WO1989003886A1 - Systemes d'expression destines a la preparation de polypeptides dans des cellules procaryotiques - Google Patents

Systemes d'expression destines a la preparation de polypeptides dans des cellules procaryotiques Download PDF

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Publication number
WO1989003886A1
WO1989003886A1 PCT/US1988/003872 US8803872W WO8903886A1 WO 1989003886 A1 WO1989003886 A1 WO 1989003886A1 US 8803872 W US8803872 W US 8803872W WO 8903886 A1 WO8903886 A1 WO 8903886A1
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sequence
gene
expression cassette
pbm11
promoter
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PCT/US1988/003872
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English (en)
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Timothy M. Rose
Thomas J. Franceschini
A. Gregory Bruce
Suo Win Liu
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Oncogen, A Limited Partnership
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Publication of WO1989003886A1 publication Critical patent/WO1989003886A1/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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/522—Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4, KC
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates to compositions and methods for preparation of novel polypeptides, in particular fusion polypeptides, using recombinant DNA techniques.
  • the expression cassettes include efficient transcriptional and translational initiation and termination regulatory regions appropriate for the host cell to provide for expression of a desired polypeptide.
  • the expression cassette preferably further includes, as appropriate for the host cell, a leader sequence for expression under the transcriptional and translational regulation of the regulatory region, sequences providing for enzymatic or chemical cleavage sites for cleavage of the leader peptide from mature polypeptide, and regulatory sequences which allow the time of expression of the gene of interest to be modulated.
  • the expression cassettes are introduced into a host cell under conditions whereby the resulting transformants stably maintain the expression cassette.
  • Naturally occuring DNA and synthetic genes may be employed for the production of a polypetide of interest.
  • expression cassettes are provided which, when inserted into a host cell, allow for the preparation of a polypeptide of interest which has enhanced stability and is either secreted in an active conformation or may be conveniently processed and renatured to an active state.
  • the nucleotides encoding the N-terminal amino acids of the polypeptide of interest are modified within the constraints of codon degeneracy to mimic those of the natural gene sequence found with the Shine-Dalgarno sequence used in the expression cassette.
  • the expression cassette thus will have the following general structure:
  • P comprises a promoter sequence including the regulatory regions occurring at about -35 and -10 nucleotides upstream from the start of the RNA chain and may also include regulatory sequences allowing for the induction of regulation;
  • S.D. comprises a Shine-Dalgarno sequence
  • met comprises a codon for the initiating methionine of the polypeptide of interest
  • G 1 comprises the gene for the polypeptide of interest wherein the first 7 to 30 codons of the gene have been modified wherever possible, using codon degeneracy to approximate the nucleotide sequence of the natural gene which would follow the Shine-Dalgarno sequence used in the expression cassette.
  • the polypeptide can be expressed as a fusion protein by including in the expression cassette a DNA sequence encoding a leader sequence peptide joined in reading frame upstream from the gene of interest. Expressing the polypeptide of interest as a fusion protein can result in up to 30% or more of the protein produced by the host cell being the polypeptide of interest.
  • the expression cassette for expressing a fusion protein will thus have the following basic structure:
  • P--S.D. metal--L--G wherein: P, S.D. and met have the meaning described above;
  • G comprises a gene for the polypeptide of interest
  • L comprises a DNA sequence encoding a leader peptide which may be an N-terminal sequence from any bacterial or bacteriophage gene, but generally is from a highly expressed gene; an amino acid sequence containing large numbers of hydrophobic amino acid residues; or an amino acid sequence containing large numbers of hydrophilic amino acid residues.
  • this sequence will preferably also function as a signal sequence, allowing secretion of the polypeptide of interest from the host cell and cleavage of the signal sequence from the polypeptide.
  • the DNA sequence coding for L may also be modified wherever possible, using codon degeneracy to approximate the nucleotide sequence of the natural gene which would follow the Shine- Dalgarno sequence used in the expression cassette.
  • the expression cassette described above provides for a fused expression product comprising the leader peptide and the polypeptide of interest. If it is desired to obtain the polypeptide of interest alone and there is no convenient cleavage site, e.g. as provided by a natural signal sequence, a cleavage site may be provided for by joining at least one codon encoding a cleavage site in reading frame upstream from the gene of interest.
  • the cassette will thus have the following structure:
  • C comprises at least one codon providing for a chemical or enzymatic cleavage site.
  • a transcriptional termination region can be included in the expression cassette downstream from the gene of interest.
  • An example of an expression cassette comprising T is as follows:
  • Design of an expression system to yield high levels of gene product must take into consideration not only the particular regions of a gene which have been determined to influence expression but also how these regions (and thus their sequences) influence each other. Where possible, choice of appropriate regulatory sequences will take into account the various factors which affect expression. Different genes have evolved a combination of all of these factors to yield a particular rate of expression; thus highly expressed genes can be considered useful models.
  • transcriptional regulation the amount and stability of messenger RNA are important factors which influence the expression of gene products. The amount of mRNA is determined by the copy number of a particular gene, the relative efficiency of its promoter and the factors which regulate the promoter, such as enhancers or repressors. Initiation is believed to occur in the region just upstream of the beginning of the coding sequence.
  • the promoter in prokaryotic cells comprises nucleotide sequences which can affect the efficiency of transcription. These sequences include the regulatory regions at about -35 and -10 nucleotides from the start of the RNA chain. Efficient promoters include those in which the nucelotide sequence of the -35 and -10 regulatory regions is substantially the same as consensus sequences for these regions in bacterial promoters from highly efficient genes. Generally these regions are about 5 nucleotides annd 6 nucleotides, respectively, in length, and each sequence may vary by about 1 nucleotide in length and/or in sequence. A. preferred sequence for the -35 consensus regulatory sequence is from the trp promoter, namely TGACA, and for the -10 consensus regulatory sequence is from the lac promoter, namely TATAAT.
  • the consensus sequences of the -35 and -10 regulatory regions are separated by about 16 to 18 nucleotides, preferably by about 17 nucleotides.
  • Ilustrative transcriptional regulatory regions or promoters which provide for efficient transcription include the B-gal promoter, lambda left and right promoters, the trp and lac promoters and trp-lac (tac) fusion promoters, and the like. Synthetic promoters having sequences substantially similar to these sequences may also find use.
  • a preferred promoter is a fusion promoter comprising the -35 regulatory region from the trp promoter and the -10 regulatory region from the lac promoter. Most preferably, the promoter is one in which the -35 trp consensus sequence is located about 17 nucleotides upstream from the -10 lac consensus sequence.
  • the transcriptional regulatory region may additionally include regulatory sequences which allow the time of expression of the gene of interest to be modulated, for example by presence or absence of nutrients or expression products in the growth medium, temperature, and the like.
  • expression of the gene of interest may be regulated by temperature of the host cell growth medium by including a regulatory sequence comprising the bacteriophage lambda P L promoter, the bacteriophage O L operator and the gene CI857 which codes for the temperature-sensitive C I repressor in the expression vector. This would allow regulation of the promoter by interaction between the repressor and the operator at low temperatures, for example about 30°C. Increasing the temperature to about 42°C would inactivate the repressor and allow expression of the gene of interest.
  • regulation of the lac or the trp-lac hybrid promoter can be accomplished by use of the gene for the Lad repressor, which binds in the lac promoter region downstream from the -10 regulatory region.
  • the Lad repressor gene may be present on an episome, preferably the lacIq enhanced mutant, or can be included in the expression cassette itself. Presence of a significant concentration of the repressor in the growth medium inhibits the promoter function in the absence of inducers. Thus addition of IPTG or lactose to the host cell growth medium enhances promoter function. When the bacterial strain is Lac + , lactose may be used as an inducer instead of IPTG.
  • the transcriptional regulatory region may additionally include regulatory sequences which terminate transcription and which provide sequences or structures which inhibit degradation of the mRNA and thus increase the stability of the mRNA species and allow for higher expression.
  • regulatory sequences which terminate transcription and which provide sequences or structures which inhibit degradation of the mRNA and thus increase the stability of the mRNA species and allow for higher expression.
  • prokaryotic sequences are known-- the trp terminator, the gene 32 (T4) terminator, or synthetic terminators which are similar in sequence to gene 32.
  • T4 terminator the gene 32 terminator
  • synthetic terminators which are similar in sequence to gene 32.
  • expression can be regulated by influencing the rate of initiation (ribosome binding to the mRNA), the rare of elongation (translocation of the ribosome across the mRNA) , the rate of post-translational modifications and the stability of the gene product.
  • the rate of elongation is probably affected by codon usage; the use of codons for rare tRNA's jnay reduce the translation rate. It is therefore preferable to use codons which frequently appear in genes normally expressed by the host cell to increase the translation rate.
  • ribosomal binding and initiation of translation can be achieved by using a ribosomal binding site functional in the host cell from a highly expressed gene.
  • the leader sequence and the Shine-Dalgarno sequence are obtained from the same gene, or where they are obtained from different genes, the codons of the leader sequence can be modified using codon degeneracy to approximate the nucleotide sequence of the natural gene that follows the leader sequence.
  • the position of the AGGA sequence with respect to the initiating ATG codon can influence expression.
  • the Shine-Dalgarno sequence is located from about 5 to 9 nucleotides from the initiating codon, although, unexpectedly, high levels of expression can be achieved using expression cassettes wherein the Shine-Dalgarno sequence is located from about 10 to 13 nucleotides, preferably 11 to 12 nucleotides from the initiating codon.
  • Stability of the mRNA is governed by the susceptibility of the mRNA to ribonuclease enzymes.
  • exonuclease digestion is inhibited by the presence of structual motifs at the ends of the mRNA; palindromic structures, altered nucleotides or specific nucleotide sequences. Endonuclease digestion is believed to occur at specific recognition sites within the mRNA and stable mRNA would lack these sites.
  • mRNAs undergoing high levels of translation are also protected from degradation by the presence of ribosomes on the mRNA.
  • Stability of the expression product is aided by expression of the desired gene product as a fused polypeptide in which the desired polypeptide is expressed in conjunction with a second polypeptide or fragment thereof, especially a bacterial polypeptide.
  • stability of the expression product is achieved by providing for synthesis of a fusion protein in which the polypeptide of interest is expressed, joined to a leader sequence.
  • a DNA sequence encoding an N-terminal amino acid sequence from, for example, a highly expressed bacterial or bacteriophage gene such as the bacteriophage lambda N protein gene or cro gene or the ⁇ -galactosidase gene is joined upstream from and in reading frame with the gene of interest.
  • the leader sequence usually includes from about 8 to about 35, preferably from about 15 to about 25, -N-terminal amino acids.
  • the presence of the N-terminal amino acids provides a means for using general purification techniques for purification of any of a variety of polypeptides.
  • the N-terminal amino acids of the N-protein are predictably antigenic, and thus specific antibodies raised against the N-terminal amino acids of the N-protein may be used for the amino purification of the fusion proteins containing the N-terminus of the N-protein.
  • the N-terminus of the N-protein has a high positive charge, which facilitates purification of the desired protein by ion-exchange chromatography, and the like.
  • the leader sequence can also be a hydrophobic amino acid sequence, which may additionally function as a signal sequence for secretion.
  • a DNA sequence encoding the signal sequence is joined upstream from and in reading frame with the gene of interest.
  • the signal sequence includes a cleavage site which is recognized by a signal sequence peptidase.
  • hydrophobic amino acid sequences include the bacterial alkaline phosphatase signal sequence; the OMP-A,B,C,D,E or F signal sequences; the LPP signal sequence, ⁇ - lactamase signal sequence; and toxin signal sequences.
  • Other leader sequences which can be used include hydrophilic sequences, for example the N-terminal 41 amino acid residues from amphiregulin which may provide for modification of the function of the polypeptide of interest.
  • cytotoxic agent such as a toxin A-chain fragment, ricin A-chain, snake venom growth arresting peptide, or a targeting molecule such as a hormone or antibody
  • a cytotoxic agent such as a toxin A-chain fragment, ricin A-chain, snake venom growth arresting peptide, or a targeting molecule such as a hormone or antibody
  • a DNA sequence encoding the leader sequence is joined upstream from and in reading frame with the gene of interest.
  • leader sequence is not a signal sequence or does not contain a convenient natural cleavage site
  • additional amino acids may be inserted between the gene of interest and the leader sequence to provide an enzymatic or chemical cleavage site for cleavage of the leader peptide, following purification of the fusion protein, to allow for subsequent purification of the mature polypeptide.
  • introduction of acid-labile aspartyl-proline linkages between the two segments of the fusion protein facilitates their separation at low pH. This method is not suitable if the desired polypeptide is acid-labile.
  • the fusion protein may be cleaved with, for example, cyanogen bromide, which is specific for the carboxy side of methionine residues. Positioning a methionine between the leader sequence and the desired polypeptide would allow for release of the desired polypeptide. This method is not suitable when the desired polypeptide contains methionine residues.
  • leader sequence comprises a signal sequence
  • genes of interest with secretory leader sequences can be expressed with or without the leader sequence under conditions where the sequence may be retained or cleaved.
  • a promoter such as the tac promoter
  • higher levels of secretion can be obtained at the lower temperatures.
  • Extremely high expression levels can prevent full translational modifications of the protein to occur, resulting in aggregation and accumulation of uncleaved precursor (i.e., structural protein and secretory leader).
  • the polypeptide of interest may be any polypeptide for which expression is desired and may be either homologous (derived from the host cell) or heterologous (derived from a foreign source or synthetic DNA sequence).
  • the polypeptide may be derived from prokaryotic sources, or eukaryotic sources, which eukaryotic sources may include fungi, protists, vertebrates, invertebrates, and the like.
  • the polypeptide of interest may include enzymes such as isopenicillin synthetase; mammalian peptides such as interleukins, cytokines, growth factors, e.g.
  • epidermal growth factor platelet-derived growth factor, oncostatin M, TGF- ⁇ and - ⁇ , viral growth factors, e.g. Vaccinia Virus, Shopes fibroma; snake venom growth-arresting peptide, brain-derivable peptides, immunoglobulins and fragments thereof, and the like.
  • the entire gene with its natural 5' and 3'-regulatory regions may be introduced into an appropriate expression vector.
  • further manipulation may be required.
  • the non-coding 5 '-region upstream from the gene of interest may be removed by endonuclease restriction, Bal31 resection, or the like.
  • the gene of interest may be restricted and an adapter employed for linking the gene of interest to the promoter region, where the adaptor provides for the lost nucleotides of the gene of interest.
  • an adapter employed for linking the gene of interest to the promoter region, where the adaptor provides for the lost nucleotides of the gene of interest.
  • the DNA sequences encoding the polypeptide of interest can be synthesized using conventional techniques giving overlapping single strands which may be ligated together to define the desired coding sequences.
  • the termini can be designed to provide restriction sites or one or both termini may be bluntended for ligation to complementary ends of an expression vector.
  • an initiating methionine is provided for expression of the sequence.
  • Expression vectors are generally available and are amply described in the literature. Instead of synthesizing the gene of interest, the gene may be isolated by various techniques.
  • mRNA from a host organism which codes for the polypeptide of interest
  • mRNA reverse transcribed the resulting single-stranded (ss) DNA used as a template to prepare double-stranded (ds) DNA and the ds DNA isolated.
  • Another technique is to isolate a piece of the host cell genomic DNA, and using a probe, appropriately degenerate, comprising a region of the most conserved sequences in the gene encoding the polypeptide of interest, identify sequences encoding the polypeptide of interest in the host cell genome.
  • the probe can be considerably shorter than the entire sequence, but should be at least 10, preferably at least 14, more preferably at least 20 nucleotides in length. Longer oligonucleotides are also useful, up to the full length of the gene encoding the polypetide of interest. Both DNA and RNA probes can be used.
  • the probes are typically labeled in a detectable manner (for example with 32 P -l abelled or biotinylated nucleotides) and are incubated with single-stranded DNA or RNA from the organism in which a gene is being sought. Hybridization is detected by means of the label after single-stranded and double-stranded (hybridized) DNA or DNA/RNA have been separated, typically using nitrocellulose paper. Hybridization techniques suitable for use with oligonucleotides are well known to those skilled in the art.
  • oligonucleotide refers to both labeled and unlabeled forms.
  • chimeric polypeptide sequences may be prepared by combining gene fragments of a least two polypeptides having sequences substantially similiar to naturally occuring polypeptide chains. It is highly desirable that the three dimensional structure of the polypeptide be retained, particularly that portion of the structure which may be responsible for biological activity of the polypeptide of interest.
  • convenient restriction sites may be designed into the synthetic genes used to construct the chimeric polypeptides. When possible the restriction site(s) leaves the amino acid sequence of the polypeptide unaltered. However, in some cases incorporation of the new restriction site(s) may yield an altered amino acid sequence without changing the activity of the protein.
  • various fragments of the DNA will usually be cloned in an appropriate cloning vector, which allows for amplification of the DNA, modification of the DNA or manipulation by joining or removing of sequences, linkers, or the like.
  • the vectors will be capable of replication in at least a relatively high copy number in bacteria.
  • a number of vectors are readily available for cloning in gram-negative bacteria, especially E. coli, including such vectors as PBR322, pACYC184, M13, Charon 4A and the like.
  • the cloning vectors are characterized by having an efficient replication system functional in the host bacteria.
  • the cloning vector will have at least one unique restriction site, usually a plurality of unique restriction sites and may also include multiple restriction sites.
  • the cloning vector will have one or more markers which provide for selection of transformants.
  • the markers will normally provide resistance to cytotoxic agents such as antibiotics, heavy metals, toxins or the like, complementation of an auxotrophic host, or immunity to a phage.
  • the plasmid After each manipulation of the DNA in the development of the cassette, the plasmid will be cloned and isolated and, as required, the particular cassette component analyzed as to its sequence to ensure that the proper sequence has been obtained. Depending upon the nature of the manipulation, the desired sequence may be excised from the plasmid and introduced into a different vector or the plasmid may be restricted and the expression cassette component manipulated, as appropriate.
  • a shuttle vector will be employed where the vector is capable of replication in different hosts requiring different replication systems. This may or may not require additional markers which are functional in the two hosts. Where such markers are required, these can be included in the vector, where the plasmid containing the cassette, two replication systems and the marker (s) may be transferred from one host to another, as required.
  • any useful marker may be used. Desirably, resistance to neomycin or tetracycline are of interest.
  • a marker for selection is highly desirable for convenience, other procedures for screening transformed cells have been described. See for example G. Reipin et al. Current Genetics (1982)
  • Transformed cells may also be screened by the specific products they make, for example, synthesis of the desired product may be determined by immunological or enzymatic methods.
  • the expression cassette may be included within a replication system for episomal maintenance in an appropriate celluar host or may be provided without a replication system, where it may become integrated into the host genome.
  • the DNA may be introduced into the host in accordance with known techniques, such as transformation, using calcium phosphate-precipitated DNA, transfection by contacting the cells with a virus, microinjection of the DNA into cells, and the like.
  • Host cells can include gramnegative organisms such as E. coli, e.g., JM109, JM101, and 107; HB101, DH1 or DH5. Particularly suitable are gram-positive organisms such as B. subtilis which have no periplasmic space and directly secrete polypeptides into the growth medium.
  • the host cell may be grown to high density in an appropriate nutrient medium.
  • permissive conditions will then be employed, for example, temperature change, exhaustion or excess of a metabolic product or nutrient, or the like.
  • the regulatory sequence comprises the bacteriophage ⁇ P L promoter, the bacteriophage O L operator, and the CI857 temperature sensitive repressor
  • the host cells may be grown at the permissive temperature, generally about 30°C, at which temperature transcription from the P L promoter is repressed and the host cells may grow unhindered by the demands of the synthesis of the foreign gene product, which additionally may be toxic to the host organism.
  • the temperature may be increased to a non-permissive temperature, for example about 42°C, at which time the CI repressor is rendered inactive, permitting transcription from the P L promoter.
  • Maximal secretion can be obtained by using the lac promoter or a trp-lac promoter and induction with a metabolic inducer such as lactose for a lac + host strain, and providing lacI q on a vector.
  • host cells which could be used with this system include DH1, DH5 or HB101.
  • the cells are harvested, lysed and the product isolated and purified by extraction, precipitation, chromotography, electrophoresis, and the like.
  • the product is secreted into the periplasmic space, the cells are harvested and the product is liberated by destruction of the cell wall, e.g., by hypotonic shock and the like.
  • the nutrient medium may be collected and the product isolated by conventional means, for example, affinity chromotography.
  • affinity chromotography To produce an active protein it may be necessary to allow the protein to refold.
  • the leader sequence may be removed by treatment with for example formic acid or cyanogen bromide. The leader sequence preferably is removed after refolding of the protein.
  • Plasmid pBM11 1. Construction of pBM4
  • Example V Expression of the Polypeptide of Interest as a Fusion Protein with the N-Protein and a Cleavage Site
  • TGF and Modified TGF a. N/TGF
  • VGF Modified and Truncated VGF a. PAD/nVGFa b. NDP/VGFa c. VGFa d. NDP/VGFA 3. Chimeric TGF/VGF Hybrids a. N/TTV b. NDP/TTV C. NDP/VTV d. NDP/TVV
  • the mitogenesis assays were performed as follows: Diploid human fibroblasts obtained from explants of newborn foreskin were seeded at a density of 3x10 4 cells/well (96-well plates, Nunclon, Roskilde, Denmark) and were grown to confluency in Dulbecco's modified Eagle's medium (GIBCO)/10% newborn calf serum. Cultures were then placed in medium containing 0.2% newborn calf serum, and two days later various concentrations of the growth factor to be tested were added.
  • Dulbecco's modified Eagle's medium GIBCO
  • EGF Receptor Binding Inhibition Assay The radioreceptor assays were performed as follows: The binding of 125 I-labeled growth factor to its receptor on monolayers of a target cells was modified from the procedure described by Cohen and Carpenter, Proc. Natl. Acad. Sci. USA (1975) 72:1317-1321. Cells (1x10 3 per well) were fixed on 24-well plates (Linbro, Flow Laboratories) with 10% formalin in phosphate-buffered saline prior to assay. Formalin-fixed cells do not slough off plates as easily as do unfixed cells, and replicate values were thus more consistent. Growth factor concentrations are expressed as ng equivalents of the native growth factor/ml i.e., the amount required to produce an inhibition of 125 ⁇ growth factor binding equivalent to that produced by a known amount of native growth factor.
  • a 5-month-old 20.5 kg micropig was anesthetized with 20 mg/kg ketamine and 2 mg/kg Rompum.
  • the dorsal thorax was shaved, prepped with betadine and thoroughly rinsed with saline.
  • a series of 6 5x5 cm donor sites were made on each side of the dorsal thorax with a Padgett dermatome at 650/1000 inch by taking two swipes at 30/1000 inch.
  • Topical therapy included 1 ml of saline in 20 gm of Silvadene ® distributed evenly between the six wounds on the left side.
  • the right side was treated with 1 ml of the growth factor to be tested, in 20 gm of Silvadene ® divided evenly between the six wounds.
  • test well Into each test well was delivered 50 ⁇ l of test compound in assay medium, while control wells received 50 ⁇ l assay medium alone. Each plate was then incubated at 37°C for 3 days. On day 4, into each well 50 ⁇ l of a solution of 125 I-iodo-2'-deoxyuridine (4 Ci/mg to 0.5 mCi/ml) (1 ⁇ l isotope/ml assay medium) was added and the plates incubated at 37°C overnight. On day 5, the medium was aspirated from the wells, and the wells washed IX with PBS. One hundred microliters of methanol were added for 10 min at room temperature.
  • mice Male nude mice (Ba1b/c-nu+/nu+) were supplied by the Fred Hutchinson Cancer Research Center, Seattle, WA. At 12 weeks of age, mice were given injections (s.c. in the neck region with approximately 1.3 x 10° human lung carcinoma cells (A549). in a volume of 0.2 ml of phosphate-buffered saline. Palpable tumors (approx. 10 mm 3 ) usually developed in 20 days. Each group contained 5 animals. Animals were injected every two or three days at the tumor site with 0.1 ml of PBS (control group) or test sample (1.2 ⁇ g/injection) resuspended in 0.1 ml of PBS. Day one post-treatment corresponds to the first day animals were injected at the tumor site with test compounds. Tumor size was measured before subsequent injection on the days indicated and represents the average size of tumor in each animal in the group.
  • PBS control group
  • test sample 1.2 ⁇ g/injection
  • Plasmid pBM11 contains the nucleotide sequence coding for the first 33 N-terminal amino acids of the bacteriophage lambda N protein. It also contains the neomycin resistance gene as a selective marker and a unique BamHI site for cloning of a foreign gene downstream from the lambda PL promotor. pBM11 was constructed as follows. 1. Construction of pBM4
  • a 2.4 kb DNA fragment containing the gene sequences coding for bacteriophage lambda cI was isolated by digesting (cleaving) 120 ug of lambda CI857S7 DNA (New England BioLabs) with 170 units of Bgl II (BRL) restriction enzyme by incubating the mixture for 2 hr. at 37°C. The digestion mixture was subjected to electrophoresis on a 1% preparative agarose gel using techniques which are conventional in recombinant DNA technology.
  • the 2.4 kb fragment (base pair 35771 to 38103; the numbering is based on Daniel, et al., pg. 519 in Lambda II, ed.
  • Plasmid pPL-lambda (5ug, Pharmacia) (a pBR322 derivative containing the lambda leftward promoter P L , the lambda N gene and the termination site for transcription of N (T L ) and 2 BamHI sites) was digested with 5 units of BamHI by incubating the mixture for 20 min. at 37°C. The digested DNA was separated by agarose-electrophoresis and the full-length DNA was recovered by electroelution using techniques which are conventional in the art.
  • the linearized pP L -Lambda was treated with alkaline phosphatase to remove the 5' phosphates and ligated to the 2.4 kb BglII fragment of CI857S7 (3 ⁇ l, about 250 ng), in the presence of ligase buffer (500mM Tris-HCl, pH 7.8, 100 mM MgCl 2 , 200 mM DTT, 10 mM ATP), and T4 DNA ligase (BRL). The resulting reaction mixture was incubated at 12°C for about 15 hr then used directly for transformation into competent E. coli HB101 cells.
  • ligase buffer 500mM Tris-HCl, pH 7.8, 100 mM MgCl 2 , 200 mM DTT, 10 mM ATP
  • T4 DNA ligase T4 DNA ligase
  • E. coli cells were made competent by a modification of a procedure described by Hanahan, J. Mol. Biol., (1983) 166 : 557-580.
  • a saturated culture of the HB101 cells was diluted 1:200 in Luria Broth supplemented with 20 mM MgCl 2 and incubated at 37 °C in a gyratory water bath until the culture reached an optical density (OD) nm A 550 , of 0.3.
  • OD optical density
  • a 20 ml portion of the culture was harvested by centrifugation at 4°C.
  • the resulting pellet was resuspended in 4 ml of ice cold 50 mM MnCl 2 /20 mM potassium acetate, pH 6.0 and kept on ice for 15 min.
  • the cells were centrifuged and the pellet was resuspended in 1.4 ml of a solution of 10 mM potassium methanesulfonate, pH 6.2, 100 mM of potassium chloride, 45 mM MnCl 2 .4H 2 O,10mMCaCl 2 , 3 mM hexamine CoCI 3 , 100 ⁇ l DMSO, and 100 ⁇ l 1M DTT.
  • Three hundred ⁇ l of the treated cells were added to 4 ⁇ l and 6 ⁇ l of the ligation mixture. The treated cells were placed on ice for 30 min., incubated at 42°C for 90 sec, and then placed on ice again for 90 sec.
  • Plasmid pBR322 (5 ⁇ g) was digested with PstI (3ul, New England BioLabs) followed by 3 successive extractions using a 1:1 (v:v) phenol:chloroform solution followed by 2 ether extractions. The DNA was precipitated completeness of digestion was analyzed by electrophoresis on 0.8% agarose gel.
  • the PstI digested pBR322 (10 ⁇ l) was treated with T4 polymerase (1 ⁇ l, BR1) to convert the 3' protruding ends to blunt ends.
  • a HindIII site was introduced at the PstI site of pBR322 by ligation of a synthetic phosphorylated
  • Hindlll* linker 5 ' -d[CAAGCTTG] ) (Pharmacia). The resulting DNA was digested with excess Hindlll. Two DNA fragments, a 3.58 Kb fragment and a 782 bp fragment were obtained. The 3.58 Kb fragment was isolated, ligated, and transformed into competent E. coli HB101. The transformants were analyzed as described above by sizing the HindIII digested DNA on agarose gels.
  • Plasmid pBM8 was digested with HindIII and BamHI to yield 2 fragments of DNA. The larger fragment, 3.23 Kb, was isolated by agarose gel electrophoresis and recovered by electroelution. Plasmid pNeo (Pharmacia) was digested with HindIII and BamHI to yield a 1.5 Kb fragment containing the gene sequences coding for neomycin resistance. The 1.5 Kb fragment was isolated and purified, then ligated to the 3.23 Kb fragment from pBM8. The resulting plasmid, pBM9, was transformed into competent E. coli HB101 and the transformants were screened as described previously. 4. Construction of pBM10
  • Plasmid pBm9 was digested with Ndel (New England BioLabs). The 5' protruding end was converted to blunt end using E. coli DNA Polymerase "Klenow" fragment and ligated to a synthetic phosphorylated EcoRI linker (5 '-d[GGAATTCC]-3') at the filled in NdeI site. The resulting plasmid, pBM10, an NdeI site and gained an EcoRI site pBMlO was transformed into competent E. coli HB101 and the transformants were screened as described previously. 5.
  • Plasmid pBM10 was digested to completion with EcoRI and BamHI. The resulting 2.8 Kb fragment containing the neomycin gene and origin of replication was isolated by agarose gel electrophoresis and recovered by electroelution. Plasmid pBM4 (described above) was digested to completion with EcoRI and BamHI. The 2.84 Kb fragment containing the DNA sequences for the cl gene and lambda P L promoter and the N gene ribosomal binding site was isolated and recovered. It was then ligated to the 2.84 Kb pBM4 fragment to form pBM11. Plasmid pBM11 transformed into competent E. coli HB101 and the transformants obtained were screened as described previously. The cells containing pBM11 were screened further by restriction enzyme analysis with BamHI.
  • Plasmid pBM11/M4 also contains an NcoI site in the neomycin gene. It was constructed as follows: 1. Construction of pBM11/M
  • Plasmid pBM11 (20 ug) was cleaved with BamHI and PvuI. After complete digestion, the DNA was electrophoresed through an 0.8% agarose gel and the large fragment (5124 bp) was isolated and recovered by electroelution. A second sample of pBM11 (10 ug) was digested to completion with SphI and the 3' protruding ends converted to blunt ends with T4 DNA polymerase. 0.3 ug of each of the two fragments were mixed together with 37.5 pmol of the phosophorylated synthetic oligodeoxynucleotide (Pharmacia P-L Biochemical)
  • the resultant plasmid was designated pBM11/M.
  • the plasmid pBM11/M was heated at 100°C for 3 minutes, then sequentially cooled as follows: 30°C for 30 minutes, 4°C for 30 minutes, and 0°C for 10 minutes.
  • the reannealed DNA was subsequently treated with T4 DNA ligase and transformed into E. coli HB101.
  • Neomycinresistant transformants were screened for plasmids containing the three new restriction sites encoded by the synthetic olignucleotide.
  • Plasmid pBM11/M was digested to completion with BamHI then electrophoresed through a 0.7% agarose gel. The large fragment (5554 bp) was isolated and recovered by electroelution. The resultant plasmid, which lacks the 100 bp region of the lambda N gene downstream from the ribosomal binding the translation initiation site, was designated pBM11/M1. Plasmid pBM11/M1 was religated and transformed into competent E. coli HB101.
  • a first sample of plasmid pBM11 (0.3 ug) was cleaved with BamHI and PvuI.
  • a second sample of plasmid pBM11 (0.3 ug) was cleaved with SphI and then treated with T4 polymerase. The two samples were combined with 3.75 pmol of the phosphorylated synthetic oligodeoxynucleotide (Pharmacia P-L Biochemical)
  • the mixture was heated and cooled as described previously, and treated with T4 DNA ligase and E. coli DNA polymerase "Klenow" fragment.
  • the resultant plasmid designated pBM11/M2
  • was transformed into competent E. coli HB101. Neomycin-resistent colonies were screened for plasmids containing the new BamHI site encoded by the synthetic oligodeoxynucleotide.
  • Plasmid pBM11/M2 was digested to completion with BamHI. The cleaved DNA was electrophoresed through a 0.7% agarose gel and the large fragment (5554 bp) isolated and recovered by electroelution. The DNA religated and designated pBM11/M2 transformed into competent E. coli HB101.
  • Plasmid pBM11/M1 (20 ug) was digested to completion with EcoRI and BamHI, then electrophoresed through a 0.7% agrose gel. The large fragment (5544 bp) was isolated and recovered by electroelution. This DNA was ligated to the following pair of phosphorylated synthetic oligodeoxynucleotides (200pmol each):
  • the resultant plasmid designated pBM11/M4 was transformed into competent E. coli HB101. Neomycin resistant colonies were screened for plasmids containing a new NcoI site.
  • Plasmid pBM11/M5 was derived from pBM11. An Ncol site present in the neomycin resistant gene has been removed by self directed mutagenesis. Plasmid PBM11/M5 is identical to pBM11/M4 except that the NcoI site in the neomycin gene has been removed by site- specific mutagenesis. Cloning of foreign genes into pBM11/M5 therefore does not require partial digestion of the vector with NcoI.
  • Plasmid pBM11 30 ug, was digested with 80 ug PvuI and 192 units BamHI. The resulting two fragments (5.1kb and 0.53kb) were separated by gel electrophoresis and isolated by electroelution. The 0.53kb fragment was then digested with HaeIII. The resulting fragments were separated by gel electrophoresis and the 324bp fragment was isolated by elect oelution. The construct pBMHC, the 5.1kb, the 324bp fragments isolated from plasmid pBM11 and a phosphorylated chemically synthesized linker.
  • Plasmid pBM11/C has restriction site BamHI as the cloning site as well as a unique SspI restriction site within the spacer region of the ribosomal binding site. It has been reported that spacer length affects the efficiency of protein translation. The presence of the SspI site allows the length of the spacer region to be changed and also allows for the insertion of other cloning sites for other genes to be expressed.
  • pBM11/C Modification of plasmid pBM11/C has also been performed.
  • pBM11/C 10 ug, was digested with restriction enzyme SspI, 45U, and the 5'phosphate was removed by phosphatase.
  • This DNA was ligated to a phosphorylated synthetic Ncol linker CCATGG.
  • the resulting plasmid, termed pBM11C/1 contains 2 NcoI sites, one in the neomycin gene and one for the cloning of the foreign gene(s).
  • the Ncol restriction site in the neomycin gene was removed by site-specific mutagensis using techniques described previously. This plasmid is termed pBM11/C2.
  • Plasmid pBM11/NDP derived from plasmid pBM11, contains the nucleotide sequences coding for the first 32 amino acids of the lambda N protein followed by nucleotide sequences coding for the acid cleavable aspartic acid-proline dipeptide.
  • the plasmid contains an NcoI site and a ClaI site for cloning a foreign gene downstream of the P L promotor.
  • This plasmid is identical to pBM11/NDP except that it lacks the NcoI site in the neomycin gene as described above (Example II.F.) and it contains the transcription terminator as described for plasmid pTNPt (see Example (II.N. for terminator sequence).
  • Plasmid pBM11/NDP/VGFa (see Example V.A.I, below) was digested with Ncol and BamHI which removes the synthetic gene TVV from the pBM16/DP plasmid fragment.
  • the pBM16/NDP NcoI-BamHI 5.5 kb plasmid fragment was then gel purified.
  • the NcoI site in this fragment is positioned downstream of the nucleotide sequences coding for the first 32 amino acids of the N- gene and directly after the sequences coding for the acid labile dipeptide Asp-Pro.
  • pBM11/PAD also called pBMHM3PAD
  • This plasmid is derived from pBM11/M3 and allows a foreign gene to be cloned at a HindIII, SmaI or BamHI site downstream from a modified alkaline phosphatase signal sequence.
  • Synthetic oligonucleotides were designed to allow insertion of DNA coding for a modified alkaline phosphatase signal peptide and a linker region with 3 cloning sites (HindIII, SmaI and BamHI) into the pBM11 expression vector downstream from the P L promotor and N gene ribosomal binding site.
  • the nucleotide sequence was optimized to be as similar as possible to the nucleotide sequence of the amino terminus of the lambda N gene as the lambda N gene sequence has evolved with that of its ribosomal binding site for efficient ribosome initiation and translation.
  • PA2 5' GATCCCGGGAAGCTTTTGTCACTGGAGTGAACAGCAGTGGGAGAAGT GCGAGGGCGATTGTAGATT 3'
  • Oligonucleotides A1 and A2 were synthesized on a Applied Biosystems Oligonucleotide Synthesizer and purified on an acrylamide gel. The oligonucleotides were phosphorylated at the 5' end using T4 polynucleotide kinase and then annealed to each other yielding a double stranded 0.067kb DNA fragment with a BamHI overhang at each end.
  • pBM11/PAD was constructed as follows: Plasmid pBM11/M3 (20ug) was digested with 30 units of BamHI to linearize the plasmid directly after the N gene ribosomal binding site and the ATG codon for the initiating methionine. The 5' phosphates were removed by digestion with calf intestinal alkaline phosphatase.
  • Plasmid pBR322 was digested with restriction enzyme PstI. The resulting 3' protruding end was converted to a blunt end by treating the digestion product with T4 DNA polymerase according to conventional techniques. Synthetic BglII linker (5'-d[CAGATCTG]) was phosphorylated and ligated onto the blunt end DNA and used to transform competent E. coli HB101. Plasmid DNA preparations were prepared from tetracycline-resistant transformants using conventional techniques digested with PstI and BglII to screen for loss of the PstI restriction site and addition of the Bglll restriction site. 2. Construction of pBM13
  • Plamid pBM4 obtained as described above, was digested with HpaI. The 3' protruding end was converted to a blunt end and the phosphorylated BglII linker added to the blunt end. The ligation reaction mixture was used to transform competent E. coli HB101. 3. Final Construction of pBM14
  • Plasmid pBM12 was digested with BglII and EcoRI. The resulting 3.6 Kb fragment containing the tetracycline gene and origin of replication was isolated and recovered. Plasmid pBM13 was digested with BglII and EcoRI and the resulting 2.8 Kb fragment containing the bacteriophage lambda cI857 DNA sequences and the lambda P L and the N ribosomal binding site was isolated and recovered. The 3.6 Kb fragment from pBM12 and 2.8 Kb fragment from pBM13 were ligated to form plasmid pBM14. The resulting ligation mixture was transformed into E. coli HB101 and the resulting transformants screened for pBM14. The putative pBM14 was analyzed further by restriction digest using conventional techniques. J. Plasmid pLEBam
  • Plasmid pLEBam was used to clone synthetic oligonucleotide fragments because of its convenient BssHII and BamHI restriction sites.
  • a plasmid with Ncol and BamHI restriction sites such as pBM11 or pBM11/NDP (described below) can be used for cloning the synthetic nucleotide fragments.
  • Plasmid plac/cro- ⁇ gal The controlling elements of the vector plac/cro- ⁇ gal consist of the operator-promoter region of E. coli lactose (lac) operon, as well as the ribosome-binding sites of lac and cro.
  • This vector is derived from plasmids pTR213 (Roberts et al., Proc. Natl. Acad. Sci. USA (1978) 76:760) and pLG300 (Guarante et al., Cell (1980) 20:543).
  • Plasmid plac/cro- ⁇ gal was constructed by ligating a 0.96 kb Pstl-BglII fragment from pTR213 and a 5.54 kb PstI-BamHI fragment from pLG300 in the presence of the oligonucleotide linker which had been digested with BamHI and BglII:
  • This linker served the following purposes: (1) to regenerate the BglII and BamHI sites from the parental plasmids, (2) to provide additional sites for the insertion of foreign DNA, and, (3) to allow the inserted DNA to be in the correct translational reading frames with respect to the cro 5'-gal coding sequence.
  • Plasmid ptac/cro- ⁇ gal allows a foreign gene to be cloned downstream of the N-terminal 21 amino acids of the bacterial Cro protein. It was constructed by inserting a 0.87 kb RsaI fragment of the plac/cro- ⁇ gal plasmid into pDR540 (Pharmacia) at the BamHI site, which was previously converted to blunt ends by the action of Klenow enzyme. The orientation of the inserted DNA was such that the ribosomal binding site and the coding sequence of Cro were located downstream from the ribosomal binding site of lac. The resulting plasmid, ptac/cro, contained ribosomal binding sites of both lac and cro, and the N-ter,minal coding sequences of Cro.
  • the second step in the construction of ptac/cro- ⁇ gal was to ligate the 1.16 kb and the 5.54 kb PstI-BamHI fragments from ptac/cro and pLG400 plasmids, respectively.
  • Expression vector ptac/cro- ⁇ gal is thus similar to plac/cro- ⁇ gal, with the exception that the promoter of ptac/cro- ⁇ gal consists of the -35 region from the promoter of the tryptophan operon and the Pribnow box (-10 region) of the lac operon. This hybrid promoter allows a higher level of, expression than plac/cro- ⁇ gal.
  • Plasmid pTCPt This plasmid is designed to have the tac promoter elements and utilize the cro SD to express the gene of interest behind the alkaline phosphatase signal sequence.
  • An example of the construction of this plasmid is given below in the construction of pTCPt/EGF.
  • pTNPt [trp-35]17bp[lac-10] [nSD] 8bp[ATG]/alkaline phosphatase signal/linker/trans. term.-NEO) This plasmid is designed to have the tac promoter elements and utilize the N-gene SD to express a given gene behind the alkaline phosphatase signal sequence. It has a pBR322 background with the Neomycin resistance gene. Plasmid pTNPt was constructed as follows:
  • Plasmid pBM16t/VGFa was digested with EcoRI and BamHI and the 2.8kb fragment was isolated. The 2.8kb fragment was ligated to an EcoRI-BamHI linker and a correct construct was isolated by restriction analysis and is referred to as Intermediate I.
  • the 2.8kb EcoRI-BamHI fragment of pBM16t/VGFa lacking the HindIII site was isolated by digesting plntermediate II with EcoRI and BamHI. The resulting 2.8kb fragment was isolated by agarose gel electrophoresis.
  • Plasmid pBM11/PAK is identical to pBM11/PAK/EGF except that it contains a linker region with HindIII, SmaI and BamHI sites downstream of the alkaline phosphatase signal sequence instead of the EGP gene.
  • pBM11/PAK was digested with BsmI and BamHI and the 150bp fragment containing the N-gene SD, the alkaline phosphatase signal sequence and the linker region was isolated.
  • Oligonucleotides TacA+ and TacA- were synthesized on an Applied Biosystems Oligonucleotide synthesizer and were designed to have an EcoRI overhang at the 5' end with the trp-35 consensus sequence separated from the lac-10 consensus sequence by 17 nucleotides within which was positioned a SstI site. The sequence also contained the 5' end of the lac mRNA, the lac repressor binding site and a BsmI overhang.
  • the 2.8kb EcoRI-BamHI fragment, the 150bp BsmI-BamHI fragment and oligonucleotides TacA+ and TacA- were ligated together using DNA ligase and the DNA was used to transform competent JM109(lacIq). A correct construct was isolated by restriction analysis and DNA sequencing.
  • Synthetic growth factor genes were designed which use host cell codons optimized for high levels of expression.
  • several convenient restriction sites were designed into the synthetic genes. When possible, the new restriction sites left the amino acid sequence of the growth factor gene unaltered, however, in some cases incorporation of the new restriction site yielded an altered amino acid sequence. These sites roughly divide the synthetic genes into thirds yielding N-terminal, middle and C-terminal domains.
  • the natural VGF gene product contains an extreme N-terminal domain which has no counterpart in mature TGF. VGF fragments lacking this domain are referred to as truncated.
  • the restriction sites were used for initial construction of the final genes from partial synthetic oligonucleotide fragments extending from one restriction site to another.
  • the oligonucleotides were synthesized on an Applied Biosystems oligonucleotide synthesizer and were purified on an acrylamide gel. The oligonucleotides were phosphorylated at the 5' end using T4 polynucleotide kinase and each oligonucleotide was then annealed to its complement.
  • TGF Synthetic Oligonucleotides a. Human TGF N-terminal domain
  • VGF Synthetic Oligonucleotides a. VGF extreme N-terminal domain
  • VGF N-terminal domain including Asp-Pro cleavage site, with the sequence HGT replacing the natural sequence HGD
  • VGF middle domain having the sequence GYAC replacing the natural sequence GMYC
  • VGF C-terminal domain 3' fragment, ending at YQR instead of PNT, the deduced C-terminus of natural secreted VGF
  • Three sets of overlapping synthetic oligonucleotides 1(A,B), 2(A,B) and 3(A,B) coding for human EGF were synthesized on an Applied Biosystems oligonucleotide synthesizer and purified on an acrylamide gel.
  • the oligonucleotides were phosphorylated at the 5' end using T4 polynucleotide kinase. Each oligonucleotide was annealed to its complement.
  • TTV The synthetic chimeric growth factor, denoted TGF/TGP/VGF) was assembled in the cloning vector pLEBam.
  • This hybrid growth factor contained the amino acid sequence of human TGF in the amino terminal two-thirds of the gene with the exception of the se quence QEEK which was altered from the natural human sequence QEDK.
  • the carboxy terminus was derived from the amino acid sequence of VGF and terminated with the sequence YQR TMR upstream of the natural sequence PNT.
  • Plasmid pLEBam was digested with BssHII and BamHI.
  • BssHII-BamHI pLEBam was then ligated to oligonucleotides TGF101, 102, 103, and 104, and VGF1, 2, 3, and 4 using DNA ligase and the resulting plasmids used to transform competent HB101.
  • the transformants were selected on ampicillin and screened by restriction analysis using EcoRI , Ncol and BamHI and by nucleotide sequencing using the Maxam-Gilbert protocol. A correct construction was isolated and denoted pLEBam/TTV.
  • the synthetic chimeric growth factor denoted TVV or (TGF/VGF/VGF) was assembled in the cloning vector pLEBam.
  • This hybrid growth factor contained the amino acid sequence of human TGF in the N-terminal domain of the gene.
  • the middle and C-terminal domains are derived from the truncated VGF sequence and end with the sequence YQR upstream of the natural sequence PNT.
  • the synthetic gene has the modification, GYACVC for GMYCRC.
  • VGFlOla and 102a were ligated to the 4.3kb Kpnl-SphI fragment of pLEBam/TTV using DNA ligase and the resulting mixture was used to transform competent HB101.
  • the transformants were selected on ampicillin and were screened by nucleotide sequencing using the Sanger-dideoxy method. A correct construct was isolated and denoted pLEBam/TVV.
  • a synthetic platelet factor 4 gene was designed which uses bacterial codons optimized for high levels of expression. Single-stranded overlapping sequences were prepared, combined in an annealing medium and ligated to provide the complete gene with appropriate termini for insertion into an expression vector in reading phase to prepare a fused protein from which platelet factor 4 could be isolated. The resulting expression vector was called pHCPF4.
  • the single-stranded segments were 5' -phosphorylated with T4 polynucleotide ligase and annealed by combining 200 pM of each segment in a 30 ⁇ l reaction volume (30 mM ATP, 10 mM DTT, 10 mM MgCl 2 1 ⁇ g/ml spermidine, 100 mM Tris-HCl, pH 7.8 and T4 DNA ligase.
  • the dsDNA was digested with BssHII and BamHI and purified on a 7% native polyacrylamide gel.
  • RNA obtained from U937 cells treated with media containing phorbol 12-myristate 13-acetate PMA (10 ng/ml) for 16, 36, and 52 hours was pooled and used for cDNA synthesis and cloning into a ⁇ gt 10 vector, essentially as described by Huynh et al., DNA
  • the sized dG tailed cDNA were ligated into EcoRI cut ⁇ gt 10 in the presence of single stranded 16 nucleotide-long linker molecule comprising, from the 5' end, AATT followed by 12 deoxycytosine residues (Webb et al., 1987).
  • the ligated DNA was packaged in vitro (Grosveld et al., Gene (1981) 13:227-237 ) and the phage was used to infect E. coli C60 Hf1 + . This technique gave 3x10° recombinants/ ⁇ g cDNA. Nitrocellulose filter plaque lifts were done in duplicate and the filters were probed using long, best guess 35 to 50 nucleotide long probes.
  • the oligonucleotide probes were derived from the peptide sequences obtained by automated repetitive Edman degradation.
  • the purified Oncostatin M sequence was either derived from the N- terminal of the protein or by sequencing the proteasegenerated lysine peptides.
  • ⁇ gt 10 clones showing positive reactivity to [ 3 P]-labeled above oligonucleotide were plaque purified. Eight clones were obtained. Southern blot analysis showed that the positively reacting cDNA inserts in the clones ranged between 600 bp to 2 Kb. Subsequently, Southern blots were done using a 35 mer oligonucleotide (encoding amino acids 53-64) and a 41 mer oligonucleotide (encoding amino acids 22-35). Only one clone showed positive reactivity with all three radiolabeled oligonucleotide probes.
  • the cDNA insert of the ⁇ gt 10 clone ( ⁇ OM) was found to be approximately 2.1 Kb.
  • the recombinant was termed pOncM46.
  • additional cDNA clones were obtained by specific priming using oligonucleotides derived from the 5' coding region of the Oncostatin M gene and a genomic clone containing the entire gene was isolated.
  • a restriction map of the clone pOncM46 coding Oncostatin M protein was obtained by standard single or double digestions of the plasmid DNA.
  • the coding region has four PstI sites, a BglII site and a SmaI ite.
  • the consensus sequence was further verified by comparison with the sequence of the genomic clone.
  • the open reading frame continues from nucleotide 1 to the stop codon at nucleotide 783.
  • the open reading frame codes for 8 amino acids upstream from the putative initiating methionine.
  • the nucleotide sequence coding for the putative initiating methionine agrees with the consensus sequence for the initiating methionine (Kozak, Cell (1986) 44:283-292).
  • the amino acid sequence of the Oncostatin M polypeptide deduced from the consensus cDNA sequence shows that Oncostatin M is derived from a 253 amino acid precursor polypeptide.
  • the amino terminal sequence of purified Oncostatin M see above and and Zarling, et al., Proc.
  • Oncostatin M is an extremely hydrophilic molecule. Twenty four base pairs of the 5' untranslated region and 1054 base pairs of 3' untranslated regions were. obtained in the different cDNA clones. However, a polyA tail and polyadenylation recognition site were not obtained. 4. Preparation of pOncMVV2
  • Oncostatin M cDNA was excised from the ⁇ phage recombinant, pOncM46, by EcoRI digestion.
  • the insert was cloned into pEMBL18 (Dente et al., Nucl. Acid Res. (1983) 11:1645-1655) at the EcoRI site to produce clone pOncM46-15 vector with the Oncostatin M coding sequence opposed to the ⁇ -gal sequence.
  • the 5' noncoding sequence of Oncostatin M was removed by Sail and BglII double digestion of pOncM46-15 and replaced by a synthetic 80 bp SalI-BglII fragment to provide new BamHI and Ncol sites.
  • the resulting clone was termed pOncMEV5.
  • the sequence of the 80 bp fragment is as follows :
  • Oncostatin M was excised from pOncMEV5 by XhoI and Sail double digestion as a 0.7 kb fragment and cloned into the pUC8 vector as the Sail site.
  • the 0.7 kb NcoI-BamHI and the 0.7 kb BamHI-BamHI fragment excised from clone pONcMVV2 were used to construct a ⁇ pL-based (pBM16/NDP/OncM) expression vector.
  • the modified human TGF was expressed in this system as part of a fusion with the 33 N-terminal amino acids of the N-gene and has the sequence QEEK replacing the human sequence QEDK.
  • a Preparation of a 780 bp SphI-PvuI fragment of pBM11/N/TTV Plasmid pBM11/N/TTV was digested with SphI and Pvul and the 780 bp SphI-PvuI fragment was gel purified. This fragment contains part of the pBM11 plasmid at the PvuI end and at the SphI end, the N-gene and N- terminal two-thirds of the human TGF gene. b.
  • pBM11/N/TTV Plasmid pBM11/N/TTV was digested with BamHI and PvuI and the 5 kb BamHI-PvuI fragment was gel purified.
  • c. Ligation and isolation of pBM11/N/TGF: Oligonucleotides TGF 205 and 206, the 780 bp SphI-PvuI fragment and the 5 kb BamHI-PvuI fragment of pBM11/N/TTV were ligated together and used to transform competent HB101. The transformants were selected on neomycin and were screened by restriction analysis using EcoRI and nucleotide sequencing following the Sanger-dideoxy method. A correct construction was isolated and denoted pBM11/N/TGP.
  • a synthetic modified TTV chimeric gene was expressed as the C-terminal portion of a fusion protein having the first 33 amino acids of the N-gene at the N-terminus.
  • This hybrid growth factor contained the amino acid sequence of human TGF in the amino terminal two-thirds of the gene with the exception of the sequence QEEK which was altered from the natural human sequence QEDK.
  • The. carboxy terminus was derived from the amino acid sequence of VGF and terminated with the sequence YQR upstream of the natural sequence PNT.
  • Plasmid pLEBam/TTV was digested with Ncol and the ends were made blunt by filling in the overhangs using the Klenow fragment of DNA polymerase. The DNA was then digested with BamHI and the 170 bp Ncol(blunt)-BamHI TTV fragment was gel purified.
  • b. Preparation of BamHI digested pBMll Plasmid pBM11 was digested with BamHI. c.
  • This plasmid was prepared as described above for pBM11/N/TTV, except that the synthetic PF4 gene was used in place of the chimeric TTV gene.
  • the nucleotide sequence and corresponding amino acid sequence of the synthetic platelet factor 4 gene in fusion downstream of the nucleotide sequences coding for the first 33 amino acids of the bacteriophage ⁇ N-gene in the expression vector pBM11 is as follows.
  • pBM11/NDP/VGFA The N-terminal sequence of the synthetic VGFA gene is a truncated version of the natural VGF sequence and begins with the sequence DIPAIR. In this plasmid the VGFA fragment is located downstream of 32 amino acids of the lambda N-protein and the dipeptide aspartic acid-proline. In order to preserve the KpnI cloning site, the synthetic sequence was altered to code for CLHCGTC instead of the natural VGF sequence CLHGDC and terminates with the sequence YQR upstream of the natural sequence PNT. In addition, the VGFA gene codes for the sequence GYACVC which replaces the natural sequence GMYCRC. a.
  • BamHI fragment of pBM11 and the 80bp KpnI-BamHI fragment of pLEBam/TVV were ligated together using DNA ligase and then used to transform competent HB101.
  • the transformants were selected on neomycin and were screened by restriction analysis using ClaI and nucleotide sequencing following the Sanger-dideoxy technique.
  • a correct construction was isolated and denoted pBM11/NDP/VGFA. This construction has the sequences GTC and GYACVC instead of the authentic VGF sequences GDC and GMYCRC.
  • the N-terminal sequence of VGFa is a truncated version of the natural VGF sequence and starts with the sequence DIPAIR.
  • the VGFa sequence contains the altered sequences GTC and GYACRC instead of the natural VGF sequences GDC and GMYCRC.
  • the VGFa gene is located downstream of 32 amino acids of the lambda N-protein and the dipeptide aspartic acid-proline.
  • Treatment of the purified fusion protein with formic acid results in cleavage at the acid labile aspartic acid-proline peptide bond allowing separation of the VGFa protein from the lambda N-protein amino-terminus. Cleavage is such that the VGFa protein is left with the proline residue at the amino terminus.
  • the 24bp fragment containing oligonucleotides VGF IA and.2A, the 5kb SphI fragment and the 70bp EcoRI-SphI fragment of pBM11/DP/VGFA were ligated together and the mixture was used to transform competent E. coli HB101 cells.
  • the transformants were screened by nucleotide sequencing using the Sangerdideoxy nucleotide method. A correct clone was isolated and denoted pBM11/NDP/VGFa.
  • the synthetic modified TTV chimeric gene is expressed as the C-terminal portion of a fusion protein having the first 32 amino acids of the N-gene at the N-terminus.
  • An acid labile aspartic acid-proline dipeptide separates the two parts of the fusion.
  • the hybrid growth factor contains the amino acid sequence of human TGF in the amino terminal two-thirds of the gene with the exception of the sequence QEEK which was altered from the natural human TMR sequence QEDK.
  • the carboxy terminus was derived from the amino acid sequence of VGF and terminated with the sequence YQR upstream of the natural sequence PNT.
  • a Preparation of 5kb Ncol pBM11 plasmid fragment Plasmid pBM11/NDP/VGFA was digested with Ncol and the 5kb NcoI plasmid fragment was gel purified. This fragment has one Ncol overhang at the aspartic acid-proline cleavage site downstream of the sequences coding for the first 32 amino acids of the N-gene. The other NcoI site is in the neomycin resistance gene. b.
  • This fragment has the Ncol overhang at the 5' end of the gene and the BamHI overhang at the 3' end of the gene.
  • d Ligation and isolation of pBM11/NDP/TTV
  • the 5kb NcoI and the 0.6kb NcoI-BamHI plasmid fragments were ligated with the 170bp NcoI-BamHI TTV gene using DNA ligase and the resulting mixture was used to transform competent HB101I
  • the transformants were selected on neomycin such that only colonies with correctly rec ructed neomycin resistance genes would survive.
  • Transformants were screened using restriction analysis with Ncol and nucleotide sequencing using the Sanger-dideoxy technique. A correct construction was isolated and denoted pBM11/NDP/TTV.
  • the synthetic modified VTV chimeric gene was expressed as the C-terminal portion of a fusion protein having the first 32 amino acids of the N-gene at the N-terminus.
  • An acid labile aspartic acid-proline dipeptide separates the two parts of the fusion.
  • the hybrid growth factor contained the amino acid sequence of human TGF in the middle domain with the amino acid sequence QEEK replacing the natural sequence QEDK.
  • the N-terminal and C-terminal domains were derived from the truncated VGF sequence and begin with the sequence DIPAIR and end with the sequence YQR which is upstream of the natural sequence PNT.
  • Plasmid pBM11/N/TTV was digested with BamHI and NcoI and the 5kb BamHI-NcoI fragment was gel purified. This fragment contains a BamHI overhang at the 3' end of the sequences coding for the first 32 amino acids of the N-gene and a NcoI site in the neomycin resistence gene.
  • Plasmid pBM11/N/TTV was digested with KpnI and NcoI and the 700 bp KpnI-Ncol fragment was gel purified. This fragment is made up of part of plasmid pBM11 containing part of the neomycin resistence gene at the NcoI overhang, and the C-terminal VGF domain of the TTV synthetic gene at the KpnI overhang.
  • c. Ligation and isolation of pBM11/NDP/VTV Oligonucleotides VGF 103a and 104a, the 5kb
  • the synthetic modified TVV chimeric gene was expressed as the C-terminal portion of a fusion protein having the first 32 amino acids of the N-gene at the N-terminus.
  • An acid labile aspartic acid-proline dipeptide separates the two parts of the fusion.
  • the hybrid growth factor contained the amino acid sequence of human TGF in the N-terminal domain.
  • the middle and C-terminal domains were derived from the truncated VGF sequence and end with the sequence YQR.
  • the synthetic gene has the modification
  • Plasmid pBM11/NDP/VGFa was digested with NcoI and BglII and the 4.3kb fragment was gel purified.
  • the NcoI overhang is positioned at the aspartic acid- proline cleavage site just downstream of the first 32 amino acids of the N-gene.
  • Plasmid pLEBam/TVV was digested with Ncol and BamHI and the 170 bp NcoI-BamHI fragment was gel purified. This synthetic gene fragment has the Ncol site at the 5' -end and the BamHI site at the 3'-end.
  • Ligation and isolation of pBM16/NDP/TW The 4.3kb NcoI-BglII fragment of pBM11/NDP/VGFa and 1.2kb BamHI-BglII fragment of pBMHM5, and the 170 bp NcoI-BamHI TVV synthetic gene fragment were ligated together using DNA ligase and the resulting mixture was used to transform competent HB101.
  • the transformants were selected on neomycin and screened by restriction analysis and nucleotide sequencing using the Sanger-dideoxy technique.
  • the plasmid is denoted pBM16 to indicate the loss of the NcoI restriction site in the neomycin resistance gene.
  • the human EGF gene is expressed as part of a fusion with the 32 N-terminal amino acids of the N-gene which is downstream of an Asp-Pro cleavage site.
  • a Preparation of a 5kb Ncol fragment of pBM11 Plasmid pBM11/DP/VGFA was digested with NcoI and the 5' phosphates were removed by treatment with calf alkaline intestinal phosphatase. The 5kb plasmid fragment was gel purified. This fragment has one NcoI overhang at the Asp-Pro cleavage site downstream of the sequences coding for the first 32 amino acids of the N-gene. The other NcoI site is in the Neomycin resistance gene.
  • b Preparation of a 0.6kb NcoI-BamHI fragment of pBM11
  • Plasmid pBM11/N/TTV was digested with NcoI and BamHI and the 0.6kb NcoI-BamHI plasmid fragment was gel purified. This fragment has the NcoI overhang in the Neomycin resistance gene.
  • c. Ligation and Isolation of pBM11/DP/EGP
  • the three sets of annealed EGF oligonucleotides with an NcoI overhang at the 5' end and a BamHI overhang at the 3' end, the 5kb Ncol fragment of pBM11 and the 0.6kb NcoI-BamHI fragment of pBM11 were ligated together using T4 DNA Ligase and the resulting mixture was used to transform competent E. coli HB101.
  • the transformants were selected on Neomycin such that only colonies with a correctly reconstructed Neomycin resistance gene would survive.
  • the transformants were screened by restriction analysis using EcoRI and BamHI and by DNA sequencing, as described above.
  • the synthetic PF4 gene is expressed as the C-terminal portion of a fusion protein having the first 32 amino acids of the N-gene at the N-terminus.
  • An acid labile aspartic acid-proline dipeptide separates the two parts of the fusion.
  • Plasmid pBM11/NDP/VGFA was digested with NcoI and the 5kb NcoI plasmid fragment was gel purified. This fragment has one NcoI overhang at the aspartic acid-proline cleavage site downstream of the sequences coding for the first 32 amino acids of the N-gene. The other NcoI site is in the neomycin resistance gene.
  • Plasmid pBM11/N/PF4 was digested with Ncol and BamHI and the 0.6kb NcoI-BamHI plasmid fragment was gel purified. This fragment has the NcoI overhang in the neomycin resistance gene.
  • c. Ligation and isolation of pBM11/NDP/PF4 The 5kb NcoI and the 0.6kb NcoI-BamHI plasmid fragments were ligated with the PF4 gene using DNA ligase and the resulting mixture was used to transform competent HB101. The transformants were selected on neomycin such that only colonies with correctly reconstructed neomycin resistance genes would survive. Transformants were screened using restriction analysis with NcoI and nucleotide sequencing using the Sanger dideoxy technique . A correct construction was isolated and denoted pBM11/NDP/PF4.
  • nucleotide sequence and corresponding amino acid sequence of the synthetic platelet factor 4 gene in fusion downstream of the nucleotide sequences coding for the f irst 32 amino acids of the bacteriophage ⁇ N-gene and the acid labile dipeptide
  • Asp-Pro ( *** ) in the expression vector pBM11 is as follows .
  • DP acid cleavable dipeptide
  • the fusion protein is characterized by having the 32 amino-terminal residues of the bacteriophage ⁇ N-gene followed by the acid labile dipeptide Asp-Pro followed by the 228 amino acids of Oncostatin M including its N-terminal methionine.
  • OncM cDNA (BglII-HindIII) isolated from pOncMVV2 were cloned into the pBM16/NDP/TVV vector prepared by Ncol and HindIII double digestion. The DNA was used to transform E. coli DH5 ⁇ . The clone pBMX was isolated and confirmed to carry the predicted sequence.
  • the recombinant protein produced by pBMX should be cleaved at the R residue of -I-E-G-R- to generate mature OncM with the authentic N-terminal sequence following treatment of activated factor X. Similar procedures were used to prepare Oncostatin M using pBMX as to prepare Oncostatin M from pBM16/NDP/OncM.
  • Synthetic oligonucleotides were designed to allow insertion of DNA coding for a modified alkaline phosphatase signal peptide and a linker region with 3 cloning sites (HindIII, SmaI and BamHI) into the pBM11 expression vector downstream of the P L promoter and N gene ribosomal binding site.
  • the nucleotide sequence was optimized to be as similar as possible to the nucleotide sequence of the amino terminus of the lambda N gene as the lambda N gene sequence has evolved with that of its ribosomal binding site for efficient ribosome initiation and translation.
  • the second amino acid of the alkaline phosphatase signal sequence the basic amino acid lysine was changed to an acidic amino acid, aspartic acid.
  • Plasmid pBM11/NDP/EGF (30ug) was digested with 30 units of EcoRI and then treated with 4 units of Klenow fragment of DNA polymerase to create blunt ends. The DNA was finally digested with 30 units of BamHI and the 0.17kb fragment of the EGF gene was recovered after electrophoresis on an agarose gel. The DNA so purified has a blunted EcoRI site at the 5' end and a BamHI overhang at the 3' end.
  • Plasmid pBM11/PAD (18ug) was digested with 30 units of HindIII and then treated with Klenow fragment to blunt the ends. The DNA was then digested with PvuI and the 0.5kb PvuI-HindIII (blunt) fragment was recovered after electrophoresis on an agarose gel.
  • Plasmid pOncMVV2 containing a modified Oncostatin M gene was digested with NcoI and the 5' overhanging bases were removed by treatment with SI nuclease leaving the fragment blunt-ended. The nuclease treatment removed the codons for the initiating methionine.
  • the plasmid was further digested wth BamHI and the 700 bp NcoI(blunt)-BamHI Oncostatin M gene fragment was gel purified. This fragment contained the NcoI blunt end at the 5' end of the gene and BamHI overhang at the 3' end of the gene,
  • Plasmid pBMHM3/PAD containing the nucleotide sequences coding for a modified alkaline phosphatase signal sequence was digested with HindIII which cuts directly downstream of the signal sequence. The overhanging ends were filled in and made blunt using Klenow fragment of DNA polymerase. The resulting DNA was further digested with PvuI and the 680 bp HindIII (blunt)-PvuI fragment was gel purified.
  • Plasmid pBM11M3/PAD was also digested with BamHI and PvuI and the 5 kb BamHI-PvuI fragment was gel purified. 3. Ligation and isolation of pBM11/PAD/OncM
  • the 700 bp NcoI (blunt)-BamHI Oncostatin M gene fragment, the 680 bp HindIII (blunt)-PvuI fragment of PBM11M3/PAD and the 5 kb BamHI-PvuI fragment of pBMHM3/PAD were ligated together using T4 ligase and transformed into competent HB101 E. coli. Correct construction was assayed by nucleotide sequencing using the Sanger-dideoxy technique. A correct colony was chosen and designated pBM11/PAD/OncM.
  • Synthetic oligonucleotides were designed to link the VGFa synthetic gene with an alkaline phosphatase modified signal sequence to provide for an optimal signal sequence cleavage site by coding for the addi- tional N-terminal residues occurring immediately downstream of the signal sequence cleavage site in the natural VGF, denoted extreme N-terminus above.
  • the nVGFa sequence contains the altered sequences GTC and GYACRC instead of the natural VGF sequences GDC and GMYCRC and terminates with the sequence YQR upstream of the natural sequence PNT.
  • the signal sequence remains attached to the nVGFa forming a fusion protein with nVGFa at the C-terminus.
  • Plasmid pBM11/NDP/VGFa was digested with Pvul and BamHI and the 5.2kb plasmid fragment was gel purified.
  • Plasmid pBM11/NDP/VGFa was digested with Ncol and the 5' overhangs were removed by treatment with S1-nuclease. This created a blunt end at the first codon of the VGFa truncated synthetic gene.
  • the DNA was then digested with BamHI and the 170bp NcoI (blunt)-BamHI fragment was gel purified.
  • HindIII-Pvul fragment of pBM11/PAD, the 5.2kb PvuI-BamHI pBM11 fragment and the 170bp NcoI(blunt)-BamHI synthetic VGFa gene were ligated together using DNA ligase and the resulting mixture was used to transform competent HB101.
  • the transformants were selected on neomycin and screened by restriction analysis and nucleotide sequencing using the Sanger-dideoxy technique.
  • a correct construct was isolated containing the modified alkaline phosphatase signal sequence in frame with the nVGFa gene.
  • nucleotide sequence and corresponding amino acid sequence of the synthetic platelet factor 4 gene in fusion downstream of the nucleotide sequences coding for a modified alkaline phosphatase signal peptide were prepared essentially as described above for pBM11/PAD/nVGFa, except that the synthetic PP4 gene was used instead of the synthetic VGFa gene in Step 3.
  • the construct isolated is as follows. Predicted cleavage site is noted with (***). Signal sequence ->
  • Plasmid pBM11/PAD/nVGF was mutagenized in vitro (Morinaga et al., Biotechnology (1984) 2:636-643) to alter the codons coding for the second amino acid in the signal sequence, namely to change the Asp (D) codon to that for Lys (K) the residue found in the natural sequence. This mutagenesis also introduced a PvuI site into the signal sequence.
  • the EGF gene is part of a fusion with the alkaline phosphatase signal sequence.
  • Plasmid pBM11/PAD/EGF was mutagenized in vitro to alter the codons coding for the second amino acid, in the signal sequence, to change the Asp (D) codon to that for Lys (K) the residue found in the natural sequence. This mutagenesis also introduced a PvuI site into the signal sequence.
  • Plasmid p135-1 was derived from plasmid pDR540
  • pDR540 is an expression vector containing the trp-lac hybrid promoter.
  • p135-1 was digested with Bglll and BamHI and treated with bacterial alkaline phosphatase.
  • Plasmid pBM11/PAK/EGF was digested with PvuII and BamHI and the -230 bp fragment coding for part of the alkaline phosphatase signal sequence and human EGF was isolated. 2. preparation of TacPak1 and TacPak2 Oligonucleotides
  • Synthetic oligonucleotides TacPak1 and TacPak2 were designed with an overhang, compatible with the Bglll site of p135-1 and a PvuII overhand, compatible with the PvuII site in the alkaline phosphatase/EGF PvuII/BamHI fragment.
  • the oligonucleotides were synthesized on an Applied Biosystems Oligonucleotide Synthesizer. BglII Pvul
  • trp-35 (16bp) lac-10 CATCCCCCTG [TTGACA] ATTAATCATCGGCTCG (TATAATG)
  • TacPak/EGF was digested with HindIII and then treated with the Klenow fragment of DNA polymerase to create blunt ends.
  • the DNA was then digested with BamHI and the 420bp fragment containing the tac promoter elements and the coding region for the alkaline phosphatase signal sequence and human EGF was isolated by agarose gel electrophoresis.
  • This plasmid has the tac promoter elements and uses the cro SD to express the modified VGF gene with the N-terminal extension downstream of the alkaline phosphatase signal sequence.
  • the plasmid has a pBR322 background with the neomycin resistance gene.
  • Plasmid pBM11/PAK/nVGFa was digested with Pvul and BamHI and the 350bp fragment was isolated by gel electrophoresis. This fragment contains most of the alkaline phosphatase signal sequence and the nVGFa gene.
  • Plasmid pTCPt/EGF was- digested with PvuI and BamHI and the 2.8kb fragment was isolated by gel electrophoresis.
  • the 2.8kb fragment and the 350bp fragment were ligated using DNA ligase and the DNA was used to transform competent JM109(lacIq). A correct construct was isolated using restriction analysis.
  • This plasmid is designed to have the tac promoter elements and utilize the N-gene SD to express human EGF behind the alkaline phosphatase signal sequence. It has a pBR322 background with the Neomycin resistance gene. 1. Preparation of 2.8kb PvuI-BamHI pTNPt
  • Plasmid pTNPt was digested with Pvul and BamHI and the 2.8kb fragment was isolated by gel electrophoresis. 2. Preparation of 300bp PvuI-BamHI fragment of pBM11/PAK/EGF
  • Plasmid pBM11/PAK/EGF was digested with PvuI and BamHI and the 300bp fragment was isolated. 3. Ligation and Isolation of pTNPt/EGF The 2.8kb fragment and the 300bp fragment were ligated using DNA ligase and the DNA was transformed into competent JM109(LacIq). A correct construct was isolated by restriction analysis and by DNA sequencing.
  • E. coli B (HB101) containing the p3M11/NDP/ growth factor plasmids were grown in Luria Broth at 30oC.
  • the density of the culture was measured at 550 nm and when the density reached an absorbance of 0.7 to 0.9, synthesis of the growth factor fusion protein was induced by increasing the temperature to 42°C.
  • the culture was incubated at this temperature for 5-20 hrs, then the bacteria were isolated by centrifugation and frozen at -70°C until use.
  • the cells were thawed into buffer containing 0.05 M NaH 2 PO 4 pH 7.2, 0.5 M NaCl, 0.01 M EDTA.
  • buffer containing 0.05 M NaH 2 PO 4 pH 7.2, 0.5 M NaCl, 0.01 M EDTA.
  • One hundred fifty ml of buffer was used for a preparation from 50 g bacteria, The cells were disrupted by sonication on ice for 15 min using a 1/4-inch probe, 50% pulse at 60 watts of power.
  • the insoluble protein was collected by centrifugation at 12,000 rpm in a GSA rotor for 90 min.
  • the pellet containing the insoluble protein was then resuspended in 50 ml of 6 M guanidine hydrochloride.
  • the insoluble material was collected by centrifugation for 2 hrs at 25,000 rpm in a Beckman-type 30 ultracentrifuge rotor. The supernatant was collected and stored at -20°C until further use.
  • fusion protein Purification of the fusion protein was carried out on either a Sephacryl S300 or Fractogel HW-55 column equilibrated with 1 M guanidine hydrochloride. Fractions containing the fusion protein were identified as those fractions containing a polypeptide having a molecular weight consistent with the molecular weight of the polypeptide encoded by the synthetic gene as determined on a 15% polyacrylamide-urea gel.
  • the fusion protein was allowed to refold by incubating it in 50 mM Tris-HCl buffer, pH 8.7, containing 1 M guanidine hydrochloride, 1.25 mM reduced glutathione, and 0.25 mM oxidized glutathione at 4°C for 3-10 days.
  • the biological activity of the growth factor was monitored by a competitive receptor binding assay as described above (see Example I.C). When a maximum level of activity was obtained, the protein was dialyzed against distilled water and lyophilized to dryness.
  • the protein was cleaved either by resuspending in 70% formic acid and incubating at 40°C for 3 days or by incubating overnight at room temperature in a 100-fold molar excess of cyanogen bromide.
  • the cleaved product was dialyzed against distilled water and lyophilized to dryness.
  • the growth factor was resuspended in 40% acetonitrile, 0.1% TFA and purified by HPLC using a BioRad-TSK-250 column. Fractions containing the growth factor were pooled and further purified using reversedphase HPLC, either Waters yBondapak C-18 or Rainin Dynamax C-8. The eluant was a linear gradient of 20- 40% acetonitrile containing 0.1% TFA. Fractions containing receptor binding activity were pooled, lyophilized and stored at -20°C until use. 1. TGF and Modified TGF a. N/TGF Recombinant modified human TGF was produced from plasmid pBM11/N/TGF and contained 33 amino acids of the N-gene at the N-terminus and the sequence modification QEEK instead of the natural human sequence QEDK.
  • Recombinant modified VGF was produced from plasmid pBM11/PAD/nVGFa containing the extreme N-terminal sequence of VGF and the modified sequences GTC and GYACRC instead of the natural VGF sequence GDC and GMYCRC.
  • the nVGFA fragment was expressed as a fusion protein with a modified alkaline phosphatase signal sequence at the N-terminus and was truncated at the sequence YQR at the C-terminus.
  • NDP/VGFa NDP/VGFa
  • VGFa Recombinant modified VGF was produced from plasmid pBM11/NDP/VGFa beginning at the DIPAIR sequence and ending at the YKQR sequence in- VGF. It has the modified sequences GTC and GYACRC instead of the natural VGF sequence GDC and GMYCRC.
  • the VGFa fragment was expressed as a fusion protein with 32 amino acids of the N-gene at the N-terminus and the acid labile dipeptide aspartic acid-proline. c. VGFa
  • the VGF fragment was prepared as described in 2.b above and, after cleavage from the fusion protein by acid treatment, was subsequently further purified by HPLC.
  • NDP/VGFA Recombinant modified VGF was produced from plasmid pBM11/NDP/VGFA beginning at the DIPAIR sequence and ending at the YKQR sequence in VGF and having the modified sequences GTC and GYACVC instead of the natural VGF sequence GDC and GMYCRC.
  • the VGFA fragment was expressed as a fusion protein with 32 amino acids of the N-gene at the N-terminus and the acid labile dipeptide aspartic acid-proline.
  • Chimeric TGF/VGF Hybrids a. N/TTV (TGF/TGF/VGF)
  • Recombinant modified TTV was produced from pBM11/N/TTV and contained the amino acid sequence of human TGF in the amino terminal two-thirds of the gene with the exception of the sequence QEEK which was altered from the natural human sequence QEDK.
  • the carboxy terminus was derived from the amino acid sequence of VGF and terminated with the sequence YQR upstream of the natural sequence PNT.
  • the TTV fragment was expressed as a fusion protein with 33 amino acids of the N-gene at the N-terminus.
  • Recombinant TTV was produced from plasmid pBM11/N/TTV and modified as described in (a) except that the TTV fragment was expressed as a fusion protein with 32 amino acids of the N-gene at the N-terminus and the acid labile dipeptide aspartic acid-proline. c. NDP/VTV
  • Recombinant modified VTV was produced from plasmid pBM11/NDP/VTV and contained the amino acid sequence of human TGF in the middle domain with the amino acid sequence QEEK replacing the natural sequence QEDK.
  • the N-terminal and C-terminal domains were derived from the truncated VGF sequence and begin with the sequence DIPAIR and end with the sequence YQR.
  • the VTV fragment was expressed as a fusion protein with 32 amino acids of the N-gene at the N-terminus and the acid labile dipeptide aspartic acid-proline. d.
  • NDP/TW Recombinant modified VTV was produced from plasmid pBM11/NDP/TVV and contained the amino acid sequence of human TGF in the N-terminal domain of the gene.
  • the middle and C-terminal domains were derived from the truncated VGF sequence and end with the sequence YQR.
  • the synthetic gene has the modification GYACVC for GMYCRC.
  • the TVV fragment was expressed as a fusion protein with 32 amino acids of the N-gene at the N-terminus and the acid labile dipeptide aspartic acid-proline.
  • the bacterial cultures were induced with 100 to 1000 mM concentrations of IPTG and were allowed to grow at 30°C for 16 to 24 hours.
  • the bacterial hosts carried an F-factor with the laclq gene, such as JM109, XL1, JM103, etc.
  • bacterial hosts are HB101, DH1, DH5, etc.
  • the expression cassette can be induced with 1% lactose. After the induction period, growth factors can be isolated from either the medium or the cell pellet.
  • Human EGF produced from the expression cassettes TacPak/EGF and pTcCPt/EGF was isolated from the medium in an active form with the alkaline phosphatase signal sequence removed. Approximately 85% of the active EGF is found in the medium, with the remainder associated with the call pellet. These expression cassettes have yielded 4 mg/1 of active EGF. The cells are removed from the medium by centrifugation and the medium is passed through an
  • the growth factors can be isolated from the cell pellet by osmotic shock or sonication and purified by essentially the same procedure.
  • nVGFa Recombinant nVGFa was produced from the expression cassette pTCPt/nVGFa.
  • the nVGFa was isolated from the cell pellet by sonication and was shown to constitute approximately 40% of the total bacterial protein.
  • Recombinant Platelet Factor 4 was isolated essentially as described above for growth factors.
  • Recombinant Platelet Factor 4 was produced from pBM11/N/PF4 as a fusion with the 33 N-terminal amino acids of the N-protein.
  • Recombinant Platelet Factor 4 was produced from pBM11/NDP/PF4 as a fusion with the 33 N-terminal amino acids of the N-protein and an aspartic-acid-proline cleavage site. Treatment of the fusion protein with formic acid released the mature PF4.
  • PNE buffer 0.5 M NaCl, 10 mM EDTA, 50 mM sodium phosphate
  • Aggregated proteins were collected from the cell lysate by centrifugation. Aggregated proteins were sequentially extracted for 16 hrs each with 120 ml of 8 M urea solutions buffered as follows: Solution 1) 20 mM Tris, pH 5; Solution 2) 20 mM Tris, pH 8; Solution 3) 50 mM Tris, pH 11. Most aggregated proteins were solubilized by Solutions 1 and 2, while recombinant Onco M remained insoluble until treatment with Solution 3. b. Refolding of Recombinant Molecules
  • the Solution 3 extract was then dialyzed for 24 hrs against a refolding buffer (1 M guanidine HCl, 1.2 mM oxidized glutathione, 0.2 mM reduced glutathionine, 20 mM Tris HCl, pH 8.0-9.0). Lowering the pH to ⁇ pH 8.0 resulted in a 100-fold reduction in yield of biologically active OncM. Following re-folding, proteins were dialyzed versus 1 N acetic acid before testing in the growth inhibitor assay (Example I.E). 2. PAD/Oncostatin M
  • the PAD/OncoM was prodduced from the expression cassette pBM11/PAD/OncoM.
  • the medium was tested for active OncoM and was shown to contain from 20 to 500 ⁇ g/l.
  • Example X The PAD/OncoM was prodduced from the expression cassette pBM11/PAD/OncoM.
  • the medium was tested for active OncoM and was shown to contain from 20 to 500 ⁇ g/l.
  • This assay determines the ability of a molecule to bind to the EGF receptor as measured by its ability to inhibit the binding of EGF to its receptor. All growth factors and chimeric growth factors, whether modified or truncated, isolated to date were active in the EGF receptor binding inhibition assay. A summary of these results is shown below:
  • Example VE1 The effect of natural or synthetic TGF, EGF and VGF, as well as recombinant growth factors on mid-dermal injuries was assessed as described in Example VE1.
  • Treatment with Silvadene alone or Silvadene with EGF resulted in approximately 50% reepithelialization, while treatment with synthetic TGF or natural VGF resulted in approximately 90% reepithelialization.
  • the optimal concentration to promote re-epithelialization of EGF was 1-10 ⁇ g/m ⁇ , while synthetic TGF and natural VGF produced a maximal response at 0.1 ⁇ g/ml.
  • Modified truncated VGFa was assayed for its ability to accelerate wound healing in a donor graft model.
  • the treatment regimen was as described above (see Example I) using 1 ml VGFa, 5 yg/ml, in 20 g Silvadene. Photographs were taken on a daily basis. A summary of the results is provided below:
  • VGFa - modified open; mostly healed healed truncated apparent healed epithelialization
  • the modified truncated VGFa accelerated healing of the wound as compared to the carrier control. Photographs (not provided) at POD 8 showed substantial differences between saline and VGFa. Example XI .
  • mice Male nude mice were injected with platelet factor 4 or phosphate buffered saline at 2- to 3-day intervals, as described in Example 1C. As shown below, factor 4 significantly inhibited tumor growth. Table IV
  • Oncostatin M Cellular proliferation in the presence of recombinant or native Oncostatin M was compared with proliferation in untreated samples, and expressed as a percentage of maximal (untreated) growth. Samples were assayed in duplicate or triplicate, and generally varied by less than 20% from each other.
  • One growth inhibition assay unit of Oncostatin M is defined as the amount of protein required to cause a 50% inhibition of the growth of A375 cells seeded at 3 to 4 x 103 during a 72-hr assay. Where indicated, concentrations required for half-maximal growth inhibition were determined by extrapolation from proliferation data after transformation as follows:
  • a modified proliferation assay was used, in whihc the target cell number and serum concentration were reduced.
  • Cells were seeded at 500 cells/well in DMEM containing 5% FBS, treated with Oncostatin M in the same medium, and incubated at 37°C until untreated cells reached confluence (generally between 6 and 10 days, depending on the cell line). Monolayers were then stained and processed as described below: Table
  • compositions of the subject invention comprise expression casettes for the efficient expression of polypeptides in prokaryotic cells.
  • the expression cassettes find use in production of large amounts of polypeptides by providing for increased stability of the expression products as well as for obtaining mature folded polypeptides secreted into the growth medium of the host cell.

Abstract

Cassettes d'expression permettant d'exalter l'expression et la production d'un polypeptide d'intérêt dans des cellules procaryotiques. Les cassettes d'expression assurent la production du polypeptide d'intérêt de sorte que ledit polypeptide peut être soit sécrété à partir de la cellule hôte dans une conformation active, soit convenablement traité et renaturé jusqu'à obtention d'un état fonctionnel. Le polypeptide d'intérêt est de préférence exprimé sous forme d'une protéine de fusion, notamment fusionnée avec une séquence guide à partir d'un gène bactérien ou bactériophage fortement exprimé. On peut par la suite cliver le polypeptide d'intérêt à partir de la séquence guide, puis le replier ou l'utiliser comme protéine de fusion.
PCT/US1988/003872 1987-10-30 1988-10-28 Systemes d'expression destines a la preparation de polypeptides dans des cellules procaryotiques WO1989003886A1 (fr)

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US24076888A 1988-09-02 1988-09-02
US240,768 1988-09-02
US115,139 1988-09-02

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FR2632321A1 (fr) * 1988-03-24 1989-12-08 Oncogen Sequence d'adn codant pour un polypeptide, cassette d'expression comprenant une telle sequence, cellule hote comprenant la cassette d'expression et procede pour produire un tel polypeptide
EP0378364A2 (fr) * 1989-01-10 1990-07-18 Repligen Corporation Analogues de PF4 et leurs fragments et compositions pharmaceutiques les contenant
EP0408945A1 (fr) * 1989-07-19 1991-01-23 Grünenthal GmbH Plasmides, leur préparation et leur utilisation pour obtenir un activateur de plasminogène
EP0423641A3 (en) * 1989-10-18 1992-01-15 Nippon Mining Company Limited Expression vector, transformed microorganism, fusion protein and method for the production of platelet factor 4 or tgfalpha
EP0467676A2 (fr) * 1990-07-18 1992-01-22 Applied Immunesciences, Inc. Vecteurs d'expression et de sécrétion inductibles par la température et leur utilisation dans des cellules procaryotiques
EP0482714A1 (fr) * 1990-10-26 1992-04-29 Johnson & Johnson Clinical Diagnostics, Inc. Augmentation de la production d'ADN de thermus aquaticus polymerase chez E. coli
EP0502637A2 (fr) * 1991-02-26 1992-09-09 Zeneca Limited Vecteur
WO2000020449A2 (fr) * 1998-10-07 2000-04-13 Stryker Corporation PROTEINES MODIFIEES DE LA SUPERFAMILLE DU TGF-$g(B)
US7029876B2 (en) 2001-03-09 2006-04-18 Genentech, Inc. Process for production of polypeptides
EP1677665A2 (fr) * 2003-09-19 2006-07-12 General Atomics Determination d'ions au moyen d'enzymes sensibles aux ions
US7153666B2 (en) 2003-07-17 2006-12-26 General Atomics Methods and compositions for determination of glycated proteins
US7368231B2 (en) 2003-09-19 2008-05-06 General Atomics Detection assay for potassium ions using a potassium-dependent urea amidolyase enzyme
US7855079B2 (en) 2006-07-25 2010-12-21 General Atomics Methods for assaying percentage of glycated hemoglobin
US7943385B2 (en) 2006-07-25 2011-05-17 General Atomics Methods for assaying percentage of glycated hemoglobin
US8673646B2 (en) 2008-05-13 2014-03-18 General Atomics Electrochemical biosensor for direct determination of percentage of glycated hemoglobin

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GR890100185A (en) * 1988-03-24 1990-01-19 Oncogen Novel polypeptides having growth factor activity and nucleic acid sequences encoding the polypeptides
FR2632321A1 (fr) * 1988-03-24 1989-12-08 Oncogen Sequence d'adn codant pour un polypeptide, cassette d'expression comprenant une telle sequence, cellule hote comprenant la cassette d'expression et procede pour produire un tel polypeptide
BE1004202A5 (fr) * 1988-03-24 1992-10-13 Oncogen Nouveaux polypeptides ayant une activite de facteur de croissance et sequences d'acides nucleiques codant pour les polypeptides.
EP0378364A3 (fr) * 1989-01-10 1992-01-15 Repligen Corporation Analogues de PF4 et leurs fragments et compositions pharmaceutiques les contenant
EP0378364A2 (fr) * 1989-01-10 1990-07-18 Repligen Corporation Analogues de PF4 et leurs fragments et compositions pharmaceutiques les contenant
EP0408945A1 (fr) * 1989-07-19 1991-01-23 Grünenthal GmbH Plasmides, leur préparation et leur utilisation pour obtenir un activateur de plasminogène
US5637503A (en) * 1989-07-19 1997-06-10 Gruenenthal Gmbh Plasmids, their construction and their use in the manufacture of a plasminogen activator
EP0423641A3 (en) * 1989-10-18 1992-01-15 Nippon Mining Company Limited Expression vector, transformed microorganism, fusion protein and method for the production of platelet factor 4 or tgfalpha
EP0467676A2 (fr) * 1990-07-18 1992-01-22 Applied Immunesciences, Inc. Vecteurs d'expression et de sécrétion inductibles par la température et leur utilisation dans des cellules procaryotiques
EP0467676A3 (en) * 1990-07-18 1992-11-19 Applied Immunesciences, Inc. Temperature-inducible secretion expression vectors and their use in prokaryotic cells
EP0482714A1 (fr) * 1990-10-26 1992-04-29 Johnson & Johnson Clinical Diagnostics, Inc. Augmentation de la production d'ADN de thermus aquaticus polymerase chez E. coli
US6083686A (en) * 1990-10-26 2000-07-04 Johnson & Johnson Clinical Diagnostic Systems, Inc. Increased production of Thermus aquaticus DNA polymerase in E. coli
EP0502637A2 (fr) * 1991-02-26 1992-09-09 Zeneca Limited Vecteur
EP0502637A3 (en) * 1991-02-26 1992-10-28 Imperial Chemical Industries Plc Vector
US5840521A (en) * 1991-02-26 1998-11-24 Imperial Chemical Industriles Plc Expression vector containing an inducible selection gene system
WO2000020449A2 (fr) * 1998-10-07 2000-04-13 Stryker Corporation PROTEINES MODIFIEES DE LA SUPERFAMILLE DU TGF-$g(B)
WO2000020449A3 (fr) * 1998-10-07 2000-10-12 Stryker Corp PROTEINES MODIFIEES DE LA SUPERFAMILLE DU TGF-$g(B)
US8518411B2 (en) 1998-10-07 2013-08-27 Stryker Corporation Modified TGF-β superfamily protein
US7029876B2 (en) 2001-03-09 2006-04-18 Genentech, Inc. Process for production of polypeptides
US7153666B2 (en) 2003-07-17 2006-12-26 General Atomics Methods and compositions for determination of glycated proteins
US8187831B2 (en) 2003-09-19 2012-05-29 General Atomics Determination of ions using ion-sensitive enzymes
EP1677665A2 (fr) * 2003-09-19 2006-07-12 General Atomics Determination d'ions au moyen d'enzymes sensibles aux ions
US7368231B2 (en) 2003-09-19 2008-05-06 General Atomics Detection assay for potassium ions using a potassium-dependent urea amidolyase enzyme
EP1677665A4 (fr) * 2003-09-19 2009-07-01 Gen Atomics Determination d'ions au moyen d'enzymes sensibles aux ions
US7855079B2 (en) 2006-07-25 2010-12-21 General Atomics Methods for assaying percentage of glycated hemoglobin
US8318501B2 (en) 2006-07-25 2012-11-27 General Atomics Methods for assaying percentage of glycated hemoglobin
US8338184B2 (en) 2006-07-25 2012-12-25 General Atomics Methods for assaying percentage of glycated hemoglobin
US7943385B2 (en) 2006-07-25 2011-05-17 General Atomics Methods for assaying percentage of glycated hemoglobin
US8557591B2 (en) 2006-07-25 2013-10-15 General Atomics Methods for assaying percentage of glycated hemoglobin
US8673646B2 (en) 2008-05-13 2014-03-18 General Atomics Electrochemical biosensor for direct determination of percentage of glycated hemoglobin

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EP0372005A1 (fr) 1990-06-13
EP0372005A4 (fr) 1990-06-26
JPH04502851A (ja) 1992-05-28
JP2970811B2 (ja) 1999-11-02

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