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WO1997004110A1 - Procedes d'accroissement de l'expression de proteines - Google Patents

Procedes d'accroissement de l'expression de proteines Download PDF

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Publication number
WO1997004110A1
WO1997004110A1 PCT/US1996/011600 US9611600W WO9704110A1 WO 1997004110 A1 WO1997004110 A1 WO 1997004110A1 US 9611600 W US9611600 W US 9611600W WO 9704110 A1 WO9704110 A1 WO 9704110A1
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Prior art keywords
expression
plasmid
host cell
gene
repressor
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PCT/US1996/011600
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English (en)
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Michael J. Weickert
Christopher B. Glascock
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Somatogen, Inc.
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Priority to AU64899/96A priority Critical patent/AU6489996A/en
Publication of WO1997004110A1 publication Critical patent/WO1997004110A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C12N15/72—Expression systems using regulatory sequences derived from the lac-operon

Definitions

  • This invention relates to expression of heterologous polypeptides. More specifically, the invention discloses novel recombinant host strains comprising high copy number cloning plasmids comprising a gene encoding the heterologous polypeptide. Expression of the heterologous polypeptide is effectively controlled by locating the regulator gene, which controls expression of the heterologous polypeptide, on the host chromosome and under control of a strong promoter.
  • heterologous polypeptides Methods for recombinantly-producing heterologous polypeptides are well-known (see, e.g., Watson et al., Recombinant DNA, 2nd ed., (1992)).
  • the most commonly used regulated heterologous gene expression systems in, for example, Escherichia coli host cells are those based on Lacl-mediated repression of a promoter which controls expression of the heterologous polypeptide. Release from repression occurs through addition of an inducer (such as isopropylthiogalactoside (IPTG)), which binds to the repressor, or through an excess of DNA binding sites titrating the repressor.
  • IPTG isopropylthiogalactoside
  • Repression of the E. coli Ptac promoter depends upon the concentration of the repressor, Lad. As set forth above, release from repression can occur through addition of inducer which reduces the affinity of the repressor for its specific DNA binding site, or through a reduction in the concentration of the repressor, relative to the molar concentration of specific DNA binding sites on the plasmid.
  • Lad gene is located on a high copy number cloning plasmid, a large amount of inducer is required to initiate expression because of the large amount of repressor produced in such a system.
  • the control of Ptac on a multi-copy plasmid is often mediated by Lad - expressed from a lad promoter mutation on the chromosome, designated f ⁇ clQ(sometimes referred to as lacH or variations thereof herein) , in a system without a corresponding lad gene on the expression plasmid.
  • the laclQ mutation is a single CG to TA change at -35 of the promoter region of lad (Calos, M. 1978.
  • Wild-type cells have a concentration of Lad of 10"8 M or about 10 molecules per cell, with 99% present as a tetramer (Fickert, R. & B. Muller-Hill 1992. J. Mol. Biol. 226:59).
  • Cells containing the laclQ mutation contain about 100 molecules per cell or IO"- 7 M Lad.
  • the plasmid pBR322 is present in 39-55 copies per cell, depending on the growth rate of the cells (Lin-Chao, S. & H. Bremer. 1986. Mol. Gen. Genet. 203:143). Therefore, pBR322-based plasmids should be controlled by chromosomal laclQ. Plasmids based on pUC, having a higher copy number (500-2,000 copies per cell), should not be controlled by laclQ alone, and expression should be leaky. Other systems have been developed, such as the T7 system of Studier and Moffatt (1986. J. Mol. Biol. 189:113-130 and Dubendorff and Studier. 1991. J. Mol. Biol.
  • the repressor gene is on a high copy number plasmid comprising the heterologous gene
  • the repressor is over-expressed relative to the amount needed to control expression of the heterologous gene controlled thereby (i.e., the stoichiometry is incorrect).
  • this system requires a large amount of inducer (e.g., IPTG) to be added to the host cell during fermentation, which is costly and not efficient.
  • inducer e.g., IPTG
  • the amount of repressor produced may be too little to control expression of the heterologous gene on the plasmid, resulting in leaky control of expression.
  • laclQ results in a ten-fold higher level of expression of Lac repressor, this is not sufficient for tightly regulated expression from Ptac on a high copy number plasmid if laclQ is located on the chromosome.
  • the present invention provides recombinant host strains for expressing heterologous polypeptides, comprising high copy number cloning plasmids comprising a gene encoding a heterologous polypeptide. Expression of the heterologous polypeptide is effectively controlled by locating the regulator gene, which controls expression of the heterologous polypeptide, on the host chromosome and under control of a strong promoter.
  • A, specific embodiment relates to an allele of lad capable of repressing expression-of genes from /ac-regulated promoters on high copy number plasmids.
  • lac ⁇ 0- (sometimes referred to as laclQl or other variations herein) is a mutation of the repressor's promoter (Carlos and Miller. 1981. Mol. Gen. Genet. 183:559-560) which results in ⁇ 170-fold more Lad than the wild type promoter and compared to a 10- fold increase reported for la .
  • the increased expression of Lac repressor allows for repression of gene expression on the high copy number plasmid.
  • IPTG IPTG
  • the present invention relates to methods and host cells for expressing heterologous polypeptides.
  • the invention relates to recombinant host cells comprising high copy number cloning plasmids comprising a gene encoding a heterologous polypeptide.
  • the invention relates to a prokaryotic host cell for producing a heterologous polypeptide comprising a high copy number plasmid comprising a regulatable expression unit encoding the polypeptide and a chromosomally-located gene encoding a regulatory protein capable of regulating the regulatable expression unit, expression of the regulatory protein being controlled by a strong promoter.
  • the invention relates to a prokaryotic host cell for producing a heterologous polypeptide comprising a high copy number plasmid comprising a repressible expression unit encoding said polypeptide and a chromosomally-located gene encoding a repressor capable of repressing said repressible expession unit, expression of said repressor being controlled by a strong promoter.
  • the host cell is a bacterium, preferably E. colij.
  • expression of the heterologous polypeptide is under control of a Lacl-repressed promoter, and expression of the Lad repressor is under control of the LaclQl promoter.
  • the invention also relates to methods for expressing heterologous proteins comprising culturing the above-described host cells and inducing expression of said genes.
  • the present invention also relates to methods for controlling plasmid expression. More particularly, the methods involve the use of a repressor together with an inducer to control the high-copy-number-plasmid expression of proteins, preferably recombinant proteins.
  • the methods can be used in any organism for which regulated promoter systems exist for heterologous gene expression, including, for example, species of the genera Escherichia, Salmonella, Bacillus, Clostridium, Streptomyces, Staphylococcus, Neisseria, Lactobacillus, Shigella, and Mycoplasma.
  • Figure 1 is the plasmid map for pSGE705.
  • Figure 2 shows a simplified schematic for the construction of expression vector, ⁇ SGE705.
  • Figures 2A shows the ligation of pBR322 origin and Tet resistance gene (PCR fragment).
  • Figure 2B shows the insert La from pRGT (PCR fragment).
  • Figure 2C shows the insert di-alpha and beta with new promoter region and shorter intergenic spacer.
  • Figure 2D shows the results of site-directed mutagenesis to optimize ribosome binding sites and several other modifications.
  • Figure 3 shows a simplified schematic for the construction of expression vector, pSGE715.
  • Figure 3A shows the ligation of pUC19 origin and Tet resistance gene (PCR fragment).
  • Figure 3B shows the insert Lad from pRGl (PCR fragment).
  • Figure 3C shows the insert Ptac, di-alpha and beta from pSGE705 into BamHI and
  • regulatory expression unit means a nucleic acid molecule comprising a unit of gene expression and regulation, including heterologous genes, regulator genes and control elements which are necessary for transcription, translation and for recognition by regulator gene products, including
  • control elements may be contiguous to the heterologous gene or not.
  • heterologous when referring to a gene, indicates that the gene has been inserted into a host cell either by way of a stable plasmid or through integration into the genome, and, when referring to a protein or polypeptide, indicates that the protein or polypeptide is the product of a heterologous gene.
  • Heterologous proteins are proteins that are normally not produced by a particular . host cell.
  • “Host cell” refers to a prokaryotic organism containing, or hosting, a plasmid artificially constructed using techniques well known in molecular biology. Examples include Escherichia, Salmonella, Bacillus, Clostridium, Streptomyces,
  • E. coli strains include BL21(DE3), C600, DH5 ⁇ F', HB101, JM83, JM101, JM103, JM105, JM107, JM109, JM110, MC1061, MC4100, MM294, NM522, NM554, TGI, ⁇ 177 6, XLl-Blue, and Y1089+, all of which are commercially available, for example, from New England Biolabs (Internet address: http://www.neb.com).
  • High copy number plasmid refers to a plasmid for use in cloning heterologous genes in a host cell that is present in greater than about 200 copies per cell.
  • Examples include pALTER®-l Vector, pALTER®-Exl Vector, pALTER®-Ex2 Vector, pGEM®-3Z vector, pGEM®-3Zf(+/-) Vectors, pGEM®-7Zf(+/-) Vectors, pGEM®-9Zf (-) Vector, pGEM®-HZf (+ /-) Vectors, and the other pGEM® vectors available from Promega, the pUC-based cloning vectors, including pUC 8, 9, 18 and 19, pBacPAK8/9, and pAcUW31, and pET all available from Clontech.
  • Medium copy number plasmid refers to a plasmid for use in cloning heterologous genes in a host cell that is present in about 25-200 copies per cell. Examples include pBR322, pMAM, pMAMneo, pEUK-Cl, pPUR, pYEUra3, pDR2*, pKK233-2, and pKK388-l (all available from Clontech).
  • “Strong promoter” refers to a promoter or promoter mutation that increases promoter activity, i.e. transcription initiation, at least 10-fold greater than the native promoter or equivalent, or stronger than the E. coli lac promoter, fully induced.
  • the strong promoter is called a "strong repressor promter.” Examples include the lac * tac, tre, trp, ara, fru, gal, and mal promoters.
  • Regulator or “regulatory protein” refers to a transeriptional regulatory protein which exerts direct control over gene expression by binding and /or releasing DNA at a specific promoter. In particular in the context of the invention, the regulator is involved in controlling expression of the heterologous gene.
  • Regulatable gene or expression unit refers to genes or expression units effected by a regulator.
  • Repressor refers to a DNA regulatory protein which exerts direct negative control over gene expression at a specific promoter. In particular in the context of the invention, the repressor controls expression of the heterologous gene. “Repressible” refers to a regulatory system in which the product of a regulator gene (the repressor) blocks transcription of a particular gene, or expression unit.
  • “Inducer” refers to an effector or molecule which inactivates the repressor and thereby causes expression of the repressible gene.
  • Recombinant prokaryotic systems particularly bacterial systems for producing heterologous proteins or polypeptides are well known in the art.
  • the genes encoding the target protein can be placed in a suitable expression vector or plasmid and inserted into a microorganism or host cell.
  • These host cells may be produced using standard recombinant DNA techniques and may be grown in cell culture or in fermentations.
  • human alpha and beta globin genes of human hemoglobin have been cloned and sequenced by Liebhaber et al. (Proc. Natl. Acad. Sci. USA 77:7054-7058, 1980) and Marotta et al. (J. Biol. Chem. 252:5040-5053, 1977) respectively.
  • Techniques for expression of both native and mutant alpha and beta globins and their assembly into hemoglobin are set forth in U.S. Patent
  • the host cells of the present invention can be cultured at a temperature of about, 20°C to about 30°C, more specifically about 24°C to about 28°C and preferably about 26°C.
  • the present invention relates to regulation of the expression of such target heterologous proteins.
  • One method for increasing the amount of heterologous protein expressed by a host cell involves using a high copy number plasmid into which is cloned the gene encoding the heterologous protein.
  • a high copy number plasmid into which is cloned the gene encoding the heterologous protein.
  • expression of the heterologous protein is not tightly controlled, then early expression of the protein can have deleterious effects on the host cells.
  • the system employs a chromosomally located gene encoding a regulator of expression of the heterologous protein which is under control of a strong promoter.
  • the system is useful in a wide variety of prokaryotic host cells as set forth above.
  • inducers can be used, including various sugars such as galactose, arabinose, maltose, raffinose, ribitol, sucrose and the like, as well as purines, nucleosides, hypoxanthine, guanine, modified amino acids referred to as opines, and heavy metals. These and other useful inducers are described in Weickert & Adhya, supra.
  • the system involves; (i) any regulatory protein which can be used to modulate the level of expression of a heterologous gene by binding, in a reversible manner, to a DNA binding site; (ii) sufficient quantity of this regulatory protein synthesized from a chromosomal gene(s) to match or exceed the number of DNA binding sites for said regulatory protein to which it can bind in the cell; (iii) promoter modifications to the promoter of the gene encoding said regulatory protein to insure production of a desired quantity of the regulatory protein; (iv) one or more DNA binding sites for said regulatory protein present on medium to high copy number plasmids (50-2,000 copies per cell), and; (v) one or more DNA binding sites for said regulatory protein near or in the nucleotide sequence encoding the heterologous gene(s), as part of the transeriptional control mechanism of the heterologous gene(s).
  • the result is an inducible expression system with many different promoter /regulator combinations, in many different prokaryotic organisms.
  • Promoters of regulatory proteins in prokaryotes are typically too weak to allow production of sufficient amounts of regulatory protein to be equal to or in excess of the DNA sites found on medium to high copy number plasmids. For example, there are only about 10 copies of the Lac Repressor in each E. coli cell, far less than sufficient to repress a plasmid of 50-2,000 copies per cell containing one DNA binding site for Lac Repressor per plasmid. Thus there would be no significant regulation of Lac Repressor-regulatable genes on these plasmids in these cells. The level of production of Lac Repressor and other regulatory proteins is typically low because the promoters directing transcription of the genes encoding the regulatory proteins are weak (Table A).
  • Promoter regions in other prokaryotic organisms may similarly be improved to make strong promoters.
  • Table A examples from Bacillus subtilis and from Klebsiella pneumonia are included.
  • the most common (vegetative) promoters for these organisms are similar to those of E. coli, although the strength of the the B. subtilis promoter is also dependent upon the DNA sequence adjacent to the -10 region, especially the proximal 5 basepairs (the extended -10 region).
  • the strength of the promoter for the gene encoding the regulatory protein of interest can be improved by i) improving the DNA sequence homology between the promoter consensus sequence and the regulatory protein gene's promoter sequence, and/or ii) improving the promoter strength by mutation of sequence outside the consensus sequences.
  • consensus sequences of promoters in organisms other than E. coli may not necessarily match the consensus -10 and -35 sequences shown in Table A. This is especially true for organisms with a substantially different nucleotide bias, for example, thermophilic organisms which have a higher G+C content than E. coli.
  • PrafR CTT ⁇ GGTGACGGAATT ⁇ TCTGGATTTCCGGCTTAGAACCACAGCAGGAGATA PlacI: CATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAG
  • PlacIQ CATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAG
  • PlacIQl CATTTACGTjrcAC CCACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAG
  • PgalR GCCACCCCTTGAACCAACGGGCGTTTTCCGTAACACTGAAAGAATGTAAGCG
  • PgalS TGACTCGATTCACGAAGTCCTGTATTCAGTGCTGACAAAATAGCCCGCCAGC
  • PccpA AGTATACGTTTJSATCATCTATAAAAACGTGTATAAT ⁇ TCATGAGAAGTAATB.S. piaci: GGGCGAAGCGCTGTT ⁇ TGTCGCGCGTTAAACATAAAATGTTAGCGCACGA K.p.
  • regulatory protein Since stoichiometry is important, too much regulatory protein needs to be avoided if possible. For example, if the strength of a promoter for a gene encoding a regulatory protein is increased too much, then the abundance of the regulatory proteins may be far greater than the number of sites to which they can bind, even on a high copy number plasmid. In this case, it may require a large amount of inducer to activate transcription of the heterologous gene(s), often an undesirable situation.
  • the heterologous protein After the heterologous protein has been expressed to the desired level, it generally should be released from the cell to create a crude protein solution. This can usually be done by breaking open the cells, e.g., by sonication, homogenization, enzymatic lysis or any other cell breakage technique known in the art.
  • the proteins can also be released from cells by dilution at a controlled rate with a hypotonic buffer so that some contamination with cellular components can be avoided (Shorr et al., US Patent 5,264,555).
  • cells may be engineered to secrete the protein of interest by methods known in the art.
  • the target protein is contained in a crude cell lysate or a crude cell broth or solution.
  • the protein then may be purified according to methods well known in the art.
  • useful purification methods for hemoglobin-like proteins are taught in PCT Publication WO 95/14038, incorporated herein by reference. Briefly, the methods described therein involve an immobilized metal affinity chromatography resin charged with a divalent metal ion such as zinc, followed by anion exchange chromatography.
  • the solution containing the desired Hb-containing material to be purified can first be heat treated to remove Protoporphyrin IX-containing Hb.
  • This basic purification method can be further followed by a sizing column (S-200), then another anion exchange column.
  • this solution can be separated into molecular weight fractions using ion exchange chromatography.
  • the resulting solution can then be buffer exchanged to the desired formulation buffer.
  • Appropriate recombinant host cells can be produced according to conventional methods or as described in the Examples below. Any suitable host cell can be used to express heterologous polypeptides. Suitable host cells include, for example, E. coli cells are particularly useful for expressing the heterologous polypeptides. The proteins, so-produced, can be used for their known purposes.
  • hemoglobin-like proteins and compositions containing the globin-like polypeptides or the multimeric hemoglobin-like proteins can be used for in vitro or in vivo applications.
  • in vitro applications include, for example, the delivery of oxygen for the enhancement of cell growth in cell culture by maintaining oxygen levels in vitro (DiSorbo and Reeves, PCT publication WO 94/22482, herein incorporated by reference).
  • hemoglobin-like protein can be used to remove oxygen from solutions requiring the removal of oxygen (Bonaventura and Bonaventura, US Patent 4,343,715, incorporated herein by reference) and as reference standards for analytical assays and instrumentation (Chiang, US Patent 5,320,965, incorporated herein by reference) and other such in vitro applications known to those of skill in the art.
  • hemolgobin-like proteins also can be formulated for use in therapeutic applications.
  • Example formulations are described in Milne, et al, WO 95/14038 and Gerber et al., PCT/US95/10232, both herein incorporated by reference.
  • Pharmaceutical compositions comprising hemoglobin-like proteins can be administered by, for example, subcutaneous, intravenous, or intramuscular injection, topical or oral administration, large volume parenteral solutions useful as blood substitutes, etc.
  • Pharmaceutical compositions can be administered by any conventional means such as by oral or aerosol administration, by transdermal or mucus membrane adsorption, or by injection.
  • hemoglobins can be used in compositions useful as substitutes for red blood cells in any application where red blood cells are used, or for any application in which oxygen delivery is desired.
  • Such hemoglobins, formulated as to red blood cell substitutes can be used for the treatment of hemorrhages, traumas and surgeries where blood volume is lost and either fluid volume or oxygen carrying capacity or both must be replaced.
  • a restriction map of the entire E. coli genome was determined from a set of ordered Lambda clones by Kohara et al, (1987). DNA sequences of known genes have been placed on this ordered map (Rudd et al, 1992. Alignment of E. coli DNA sequences to a revised, integrated genomic restriction map, p. 2.3-2.43. In J. Miller (ed.), A short Course in Bacterial Genetics: a Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor Prss, Cold Spring Harbor, NY). The combination of known restriction site sequence with known gene sequences was used to create a variation on anchored PCR, which was then used to clone the DNA upstream of the lac operon in E. coli.
  • Anchored PCR relies on two primers, one of known sequence and one of random sequence. Because the sequence of the flanking restriction sites is known in E. coli, a portion of the sequence of the second, random PCR primer could be specified. The size of the expected fragment could be predicted, depending upon the position of the second primer designed from the known DNA sequence.
  • the sequence upstream of the lad gene in E. coli was determined because of its potential role regulating the amount of Lac repressor synthesized within the cell.
  • the sequence of the lad gene is known (Calos, 1978; Farabaugh, 1978), but the published sequence ends just beyond the lad promoter region.
  • a region from a wild type E. coli was cloned and sequenced to establish sequence from which primers could be designed that would allow PCR amplification of lad alleles from several strains.
  • An anchored PCR technique was developed in which one primer contained a known sequence internal to lad and primed toward the promoter region, and a second primer was partially random and primed upstream of the promoter region, towards the promoter.
  • the second primer, 12 nucleotides long contained a fixed half and a random half.
  • the 3' (priming) end contained 6 nucleotides from a restriction site known to be upstream of PlacI, and the 5' 6 nucleotides were composed of equal proportions of each nucleotide at each of the six positions.
  • a product was obtained and re-amplified by PCR using a second primer homologous to a different lad sequence from the first primer, but also containing a second site for subcloning into pUC19.
  • the insert was sequenced and contained the expected lacl sequence and 675 bp upstream of the lacl promoter region, which was not homologous to any sequence in the nucleotide databases. A large open reading frame, divergent from lacl , was also observed. Using this sequence, a specific nucleotide primer was designed, synthesized, and used to amplify PlacI from the E. coli chromosome of
  • This technique may be used to close small sequencing gaps when a map is known, and quickly extend cloning and /or sequencing into mapped regions adjacent to known sequence, from any DNA source.
  • Recombinant hemoglobin-like protein (rHbl.l, described in, for example, PCT publication WO 90/13645, incorporated herein by reference) was produced by fermentation of several E. coli strains containing modifications of the lac promoter region and/or location. Construction of the parent E. coli strain 1661 carrying the plasmid pSGE705 is described below. On January 20, 1994 E. coli strain SGE1661 was deposited with the American Type Culture Collection (ATCC Accession Number 55545). Note that Strain SGE1661 carrying the plasmid pSGE705 was denoted SGE1662 (described in PCT publication number WO 95/14038, incorporated herein by reference). pSGE705 was a medium copy number plasmid because it is present in approximately 100 copies per cell. The plasmids used in preparing pSGE705 are identified in Table 1, which also provides a brief description of each.
  • pBR322, pUC19 and ⁇ NEB193 were purchased from New England Biolabs, Beverly, Massachusetts. Oiigonucleotides were synthesized on an Applied Biosystems DNA Synthesizer Model 392. The oiigonucleotides used in preparing pSGE705 are listed in Table 2. Restriction endonucleases were purchased from New England Biolabs (Beverly, Massachusetts) and used according to manufacturer's directions. T4 DNA Ligase was purchased from either New England Biolabs or Gibco-BRL (Gaithersburg, Massachusetts) and used according to manufacturer's directions. Pfu polymerase was purchased from Stratagene (La Jolla, California) and used according to manufacturer's directions.
  • pSGEl.lE4 rHbl.l expression plasmid containing di-alpha and beta globin genes and resistant to both ampicillin and tetracycline pSGEl.lE ⁇ like pSGEl.lE4 is ampicillin resistant only pSGE490 pUC19 lad on a B ⁇ mHI-Hz ' ndlll fragment pSGE491 pUC19 oc on an EcoRl-Xba ⁇ fragment pSGE492 pNEB193 Ptac- ⁇ pSGE493 pUC19 ⁇ on an Xba I-HmdIII fragment pSGE500 pUC19 ⁇ ⁇ on a B ⁇ mHI-H dIII fragment pSGE504 pSELECT-1 replace Sty I with a Pme I site pSGE505 pSGE504 rrnB Tl transeriptional terminator in the Eco RI- C l sites pSGE507 ColEl
  • Plasmid DNA Transformation Plasmid DNA Transformation. DNA transformations were performed by the procedure described in Wensick et al., Cell 3: 315-325 (1974). Briefly, cells were grown to mid log phase and then pelleted, resuspended in an equal volume of 10 mM MgS ⁇ 4 and incubated on ice for 30 minutes. The cells were centrifuged and the pellet resuspended in 1/2 the original volume of 50 mM CaCl2 and placed on ice for
  • Plasmid DNA was added to the competent cells in a solution of 10 mM Tris-HCl, pH 8.0, 10 mM MgCl2 and 10 mM CaCl2* The mixture was incubated on ice for 15 minutes and then incubated at 37°C for 5 minutes. One milliliter of LB medium was added and the mixture incubated with shaking for 30-60 minutes. The culture was then centrifuged, resuspended in 0.1 ml of LB medium and plated on the appropriate selective medium.
  • DNA fragments were purified from an agarose gel using the Geneclean system (Bio 101, Inc., La Jolla, CA) according to the method provided with product.
  • PCR products were prepared and cleaved with restriction endonucleases using the Double Geneclean system. (Bio 101, Inc., La Jolla; method provided with product.) Briefly, the PCR product was purified away from the PCR primers, then the PCR product was cleaved with restriction endonuclease(s) and purified from the restriction endonuclease and buffer. The PCR product was then ready for a ligation reaction.
  • oiigonucleotides were annealed according to the following procedure. Equimolar amounts of each oligonucleotide were mixed in 15-25 ⁇ l of 10 mM Tris-HCl, pH 8.0/1 mM EDTA and incubated at 65°C for 30 minutes. The sample was transferred to a 37°C water bath for 30 minutes. Finally, the sample was incubated on ice for 60 minutes or in the refrigerator overnight.
  • Oligonucleotide directed mutagenesis was performed with the Muta-gene phagemid in vitro mutagenesis kit (Bio-Rad, Hercules, California) according to the manufacturer's instructions which are based on the method of Kunkel (Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82: 488; Kunkel et al., (1987) Methods Enzymol. 154: 367).
  • Kunkel Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82: 488; Kunkel et al., (1987) Methods Enzymol. 154: 367).
  • the rHbl.l region of pSGE515 was cloned into pTZ18U (Bio-Rad, Hercules, CA or U.S.
  • PCR primers were designed to amplify the pBR322 origin of replication. These primers, TG62 and TG63, annealed to the positions 2380-2404 and 3170-3148 on the pBR322 DNA sequence (Sutcliffe, J. G. (1979) Cold Spring Harbor Symp. Quant. Biol. 43: 77-90).
  • the PCR product was digested with NotI and Pmel. The DNA fragment was purified according to the Geneclean procedure.
  • This plasmid has a number of restriction endonuclease sites, such as BamHI, Hindlll, Sail and SphI removed from the tet gene (Lewis and Thompson (1993) Nucleic Acids Res. 18:3439-3443).
  • a Pmel linker was inserted into the Styl site of pSELECT-1. This plasmid was designated pSGE504.
  • pSGE505 The resulting plasmid, pSGE505, was shown to have the expected restriction endonuclease sites and to have lost the sites present in the multicloning site of pSELECT-1.
  • pSGE505 was digested with NotI and Pmel. The 1417 bp fragment was purified according to the Geneclean protocol.
  • the lacl gene was isolated by amplifying the gene sequence from pRGl (Dana-Farber Cancer Inst., Boston) that carried the lad gene.
  • the PCR primers, TG59 and TG60 were designed to generate a wild type lacl promoter (Farabaugh, P. J. (1978) Nature 274:765), upstream of the gene and to place the trp terminator sequence (Christie et al., (1981) Proc. Natl. Acad. Sci. USA 78:4180- 4184) downstream of the gene.
  • the same step could be carried out using Y1089
  • telomere sequence from any E. coli strain carrying the lac region, such as MM294 (ATCC 33625.)
  • the PCR product was gel purified and isolated according to the Geneclean procedure and cloned into BamHl-Hindl ⁇ l digested pUC19 DNA to make pSGE490.
  • PCR primers EV29 and EV18 were chosen to amplify the alpha globin gene from pDLII-91F (Hoffman et al., WO 90/13645).
  • the purified PCR product was cleaved with the restriction endonucleases EagI and Xbal.
  • Primers EV30 and EV31 were used to amplify the ⁇ globin gene from pSGEl.lE4 by PCR.
  • the purified ⁇ gene fragment was digested with Xbal and Hindlll and then mixed with Xbal-Hindll digested pUC19 DNA and treated with T4 DNA ligase.
  • the ligation mixture was used to transform competent SGE476 (equivalent to MM294, ATCC 33625) and transformants were selected on LB + ampicillin (100 ⁇ g/ml) plates.
  • An isolate that contained the appropriate restriction endonuclease fragments was chosen and designated pSGE493.
  • the ⁇ gene was confirmed by DNA sequencing.
  • the ⁇ gene was isolated from pSGE493 by restriction with Xbal and HindlU followed by purification according to the Geneclean method. This DNA fragment was then ligated to Xbal-Hindlll restricted pSGE492 DNA and transformed into SGE713. (Any da r strain such as JM110 (ATCC 47013) or GM119 (ATCC 53339) could also be used.) An ampicillin resistant transformant that carried a plasmid that had the appropriate restriction fragments (consistent with Figure 1) was chosen and designated pUC19 ⁇ (pSGE500).
  • the BamH ⁇ -Hind III fragment that contained the ⁇ and ⁇ globin genes of pSGE500 was purified according to the Geneclean method.
  • An Xhol fragment that carried a portion of the di- ⁇ gene containing the glycine linker region was gel purified from pSGEl.lE5.
  • pSGEl.lE5 (described in Hoffman et al., United States Serial Number 789,179, filed November 8, 1991) is a tetracycline sensitive analogue of pSGEl.lE4 (Hoffman et al., WO 90/13645), which could also have been used.
  • pBR322 origin of replication region (pBR322 ori, above) was ligated to the tet gene fragment (above) and the ligation mixture was transformed into SGE476. (Transformation into MM294, above, would yield equivalent results.) Tetracycline resistant transformants were selected and plasmid DNA was isolated and analyzed. An isolate that contained the appropriate restriction endonuclease fragments was chosen and designated pSGE507.
  • pSGE507 and SGE490 were digested with BamHI and NotI and the appropriate fragments were purified. The two purified fragments were ligated together and the ligation mixture was used to transform competent SGE713. (Any dam' strain could also be used; see above.) Tetracycline resistant transformants were
  • plasmid DNA was isolated and analyzed.
  • a plasmid that had the appropriate restriction fragments was chosen and designated pSGE509.
  • the purified BamHl-HindUl fragment of pSGE500 that contained the ⁇ and ⁇ globin genes was ligated to BamHl-Hindl ⁇ l digested pSGE509.
  • the ligation mixture was used to transform pSGE713 (see above for equivalent strains) and tetracycline resistant transformants were selected and characterized.
  • An isolate yielding the correct size plasmid with the expected restriction endonuclease fragments was chosen and designated pSGE513.
  • the Xhol fragment of pSGEl.lE5 (described in Hoffman et al., United States Serial Number 07/789,179, filed November 8, 1991, incorporated herein by reference) that contained the di- ⁇ glycine linker sequence was ligated to Xhol digested ⁇ SGE513 to create a plasmid that contained the di- ⁇ gene.
  • SGE753 was transformed with the ligation mixture and tetracycline resistant transformants were selected. (Transformation into SGE800 would have yielded equivalent results.) Isolates were screened to identify those that contained the Xhol fragment inserted into pSGE513 in the correct orientation.
  • MW007 introduced the coding sequence for the last three amino acids of the beta gene as shown above. ( I - indicates identity, * - indicates a change)
  • Putative mutants were screened for loss of a Bglll restriction endonuclease cleavage site (introduced by MW008). Seventeen of 24 had lost the site and were further characterized by DNA sequencing at the other two mutagenized sites. One of the 17 had incorporated all the modifications from the three oiigonucleotides. These changes were verified by DNA sequencing and the rHbl.l genes were cloned into BamHI-Hmdlll digested pSGE509. An isolate that had the correct restriction endonuclease fragments was designated pSGE705. A plasmid map of pSGE705 is shown in Figure 1.
  • the plasmid map indicates many of the restriction endonuclease cleavage sites.
  • pSGE705 is smaller than its counterpart pSGEl.lE4, and the placement of its restriction sites facilitates modular alterations of the sequence.
  • An unused antibiotic resistance marker was removed, and a promoter was added to the lacl gene that would allow tighter control of rHbl.l expression.
  • a new sequence upstream of the ⁇ gene minimized the distance between the tac promoter (De Boer et al, Proc. Natl. Acad. Sci. USA 80, 21-25, 1983) and the first codon of the alpha gene.
  • the intergenic region between the di- ⁇ gene and the ⁇ gene was also designed to contain the minimum sequence that contained a restriction endonuclease site and the ribosome binding site for the ⁇ gene.
  • pSGE720 The construction of pSGE720 was performed in two stages. First, the pUC origin of replication was introduced into PSGE705 to create plasmid pSGE715, which is similar to pSGE705 in that it includes the lacl gene. Then, the lacl gene was deleted from the plasmid and replaced with a short oligonucleotide containing several convenient restriction sites to create plasmid ⁇ SGE720.
  • the pUC origin of replication was introduced to create plasmid pSGE715 through pSGE508, which is identical to pSGE509 with the exception of a single basepair substitution at base 602 (G- ⁇ A).
  • the substitution changes the pBR322 origin of replication to a pUC19 origin of replication.
  • Plasmids pSGE508 and pSGE705 were digested to completion with restriction enzymes BamHI and Hmdlll, according to the manufacturer's instructions (New England Biolabs.).
  • the plasmid, pSGE508, was digested first with BamHI to completion, then Hmdlll was added, and the digestion continued.
  • the pSGE705 digest was purified with Promega Magic DNA Clean-up protocols and reagents (Promega, Madison, WI) and further digested to completion with Bgll, according to the manufacturer's instructions (New England Biolabs).
  • the enzymes in both the pSGE508 and pSGE705 digests were inactivated by heating at 67°C for 10 minutes, then the DNA was pooled and purified together using Promega Magic DNA Clean ⁇ up protocols and reagents.
  • the DNA was suspended in ligation buffer, T4 DNA ligase was added to one aliquot, and the DNA was incubated overnight at 16°C.
  • SGE1661 cells were made competent by the method of Hanahan, using Rubidium Chloride (Hanahan, D., In DNA Cloning; A Practical Approach (Glover, D. M., ed.) vol. 1, pp.109-135, IRL Press, Oxford, 1985), and transformed with the ligation mix according to the Hanahan protocol.
  • Transformants were selected by plating the cells on LB plates containing 15 ⁇ g/ml tetracycline. Candidates were screened by restriction digestion to determine the presence of the rHbl.l genes, and sequencing to detect the pUC origin of replication. Several candidates were identified, and the resulting plasmid was named pSGE715. pSGE715 in SGE1661 was called SGE1453. The copy number of pSGE715 is about four-fold higher than pSGE705, measured to be about 460 plasmids per cell. As noted above, the difference between pSGE705 and pSGE715 is a single basepair change in the origin of replication region, which has been confirmed by sequencing.
  • the lad gene was deleted from pSGE715, replacing it with a short oligonucleotide containing several convenient restriction sites, by the following steps.
  • plasmid pSGE715 was digested to completion with restriction enzymes Bflr ⁇ HI and NotI, according to the manufacturer's instructions (New England Biolabs).
  • the pSGE715 digest was purified with Promega Magic DNA Clean-up protocols and reagents.
  • the DNA was mixed with annealed, kinased oiigonucleotides, CBG17 + CBG18, and suspended in ligation buffer.
  • CBG17 5'-GGCCGCCTTAAGTACCCGGGTTTCTGCAGAAAGCCCGCCTA
  • CBG18 5'-GATCCCCTAAGGAAAAAAAAGCCCGCTCATTAGGCGGGCTTT
  • SGE1821 cells SGE1661 + pRGl (pACYC177 with laclQ) (Dana- Farber Cancer Institute, Boston, MA) were made competent by the method of Hanahan, using Rubidium Chloride, and transformed with the ligation mix according to the Hanahan protocol.
  • SGE1821 contains pRGl plasmids in addition to pSGE720.
  • pRGl is a low copy number plasmid containing Lac .
  • Transformants were selected by plating the cells on LB plates containing 15 ⁇ g/ml tetracycline.
  • pSGE720 The plasmid, pSGE720 in SGE1675 is called SGE1464.
  • Strains SGE1494 and SGE1495 which contained lacH on the chromosome were purchased from ATCC, (ATCC accession numbers 47041 and 47043 respectively).
  • SGE77 was purchased from Stratagene, Inc. (Catalogue number D1210) and also contained a lacN on an F' episome.
  • Strain SGE1661 contained a wild type chromosomal lad allele. This strain was used as the starting material for the construction of strains containing alternative chromosomal lad alleles.
  • strain SGE1670 was constructed.
  • Strain SGE1670 containing the laclfl allele was constructed from SGE1661 by Pl bacteriophage transduction using a lysate grown on SGE299, selecting for kanamycin resistant transductants.
  • Strain SGE299 contained a putative lacll ⁇ allele on an F' episome adjacent a l ⁇ cZ gene into which a Tn5 transposon has been inserted to inactivate l ⁇ cZ. The transposon insertion conferred kanamycin resistance to the cells.
  • SGE299 is also know as
  • strain SGE1675 the l ⁇ c operon functionality on the chromosome was restored without affecting the l ⁇ cl allele.
  • the presence of the transposon insertion in l ⁇ cZ in SGE1670 resulted in a polar mutation that destroyed the function l ⁇ cY and l ⁇ cA involved in lactose metabolism and transport, which may have affected the ability to induce expression of Pt ⁇ c on the plasmid with IPTG, and thus may have affected beta-galactosidase or hemoglobin production from a given plasmid.
  • SGE1675 was constructed by transduction of SGE1670 from a Pl lysate prepared on SGE765.
  • SGE765 was a strain containing a Tn5 insertion into the l ⁇ cl gene conferring kanamycin resistance to the cells. In addition, SGE765 contained a wild type copy of l ⁇ cZ. Strain SGE765 was made by Pl transduction from a lysate made on MS24 into strain C3000. MS24 is also known as MG1655 lacI3098::lnl( n , acZAll ⁇ , and is described in Singer, et al. (Microbiol. Rev. 53: 1-24, 1989). Strain C3000 is available from the American Type Culture Collection, ATCC # 15597.
  • Transductants were selected for their ability to grow on minimal medium containing lactose as the sole carbon source and screened for sensitivity to kanamycin. Transductants that were sensitive to kanamycin and were lac + , were screened for their ability to regulate (repress) the expression of beta-galactosidase from plasmid pSGE714 (see below). Note: pSGE714 does not contain a lad on the plasmid and was examined for repression of lacZ as described for strain SGE1670.
  • pSGE712 containing the lad gene and a fusion of the lacZ gene to the 5' end of the beta globin gene was constructed as described below. This construct provided a convenient tool for screening control of expression as a function of beta- galactosidase activity in the cells. Thus in the absence of inducer, the expression of beta-galactosidase provided a sensitive measure of control of expression by the product of the lac repressor gene expressed primarily from the plasmid.
  • pSGE705 was digested with SaR and Bglll.
  • pMBL1034 obtained from Sankar Adhya, NIH, and described in Miller, G.
  • pSGE714 containing a fusion of the lacZ gene to the 5' end of the beta globin gene was constructed as described in Example 4, except that pSGE654 was used instead of pSGE705 as the starting material. Note that unlike pSGE712, pSGE714 did not contain lad. This construct provided a convenient tool for screening control of expression as a function of beta-galactosidase activity in the cells. Thus, in the absence of inducer, the expression of beta-galactosidase provided a sensitive measure of control of expression by the product of the lac repressor gene expressed from the chromosome of the cell. EXAMPLE 6
  • pSGE721 containing a fusion of the lacZ gene to the 5' end of the beta globin gene was constructed as described in Example 4, except that pSGE720 was used instead of pSGE705 as the starting material.
  • This construct provided a convenient tool for screening control of expression as a function of beta-galactosidase activity in the cells.
  • the expression of beta-galactosidase provided a sensitive measure of control of expression by the product of the lac repressor gene expressed from the chromosome of the cell.
  • This plasmid results in approximately 500 copies of the plasmid per cell.
  • more repressor must be expressed from the chromosomal copy of lad per cell than there are plasmids per cell.
  • pSGE728 was performed by digesting plasmid pSGE720 with an enzyme that cuts only within each of the two alpha subunits of the di-alpha gene encoding the globin-like protein, followed by ligation, to preferentially reconstruct deletions of one alpha subunit, and the di-alpha glycine linker.
  • the resulting plasmid, pSGE726, contains a single alpha gene rather than a di-alpha gene.
  • the Presbyterian mutation of human hemoglobin in the beta globin gene was replaced by a second digestion and ligation that introduced the wild-type beta and created pSGE728.
  • pSGE726 pSGE720 was digested with restriction enzyme Xhol, according to the manufacturer's instructions (New England Biolabs). The pSGE720 digest was purified with Promega Magic DNA Clean-up protocols and reagents (Promega, Madison, WI) and suspended in ligation buffer. T4 DNA ligase was added and the DNA was incubated overnight at 16°C. SGE1675 cells were made competent by the method of Hanahan (Hanahan, D, ibid), and transformed with the ligation mixture according to the referenced protocol. Transformants were selected by plating the as cells on LB plates containing 15 ⁇ g/ml tetracycline.
  • the small DNA band of about 300 basepairs (bp) from the pSGE0.0E4 digest, and the large band of about 3,400 bp from the pSGE726 digest, were purified by excising them from the agarose gel, pooling them into a 1.7ml tube and purifying them with GeneClean Kit ptotocols and reagents (BIO 101, La Jolla, CA). Purified, pooled DNA fragments were suspended in ligation buffer. T4 DNA ligase was added and the DNA was incubated overnight at 16°C.
  • SGE1675 cells were made competent by the method of Hanahan (Hanahan, D, ibid), and transformed with the ligation mixture according to the referenced ptotocol. Transformants were selected by plating the cells on LB plates containing 15g/ml tetracycline. Candidates were screened by restriction digestion with Sea I according to the manufacturer's instructions (New England Biolabs), to determine the presence of a wild type beta globin gene, which is not susceptible to Seal digestion, rather than the betaP- res by ter i an gene which is cleaved by Scfll. Several candidates were identified. The resulting plasmid was named pSGE728. pSGE728 in SGE1675 was called SGE1483. This plasmid expressed rFIbO.O, mono-alpha plus wild type beta.
  • Plasmid copy-number was determined by comparison of the band intensity of linearized plasmids to that of known quantities of HmdIII-digested lambda DNA (New England Biolabs) on Ethidium Bromide-stained TAE agarose gels. Plasmid DNA was extracted from bacterial cultures using the Wizard Miniprep Kit (Promega). The OD600 of these cultures was determined before plasmid purification. Using the conversion factor of 8 x IO 8 cells/OD*ml, the OD600 the culture volume used, and the mass of DNA determined by comparison to the standard, the copy number was calculated.
  • Beta-galactosidase was measured using the technique described in Miller. Cells were grown with agitation in supplemented minimal medium (M63 + casamino acids) at 37 or 30°C in 25 ml flasks or 20 ml test tubes. Expression was induced by the addition of various concentrations of IPTG to the cells in mid-log phase (OD 600 nm >0.2, but ⁇ 0.8) and incubation was continued for 3-5 hours before sampling for beta-galactosidase analysis.
  • Hemoglobin-like proteins are expressed in the strains described above using a standard fermentation protocol. First, a fermentor inoculum is grown from seed stock. The inoculum is then transferred to a 15 liter fermentor and induced. The details of the fermentation process are described below. Seed Stock
  • Seed stock is grown up in LB broth containing 10 g/L BactoTryptoneTM, 5 g/L yeast extract, 5 g/L NaCl, 0.2 g/L NaOH, and 10 ug/ml tetracycline to an optical density of 1.5 - 1.7 at 600 nm. The solution is then, made up to 10% glycerol and stored at -80°C until required.
  • Fermentor Inoculum 500 ml broth in 2 L shake flasks
  • seed stock is thawed and 0.1-0.4 ml of seed stock are inoculated into 500 ml of a solution containing approximately 4 g/L KH 2 P0 , 7 g/L K 2 HP0 , 2 g/L (NH 4 ) 2 S0 4 1 g/L Na 3 Citrate-2H 2 0, 153 mg/L MgS ⁇ 4 -7H 2 ⁇ , 2.3 g/L of L-proline, 2 g/L yeast extract, 4.8-5 g/L glucose, 75 mg/L thiamine HCl, 12 mg/L tetracycline, 81 mg/L FeCl -6H 2 0, 4 mg/L ZnCl / 6 mg/L CoCl 2 -6H 2 0, 6 mg/L Na 2 Mo0 4 -2H 2 0, 3.1 mg/L CaCl 2 -2H 2 0, 3.9 mg/L Cu(II)S0 -5H 2 0, 1.5 mg/L H 3 B ⁇ 3
  • the entire fermentor inoculum is then asceptically transferred to a 20-liter fermentor containing 10 liters of the following: 1.8 g/L KH 2 P0 4 , 3.3 g/L K 2 HP0 4 ,1.8 g/L (N ⁇ L 4 )2S0 4 , 155 mg/L thiamine HCl , 10.3 mg/L tetracycline, 3.1 g/L proline, 1.9 g/L MgS0 -7H 2 0, 1.9 g/L Na 3 -citrate-2H 2 0, 133 mg/L FeCl 3 -6H 2 0, 6.4 mg/L ZnCl 2 , 9.9 mg/L CoCl 2 -6H 2 0, 9.9 mg/L Na 2 Mo0 -2H 2 0, 5 mg/L CaCl 2 -2H 2 0, 6.3 mg/L Cu(H)S0 4 -5H 2 0, 2.5 mg/L H3BO3, and 494 ⁇ l/L HCl.
  • the entire seed fermentor inoculum is then asceptically transferred to a 600- liter fermentor containing approximately 375 liters of the solution containing: 1.8 g/L KH 2 PO4, 3.3 g/L K 2 HP0 , 1.8 g/L (NH ) 2 S0 3.3 ml/L polypropylene glycol- 2000, 220 g/L glucose, 143 mg/L thiamine HCl, 9.4 mg/L tetracycline, 1.4 g/L MgS0 -7H 2 0, 1.4 g/L Na 3 -citrate-2H 2 0, 2.9 g/L L-proline, 99 mg/L FeCl 3 -6H 2 04, .8 mg/L ZnCl 2 , 7.3 mg/L CoCl 2 -6H 2 0, 7.3 mg/L Na 2 Mo0 -2H 2 0, 3.7 mg/L CaCl 2 -2H 2 0, 4.7 mg/L Cu(II)S0 -5H 2 0, 1.8 mg
  • the pH is maintained at 6.8 - 6.95 by addition of 15% to 30% NHjOH, dissolved oxygen is maintained at or above 20%, and 50-70% glucose is added throughout the growth period, sufficient to maintain low but adequate levels of glucose in the culture (0.1 g/L-10 g/L).
  • the culture is grown between 24 and 30°C to an OD 60 0 ⁇ 10-40 prior to induction with 10-1000 ⁇ M IPTG.
  • the E Upon induction of hemoglobin synthesis, the E.
  • coli heme biosynthesis is supplemented by addition of hemin dissolved in 1 N NaOH, either by addition of the total mass of hemin required at induction, by continuous addition of hemin throughout the induction period, or by periodic addition of hemin dissolved in 50 mM to 1 M NaOH (e.g. one third of the total mass of hemin to be added to the fermentor is added at induction, another third is added after 1/4 of the total time
  • Total hemin added ranges from 50 to 300 mg/L.
  • the fermentation is allowed to continue for 8-16 hours post-induction. At the end of this period, several 1 ml aliquots are removed from the broth for determination of hemoglobin production.
  • the fermentor is run at several temperatures (see further examples below), controlling dissolved oxygen at 20% and glucose between 0-6 g/L.
  • induction occurs by adding various amounts of 100 mM IPTG to yield several different concentrations of inducer, as further described below.
  • 10 mL of 50 mg/mL hemin is added along with the appropriate volume of IPTG.
  • 13 mL of 50 mg/mL hemin is added and at 6 hours post induction, 17 mL of 50 mg/mL hemin is added.
  • Harvest and further purification occurs at 10-16 hours post induction.
  • the samples were placed on ice and protected from light. 1.00 ⁇ 0.01 mL of 25.0mM Na2B4 ⁇ 7*10H2 ⁇ was added to the cell pellet and the sample was vortexed until the contents were resuspended. 30 ⁇ 1 ⁇ L of Lysozyme NaCl solution was added to the resuspended pellet and the sample was re-vortexed. The samples were incubated on ice for 30 - 45 minutes while protected from light. Following this incubation, the samples were mixed at least three times and placed in a 37° ⁇ 1°C water bath for 2.25 ⁇ 0.25 minutes. After incubation at 37°C, the samples were again mixed.
  • Hemoglobin was extracted from the samples after cell lysis by first freezing at
  • hemoglobin solutions Five microliters of hemoglobin solutions were added to 500 ⁇ l of 0.1 M Tris, pH 8.0. 200 ⁇ l of the diluted hemoglobin solution was then added to 2.8 ml of 0.1 M Tris, pH 8.0, in a 4.5 ml cuvette for a final dilution of 1:1500.
  • the oxygenated sample (Hb02) was then analyzed by spectrophotometry in a Hewlett-Packard model HP 8452A spectrophotometer. Absorbances at 436, 425, 420, 404, 400 nm were collected and stored in a data storage system. The cuvette was then removed from the spectrophotometer and sparged with carbon monoxide two times for 15 seconds each time.
  • the cuvette was inverted 5 times between each sparge. The sample was then re-inserted into the spectrophotometer, and a second set of spectra were collected that corresponded to carbonmonoxy hemoglobin (HbCO). The cuvette was then again removed from the spectrophotometer and 30 ⁇ l of 0.1M KCN in 0.1 M Tris, pH 8.0, was added to the sample. The sample was then inverted three times, allowed to incubate for 5 minutes, and re-inserted into the spectrophotometer for a final spectrophotometric analysis (HbCN). The concentration of total hemoglobin was determined using the following quantities:
  • A the absorbance at the susbcripted wavelength for the superscripted hemoglobin species.
  • the pellet (insoluble fraction) was resuspended with 1 ml of 1% SDS in 25 mM sodium tetraborate and diluted such that the same volume of solublized pellet and water were used as were used to dilute the corresponding soluble fraction.
  • Each fraction was diluted 1:1 with 2x SDS sample buffer (125 mM Tris-HCl, 20% glycerol, 2% SDS, 2% beta-mercaptoethanol, 0.01% bromophenol blue). 10ml of each sample was applied to a 12% SDS-PAGE gel and run overnight (approximately 14 hours) at 80 N constant voltage.
  • the gel was transferred onto ProBlott Membrane (Applied Biosystems #400994) in 10 mM CAPS, pH 11, 10% MeOH for two hours at 400 mA (Hoefer TE22).
  • the blots were blocked for one hour to overnight in Tris Buffered Saline plus Tween 20 (TBST, 10 mM Tris pH 8.2, 150 mM ⁇ aCl, 0.05% Tween 20) plus 5% Food Club brand ⁇ on Fat Dry Milk ( ⁇ FDM).
  • the first antibody was an affinity purified goat anti- rHb antibody.
  • the antibody was diluted 1:2500 in TBST plus 1% ⁇ FDM and rocked at room temperature for one hour.
  • HRP Horseradish peroxidase
  • Protein G (Calbiochem catalogue #539322, San Diego, California).
  • Stock protein G-HRP was diluted 1:5000 with TBST plus 0.5% ⁇ FDM. Blots were incubated with rocking for 1- 2 hours. Three final TBST washes of five minutes each were performed. Blots were developed for one minute with LumiGLO Substrate (Kirkegaard & Perry Laboratory, #54-61-00), covered with plastic wrap and exposed to X-Ray film (Amersham HyperMm-ECL #RP ⁇ . 2103).
  • Beta-galactosidase expression in the IPTG treated fractions was monitored to ensure that expression levels were consistently inducible. Beta-galactosidase activity was also determined from a sample of the cultures that were not treated with IPTG. In the absence of inducer, beta-galactosidase expression was lowest in strains that contained a lad gene on the plasmid or in any of the strains derived from strain SGE1675 which contained a chromosomal lacV - allele (Table 3). Thus, sufficient Lac repressor was produced from the chromosomal lad allele to control expression from Ptac on both medium and high copy number plasmids which did not contain lacl.
  • laclQ on the chromosome did not control expression of a beta-LacZ fusion from high copy number (-500 copies per cell) plasmid (pSGE721).
  • laclQ on the chromosome was able to control expression of a medium copy plasmid (-100 copies per cell; pSGE714) fairly well.
  • a medium copy number plasmid which contained its own lad gene with the native lad promoter (pSGE712) was capable of controlling expression of this well (-100 copies per cell).
  • lacl+ or laclQ on the chromosome did not control expression of a beta-LacZ fusion from high copy number (-500 copies per cell) plasmid (pSGE721 in SGE77, SGE1469 and SGE1495 above).
  • laclQX on the chromosome was capable of controlling expression of the high copy plasmid the best of the strains above. Note that SGE1674 and 1675 are sibs from the same transduction.
  • SGE1662 does not produce insoluble rHbl.l. All the globin synthesized is soluble. SGE1464 produces a substantial proportion of insoluble globin protein, usually approximately equivalent to the amount of soluble protein. Therefore, temperature, which is known to improve solubility and /or stability of some proteins, can only affect the strain with a reservior of insoluble rHbl.l, SGE1464. Overproduction of the protein through increased gene dosage and improved inducibility created an opportunity for increased soluble globin expression that lower temperatures allowed further exploitation.
  • SGE1483 is the same as strain SGE1464 except SGE1483 contains pSGE728 instead of pSEG720.
  • Soluble hemoglobin was measured using the IMAC procedure described above. Soluble hemoglobin expression was enhanced when the cultures were grown at lower temperatures. 28°C significantly improved soluble hemoglobin expression for the high copy number system (SGE1483 -Table 8).
  • the proportional relationship (stoichiometry) of the regulatory protein and the DNA binding site to which it binds helps determine the level of control of gene expression in the absence of inducing conditions, and the amount of inducer required for full induction of gene expression from the promoter so-controlled.
  • Lad is the regulatory protein and the plasmid contains one DNA binding site for Lad on each copy in the cell.
  • the plasmid copy number is estimated as described (supra), and the Lad level is estimated from relative transcription levels (infra).
  • the ratio of Lad to plasmid must exceed 1:1 in order to control expression of gene(s) whose transcription is regulated by Lad, on every plasmid in the cell.
  • LacZ An excess of Lac Repressor is produced by plasmids containing a lad gene, due to the increased dosage of the gene.
  • Table 10 the chromosomal expression of LacZ, also controlled by Lac Repressor, indicates the induction achieved by IPTG addition in the same way as globin gene expression, but is a much more sensitive and easily assayed indicator than globin expression.
  • the LacZ expression as a percent of that achieved by 1,000 ⁇ IPTG addition for SGE1662 versus SGE1464, demonstrates the induction of expression by low IPTG concentrations when lower LacLplasmid ratios exist.
  • Genomic DNA from strain C600 E. coli Genetic Stock Center
  • Genomic DNA from strain C600 E. coli Genetic Stock Center
  • SDS and proteinase K treatment DNA was purified by phenol extraction and EtOH precipitation. The DNA was spooled from the EtOH solutions and rinsed.
  • a map-based Polymerase Chain Reaction (PCR) cloning method was used to amplify the region upstream of the lad gene. The Kohara map predicts that there is a PstI site at about 600 bp upstream from the lad promoter.
  • a 12 nucleotide-long oligo with a degenerate 5' half and the recognition site for Pst I at it's 3' end, and an oligonucleotide complementary to a region just downstream from the initiatation of translation site in the lad gene (CBG23): AGT CAA GCT TAA CGT GGC TGG CCT GGT T were used in the PCR reaction.
  • CBG23 contained the recognition site for Hindlll. 50 pmole of CBG24 (5'-NNNNCTGCAG-3') and 10 pmole of TG45 (5'-CTGGCACCCAGTTGATCG-3') were used per 100 ⁇ l reaction. Pfu polymerase (Stratagene, La Jolla, CA) was used.
  • SGE1661, SGE1670, and SGE1675 was prepared as above.
  • CBG23 and CBG30 were used to amplify the lad promoter region.
  • the PCR products were cloned as before into the PstI and Hindlll sites of pUC19 or pBC SK+ (Stratagene; La Jolla, CA). Insert-containing clones were identified by restriction digest and inserts were sequenced as before.
  • RNA Isolation and Primer-Extension Analysis were performed by a hot-phenol extraction method.
  • RNA and labeled primer were mixed in annealing buffer and heated to 65°C for 10 minutes, followed by 50°C for 40 minutes. The mixture was then placed on ice and incubated for 15 minutes.
  • Superscript II (Stratagene, Inc.) was used to synthesize cDNA according to manufacturer's instructions except for the following: 100 ⁇ g/ml vanadyl-ribonucleoside complex, 10 mM actinomycin D, RNasin were added. Reactions were precipitated with sodium acetate and ethanol.
  • Lad promoter region PCR products from strains SGE1661 an SGE1675 show two and sometimes three bands for most strains.
  • RV308 has a large deletion of the Lac operon region but still has the upper band which was therefore believed to be an artefact of the PCR.
  • Sequence analysis of this band revealed that it had a sequence unrelated to Lad. No sequence matches were found in database comparisons. The lowest bands are also believed to be artefacts. Two sizes of intermediate bands were observed. The lower was estimated to be 10 to 15 basepairs shorter than the upper. All strains which exhibited good control of expression had the lower band.
  • the upper, intermediate PCR band had a sequence similar to the wild-type with two minor, probably strain- related, differences.
  • the lower band from SGE1670 and SGE1675 had a 15 basepair deletion. This creates a new -35 region which has the canononical sequence. This sequence is nearly identical to the published LaclQl allele.
  • Oligonucleotide 2 (CBG24) had the sequence 5 * NNNNNNCTGCAG3' and should prime on any PstI site in the genome. Oligonucleotide 1 (TG45) corresponded to known lad sequence near the 5' end of the gene. In the first step, these two oligos were used to amplify a fragment of about 800 base pairs from E. coZz strain C600 genomic DNA. Oligo 1 determines the specificity in this case. It is believed important to use a larger amount (50 pmole/ 100 ⁇ l reaction) of oligo 2 in order to compensate for the redtmdancy. 10 pmole/100 ⁇ l reaction of oligo 1 were used.
  • This first PCR product was used in a second round of PCR which provides a second test of specificity.
  • Oligo 2 plus a second internal lad oligo, oligo 3 (CBG23) were used to amplify a subset of this region.
  • Oligo 3 has a Hindlll recognition site to allow cloning of this reaction product.
  • PCR products were digested with PstI and Hindlll and cloned into pUC19 which was also digested with these enzymes. Clones with inserts were identified and sequencing was begun. Primers complementary to the vector and to known lad sequence were used for the first reactions. Sequence determined from these reactions was used to design more oiigonucleotides in order to complete the sequence.
  • Genomic DNA was then purified from SGE1661, SGE1670, and SGE1675.
  • a primer that is 200 bp upstream from the lad promoter was designed.
  • a Hindlll site was added to this oligonucleotide (CBG30).
  • CBG23 and CBG30 were used to amplify the Lad promoter region from these three strains.
  • PCR products were analysed by agarose gel electrophoresis where it was observed that the product from i SGE1670 had a small deletion relative to the product from SGE1661 (wild-type). In addition, two products, one of each size, were obtained from SGE1675.
  • PCR products were digested with PstI and Hindl ⁇ l and cloned into pBC SK+ at the PstI and Hindlll sites. Candidates with inserts were identified and these were sequenced. It was determined that the smaller PCR product had nearly the same sequence as the LaclQl allele reported in 1981 by Calos and Miller. The larger product had the wild-type sequence.
  • Lad transcript levels were assessed in JM109, SGE299, SGE1661, SGE1662, SGE1670, SGE1675. The results show that transcription levels in SGE299, SGE1670, and SGE1675 are similar to each other and are greater than in SGE1661, JM109 and SGE1662.
  • the Lad transcript initiates at the same point in all strains. Quantitation of autoradiograms showed that the relative abundance of transcript of Lad in
  • JM109 contains laclQ on an F' factor which, is reported to result in -100 copies of Lad per cell (Calos, M.P. ibid).
  • SGE299 contains laclQX on an F' factor, which since it has 5- fold more transcript than JM109, must therefore contain -500 copies of Lad per cell.
  • SGE1662 contains a lad gene on the chromosome and on each of the estimated 100 copies of the expression plasmid, and would be estimated from the literature to have -1,010 copies of Lad per cell.
  • SGE1483 was examined by growing cells in 15 L fermentations for extended periods of time post-induction, with two different hemin feeding strategies. Soluble hemoglobin was measured using the IMAC procedure described above. Soluble hemoglobin was enhanced when additional hemin supplementation was included during extended incubation of the culture post addition of IPTG inducer (Table 11). Induction of hemoglobin expression in 15L fermentations is normally supplemented by hemin additions at the time of induction, and at three and six hours post-induction. The volumes of hemin solution at each supplementation are 10, 13 and 17mls respectively, which result in a total hemin addition equal to 0.2g/L hemin in the fermentation tank.
  • Additional hemin supplementation was achieved by a second and third 17ml addition at 9 and 12 hours post-induction during 16 hour induction incubations, resulting in a total of 0.37g/L hemin in the fermentation tank.
  • the difference in soluble rHbO.O levels at 10 hours post induction was not significant (Table 11), only after 16 hours post-induction did the additional hemin supplementation manifest a significant improvement in soluble rHbO.O accumulation (Table 11).
  • Hemin is hydrophobic, and known to precipitate in aqueous solutions. Therefore, the additional supplementation may help accumulate higher levels of rHbO.O by increasing the total concentration, by maintaining a consistent soluble hemin level throughout the fermentation, or through other mechanisms.
  • Supplementation strategies for hemin might also include , among the options, different bolus additions and timing of additions, continuous feeding, very high initial bolus additions, and alternative chelated hemin for improved solubility.
  • the volumes of hemin solution at each supplementation are 10, 13 and 17mls respectively, which result in a total hemin addition equal to 0.2g/L hemin in the fermentation tank. Additional hemin supplementation was achieved by a second and third 17ml addition at 9 and 12 hours post-induction during 16 hour induction incubations, resulting in a total of 0.37g/L hemin in the fermentation tank.
  • the difference in soluble rHbl.l levels at 10 hours post-induction was not significant (Table 12), only after 16 hours post-induction did the additional hemin supplementation manifest a significant improvement in soluble rHbl.l accumulation (Table 12), as seen for rHbO.O above.

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Abstract

La présente invention se rapporte à des cellules hôtes et concerne des procédés d'expression de polypeptides hétérologues. La cellule hôte comporte un plasmide possédant un nombre de copies élevé et codant ledit polypeptide, ainsi qu'un régulateur d'expression dudit polypeptide, lequel régulateur est situé dans le chromosome et se trouve sous le contrôle d'un promoteur puissant.
PCT/US1996/011600 1995-07-14 1996-07-12 Procedes d'accroissement de l'expression de proteines WO1997004110A1 (fr)

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Cited By (17)

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WO1998050430A3 (fr) * 1997-05-02 1999-04-01 Somatogen Inc Mutants d'hemoglobine avec expression soluble accrue et/ou evacuation reduite d'oxyde nitrique
WO1999027108A1 (fr) * 1997-11-20 1999-06-03 William Marsh Rice University Proteine represseur du lactose a sensibilite du ligand modifiee
WO1999038985A3 (fr) * 1998-01-28 1999-11-18 Amgen Inc EXPRESSION REGULEE HAUTEMENT EFFICACE DE GENES EXOGENES DANS $i(E. COLI)
US6022849A (en) * 1987-05-16 2000-02-08 Baxter Biotech Technology Saarl Mutant recombinant hemoglobins containing heme pocket mutations
WO2000022112A1 (fr) * 1998-10-13 2000-04-20 The University Of Georgia Research Foundation, Inc. Peptides bioactifs stabilises, procedes d'identification, synthese et utilisation
US6171826B1 (en) 1996-08-02 2001-01-09 Baxter Biotech Technology Sarl Methods of controlling beta dimer formation in hemoglobin
US6204009B1 (en) 1988-05-16 2001-03-20 BAXTER BIOTECH TECHNOLOGY SàRL Nucleic acids encoding mutant recombinant hemoglobins containing heme pocket mutations
WO2005052151A1 (fr) 2003-11-19 2005-06-09 Dow Global Technologies Inc. Systemes d'expression de proteine ameliores
WO2006051127A1 (fr) * 2004-11-04 2006-05-18 Universidad Pablo De Olavide Commande de l'expression genique au moyen d'un attenuateur de transcription
US7365162B2 (en) 1998-10-13 2008-04-29 University Of Georgia Research Foundation, Inc. Stabilized bioactive peptides and methods of identification, synthesis, and use
US7390645B2 (en) 1997-11-20 2008-06-24 William Marsh Rice University Lactose repressor proteins with increased operator DNA binding affinity
EP1950298A2 (fr) 1997-05-02 2008-07-30 Baxter Biotech Technology S.A.R.L. Mutants d'hémoglobine dotés d'une expression soluble accrue et/ou d'une évacuation réduite de l'oxyde nitrique
EP2298795A1 (fr) 2005-02-18 2011-03-23 Novartis Vaccines and Diagnostics, Inc. Immunogènes d'E. coli uropathogène
EP2351772A1 (fr) 2005-02-18 2011-08-03 Novartis Vaccines and Diagnostics, Inc. Protéines et acides nucléiques d'Escherichia coli associé à la méningite/sepsie
EP2586790A2 (fr) 2006-08-16 2013-05-01 Novartis AG Immunogènes d'Escherischia coli pathogènes des voies urinaires
US8623652B2 (en) 2009-04-06 2014-01-07 Lucigen Corporation Host-vector system for cloning and expressing genes
WO2022175440A1 (fr) 2021-02-18 2022-08-25 Novozymes A/S Polypeptides d'hème inactifs

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EP0345615A2 (fr) * 1988-06-08 1989-12-13 BEHRINGWERKE Aktiengesellschaft Vecteurs d'expression pour la production de proteines non fusionnées dans des micro-organismes
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Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6022849A (en) * 1987-05-16 2000-02-08 Baxter Biotech Technology Saarl Mutant recombinant hemoglobins containing heme pocket mutations
US6204009B1 (en) 1988-05-16 2001-03-20 BAXTER BIOTECH TECHNOLOGY SàRL Nucleic acids encoding mutant recombinant hemoglobins containing heme pocket mutations
US6171826B1 (en) 1996-08-02 2001-01-09 Baxter Biotech Technology Sarl Methods of controlling beta dimer formation in hemoglobin
WO1998050430A3 (fr) * 1997-05-02 1999-04-01 Somatogen Inc Mutants d'hemoglobine avec expression soluble accrue et/ou evacuation reduite d'oxyde nitrique
AU750295B2 (en) * 1997-05-02 2002-07-11 Baxter Biotech Technology S.A.R.L. Hemoglobin mutants with increased soluble expression and/or reduced nitric oxide scavenging
EP1950298A3 (fr) * 1997-05-02 2008-12-24 Baxter Biotech Technology S.A.R.L. Mutants d'hémoglobine dotés d'une expression soluble accrue et/ou d'une évacuation réduite de l'oxyde nitrique
EP1950298A2 (fr) 1997-05-02 2008-07-30 Baxter Biotech Technology S.A.R.L. Mutants d'hémoglobine dotés d'une expression soluble accrue et/ou d'une évacuation réduite de l'oxyde nitrique
US7390645B2 (en) 1997-11-20 2008-06-24 William Marsh Rice University Lactose repressor proteins with increased operator DNA binding affinity
WO1999027108A1 (fr) * 1997-11-20 1999-06-03 William Marsh Rice University Proteine represseur du lactose a sensibilite du ligand modifiee
WO1999038985A3 (fr) * 1998-01-28 1999-11-18 Amgen Inc EXPRESSION REGULEE HAUTEMENT EFFICACE DE GENES EXOGENES DANS $i(E. COLI)
US6180391B1 (en) 1998-01-28 2001-01-30 Amgen Inc. Highly efficient controlled expression of exogenous genes in e. coli
US8030464B2 (en) 1998-10-13 2011-10-04 The University Of Georgia Research Foundation, Inc Stabilized bioactive peptides and methods of identification, synthesis, and use
WO2000022112A1 (fr) * 1998-10-13 2000-04-20 The University Of Georgia Research Foundation, Inc. Peptides bioactifs stabilises, procedes d'identification, synthese et utilisation
US7365162B2 (en) 1998-10-13 2008-04-29 University Of Georgia Research Foundation, Inc. Stabilized bioactive peptides and methods of identification, synthesis, and use
US10018618B2 (en) 1998-10-13 2018-07-10 Peptide Biosciences, Inc. Stabilizied bioactive peptides and methods of identification, synthesis and use
US9322829B2 (en) 1998-10-13 2016-04-26 Peptide Biosciences, Inc. Stabilized bioactive peptides and methods of identification, synthesis, and use
US7122516B2 (en) 1998-10-13 2006-10-17 University Of Georgia Research Foundation, Inc. Stabilized bioactive peptides and methods of identification, synthesis and use
US6818611B1 (en) 1998-10-13 2004-11-16 University Of Georgia Research Foundation, Inc. Stabilized bioactive peptides and methods of identification, synthesis and use
US8440201B2 (en) 1998-10-13 2013-05-14 University Of Georgia Research Foundation, Inc. Stabilized bioactive peptides and methods of identification, synthesis, and use
WO2005052151A1 (fr) 2003-11-19 2005-06-09 Dow Global Technologies Inc. Systemes d'expression de proteine ameliores
US8288127B2 (en) 2003-11-19 2012-10-16 Pfenex, Inc Protein expression systems
ES2299284B1 (es) * 2004-11-04 2009-04-16 Universidad Pablo De Olavide Control de la expresion genica mediante el uso de un atenuador de la transcripcion.
WO2006051127A1 (fr) * 2004-11-04 2006-05-18 Universidad Pablo De Olavide Commande de l'expression genique au moyen d'un attenuateur de transcription
ES2299284A1 (es) * 2004-11-04 2008-05-16 Universidad Pablo De Olavide Sistema de expresion de genes heterologos controlado por un atenuador de la transcripcion.
EP2351772A1 (fr) 2005-02-18 2011-08-03 Novartis Vaccines and Diagnostics, Inc. Protéines et acides nucléiques d'Escherichia coli associé à la méningite/sepsie
EP2298795A1 (fr) 2005-02-18 2011-03-23 Novartis Vaccines and Diagnostics, Inc. Immunogènes d'E. coli uropathogène
EP2586790A2 (fr) 2006-08-16 2013-05-01 Novartis AG Immunogènes d'Escherischia coli pathogènes des voies urinaires
US8623652B2 (en) 2009-04-06 2014-01-07 Lucigen Corporation Host-vector system for cloning and expressing genes
WO2022175440A1 (fr) 2021-02-18 2022-08-25 Novozymes A/S Polypeptides d'hème inactifs

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