleotides 5'- GAAACCATTATTATCATGACA-3' (SEQ ID nr 4) and 5'- ACGACTGCTCCGCGTGGGTCGGTGC-3' (SEQ ID nr 5) were used to amplify the Alcaligenes faecalis secretion signal coding region including the modified trp promoter from a plasmid essentially identical to pKECtrp except that the expression cassette contained the Alcaligenes faecalis Penicillin G acylase (R.M.D.
  • the aro promoter (G.R. Zurawski, R. P . Gunsalus , K.D. Brown, C.Yanofsky. 1981. J.Mol.Biol. 145: 47-73) was linked to the A. faecalis secretion signal sequence fused to the E . coli Penicillin G acylase mature protein coding region (AFssEC Penicillin G acylase) in an analogous way with the fusion PCR methodology as described in Example 3.
  • oligonucleotides 5 ' -TCGACTGAATTCTCGATATCATGGGCCTTAGT- 3' SEQ ID nr 8 and 5 ' -TGAACTTGCGTAGCATGATAACAAA-3 ' (SEQ ID nr 9) were designed to obtain the aro promoter by PCR on chromosomal DNA from E.coli strain RR1.
  • Two other oligonucleotides 5 ' -TATCATGCTACGCAAGTTCACGTAAAAAGGAGG-3 ' (SEQ ID nr 10) and SEQ ID nr 7 Example 3 were used to amplify the N-terminal coding region of the AFssEC Penicillin G acylase. Both resulting fragments were subsequently used as templates for oligonucleotides SEQ ID nrs 7 and 8 only, yielding a new DNA fragment of 417 basepairs.
  • This 417 basepairs fragment was digested with EcoRI and Sphl and used to replace the corresponding fragment in pKAFssECtrp by ligation and transformation to E.coli strain HB101, yielding plasmid pKAFssECaro ( Figure 2) .
  • the following medium was prepared: Yeast extract (total nitrogen content of 2.1 g/1) 19.0 g Na 2 HP0 4 .2H 2 0 8.9 g KH 2 P0 4 6.8 g
  • the production medium had the following composition: per kg medium Low salt yeast extract with a free L-tryptophan contents of about
  • Antifoam agent e.g Basildon
  • Neomycin 0.01 g
  • Necessary nutritional supplements e.g. vitamin Bl, amino acids like leucine and proline
  • Heat labile components were filter sterilised.
  • the assay of E . coli Penicillin G acylase was based on kinetic, photometric measurement at 405 nM of 3- amino-6-nitrobenzoicacid formed from 6-Nitro- 3 (PhenylAcetamido) benzoic acid. Reaction temperature was 37°C. Cell culture suspension were first sonicated. Recovery samples in which the Penicillin G acylase was liberated from the biomass, were measured directly.
  • E.coli Penicillin G acylase (as produced in Example 5) were killed by adding 1-octanol to a final concentration of 4 g/1. The mixture was incubated for 4 hours and after this cooled to 10-15°C. The cells were disrupted by homogenisation, using two passes through a high pressure slit (600-700 bars) . Temperature was maintained at 15 °C by cooling. Alternatively, cell mass removal is possible by microfiltration followed by diafiltration.
  • the mixture was collected in a vessel and the pH was adjusted to a pH of 7.
  • Flocculant was added in a concentration of 4-8 g/1 depending on filterability and stirred for 1.5 hours. After this 10 wt% of dicalite 4108 was added. The solids were filtered off by means of a membrane filter press. After filtration the cake was washed with 2.5 cake volumes of water.
  • the octanol was removed by adding active carbon CAl at 2-6 g/1 of filtrate and 1-3% Dicalite 4108. After 1.5 hours of stirring the solids were filtered off using a membrane filter press. After filtration washing was performed with 2,5 cake volumes of water. The filtrate was filtered over a K700 filter followed by an EKS filter to gain a resulting filtrate low in germs.
  • This filtrate was adjusted to pH 6-8 by using 25% ammonia, and was concentrated by ultrafiltration using Polysulfonic membranes of 50 kD. After sufficient concentration (6 times) diafiltration was performed to lower the conductivity to ⁇ 2 mS/cm. The resulting filtrate was germ filtrated over K700 and EKS filtration plates . The filtrate was purified by adsorption onto a
  • Sepharose S gel at pH 5.5 (2.5 mS/cm) .
  • the column was rinsed with 20 mM NaAc/HAc buffer at pH 5.0 (1.5 mS/cm) .
  • Elution was performed using 10 mM NaAc/30 mM NaCl at pH 6.0 (4.4 mS/cm) , followed by conditioning of the resin with 10 mM NaAc/2.0 mM NaCl at pH 5.5 (150 mS/cm) and washing with 10 mM NaAc at pH 5.5 (0.8 mS/cm) .
  • Pro Glu Asp Met Ser lie Leu Gin Gly Tyr Ala Asp Gly Met Asn Ala 115 120 125
  • Trp lie Asp Lys Val Asn Thr Asn Pro Glu Thr Leu Leu Pro Lys Gin
  • aac cca tea gcg cca ace act att gcc gta caa gag agt aac tac cca 672 Asn Pro Ser Ala Pro Thr Thr He Ala Val Gin Glu Ser Asn Tyr Pro 210 215 220
  • gac cag acg aca caa acg get tac get aaa tec cgc gca tgg 1296 Gin Thr Asp Gin Thr Thr Gin Thr Ala Tyr Ala Lys Ser Arg Ala Trp 420 425 430
  • ace ate aac tgg tac tat get gat gta aac ggc aat att ggt tat gtt 1440 Thr He Asn Trp Tyr Tyr Ala Asp Val Asn Gly Asn He Gly Tyr Val 465 470 475 480 cat act ggt get tat cca gat cgt caa tea ggc cat gat ccg cga tta 1488 His Thr Gly Ala Tyr Pro Asp Arg Gin Ser Gly His Asp Pro Arg Leu 485 490 495
  • gag caa aag cca cgc tta act get gat cag gca tgg gat gtt att cgc 1728 Glu Gin Lys Pro Arg Leu Thr Ala Asp Gin Ala Trp Asp Val He Arg 565 570 575
  • gag act ctt tec aaa cgc tat ggc aat aat gtg agt aac tgg aaa aca 2208
  • gag gcg cat aag gag teg cag gaa gtg ttg cac gtt cag aga taa 2541 Glu Ala His Lys Glu Ser Gin Glu Val Leu His Val Gin Arg

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Abstract

Cette invention concerne une cassette d'expression comprenant une séquence d'ADN d'une espèce codant une protéine qui est combinée à une séquence d'ADN codant le peptide du signal de sécrétion d'une espèce A. faecalis. La séquence de l'espèce codant la protéine ne provient pas de l'A. faecalis. Cette invention concerne également un procédé de préparation et d'isolation de cette protéine, ainsi qu'un procédé de préparation d'antibiotiques à base de β-lactame où l'on utilise ladite protéine.
PCT/NL2000/000261 1999-04-29 2000-04-25 Cassette d'expression servant a la production efficace d'une proteine WO2000066751A1 (fr)

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EP99201373 1999-04-29

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1186668A1 (fr) * 2000-09-08 2002-03-13 Dsm N.V. Procédé enzymatique de préparation de composés de bêta-lactame
US7241602B2 (en) 2001-07-23 2007-07-10 Dsm Ip Assets B.V. Nucleic acid sequences encoding enantioselective amidases
US7371555B2 (en) 2002-06-14 2008-05-13 Dsm Ip Assets B.V. Polypeptides having α-H-α-amino acid amide racemase activity and nucleic acids encoding the same
EP2267144A2 (fr) 2006-12-04 2010-12-29 DSM IP Assets B.V. Système catalytique à cellules entières renfermant une hydantoinase, une racemase et une carbamoylase
WO2011003702A1 (fr) 2009-07-09 2011-01-13 Dsm Ip Assets B.V. Compositions d'enzyme stabilisées
WO2013178809A2 (fr) 2012-05-31 2013-12-05 Dsm Ip Assets B.V. Préparation orale

Citations (5)

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EP0107823A2 (fr) * 1982-10-21 1984-05-09 Gesellschaft für Biotechnologische Forschung mbH (GBF) Séquences d'ADN et structures d'ADN ainsi que procédé de préparation de pénicillinacylase les mettant en oeuvre
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EP1186668A1 (fr) * 2000-09-08 2002-03-13 Dsm N.V. Procédé enzymatique de préparation de composés de bêta-lactame
WO2002020819A3 (fr) * 2000-09-08 2002-10-24 Dsm Nv Procede enzymatique pour l'elaboration de composes a base de beta-lactamines
US7241602B2 (en) 2001-07-23 2007-07-10 Dsm Ip Assets B.V. Nucleic acid sequences encoding enantioselective amidases
US7371555B2 (en) 2002-06-14 2008-05-13 Dsm Ip Assets B.V. Polypeptides having α-H-α-amino acid amide racemase activity and nucleic acids encoding the same
EP2267144A2 (fr) 2006-12-04 2010-12-29 DSM IP Assets B.V. Système catalytique à cellules entières renfermant une hydantoinase, une racemase et une carbamoylase
WO2011003702A1 (fr) 2009-07-09 2011-01-13 Dsm Ip Assets B.V. Compositions d'enzyme stabilisées
WO2013178809A2 (fr) 2012-05-31 2013-12-05 Dsm Ip Assets B.V. Préparation orale

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