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WO2001031038A1 - Immobilisation de peptides heteromeres par genie genetique - Google Patents

Immobilisation de peptides heteromeres par genie genetique Download PDF

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Publication number
WO2001031038A1
WO2001031038A1 PCT/JP2000/007275 JP0007275W WO0131038A1 WO 2001031038 A1 WO2001031038 A1 WO 2001031038A1 JP 0007275 W JP0007275 W JP 0007275W WO 0131038 A1 WO0131038 A1 WO 0131038A1
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Prior art keywords
peptide
chitin
seq
cellulose
heteromeric
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English (en)
Japanese (ja)
Inventor
Atsuo Tanaka
Mitsuyoshi Ueda
Koji Nagao
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Fujisawa Pharmaceutical Co Ltd
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Fujisawa Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/10—Peptides being immobilised on, or in, an organic carrier the carrier being a carbohydrate
    • C07K17/12—Cellulose or derivatives thereof
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • C12N11/12—Cellulose or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the immobilization of heteromeric peptides. Specifically, the present invention relates to a heteropeptide fused with a chitin cellulose-binding domain, and a method for immobilizing the heteropeptide to chitin-cellulose. Background art
  • Enzymes may be immobilized by physical adsorption or covalent bonding.
  • a method in which a peptide having a binding activity to a carrier or a certain protein is fused to a target enzyme may be used.
  • cellulose chitin-binding domain eel lulose or chitin binding domain (CBD) j
  • CBD chitin binding domain
  • Cellulose ⁇ chitin is inexpensive, non-toxic, and chemically stable, so it is suitable as a carrier for immobilizing enzymes. It is also easy to process into membrane, powder, bead, etc. In fact, technology has been developed to immobilize enzyme proteins fused with CBD while maintaining their activity (E Ong et al., 1991, Enzyme Microb. Technol.
  • Enzymes composed of heterogeneous proteins composed of multiple subnetts are often industrially important.
  • 7-?-(4-carboxybutanamide) -cephalosporanic acid acylase (abbreviated as "GL-7ACA acylase"), which is a heteroprotein composed of spikelets and subunits, is used.
  • No. 7-Aminocephalosporanic acid (7-ACA) is an important intermediate of cephalo antibiotics, and is based on cephalosporanic acid (CC) produced by microorganisms as a starting material for chemical synthesis or enzymatic reactions.
  • GL-7ACA acylase like benicillin acylase and other cephalosporanic acylases, is an enzyme composed of heteropeptides.After the precursor gene is synthesized by the expression of the enzyme gene, GL-7ACA acylase is a specific enzyme. It is known that peptides are cleaved into multimers by a processing mechanism.
  • An object of the present invention is to provide a heteromerbide fused with a chitin'cellulose binding domain (CBD) and use thereof.
  • the present invention also provides a method for producing the heteromerbide peptide, a method for immobilizing the heteromerbide peptide, and the like. The task is to
  • the present inventors have conducted intensive studies with the example of GL-7ACA acylase, aiming at immobilization of heteropeptides by applying a genetic engineering technique. Specifically, cellulose-chitin binding domain (CBD) was fused to the GL-7ACA acylase gene, this gene was expressed using Escherichia coli as a host, and GL-7ACA acylase was immobilized on chitin beads by the action of CBD. .
  • CBD cellulose-chitin binding domain
  • a gene in which CBD was fused to three different sites of GL-7ACA acylase by genetic engineering was produced, and the expression thereof was attempted. The activity was successfully achieved in the adsorption state.
  • heteropeptide of the present invention can be applied to various other acylases including cyclic lipopeptide acylase which mediates acylation of cyclic lipopeptide.
  • cyclic lipopeptide acylase which mediates acylation of cyclic lipopeptide.
  • the present inventors have prepared a precursor peptide having a chitin-cellulose binding domain so that it can be immobilized on chitin or a cell mouth while maintaining its activity.
  • the present inventors have found that it is possible to produce merbeptides and have completed the present invention.
  • the present invention provides a heteromeric peptide fused with a chitin / cellulose binding domain, a method for producing the heteromeric peptide, a method for immobilizing the heteromeric peptide, and a use of the heteromeric peptide. More specifically,
  • Aminoacylase is 7-/?-(4-carboxybutane amide)
  • the peptide according to (3) which is a rosporan acid acylase
  • the cyclic lipopeptide acylase has the formula:
  • R 1 is Ashiru group
  • R 2 is hydroxy or Ashiruokishi group
  • R 3 is hydrogen or hydroxy group
  • R 4 is hydrogen or hydroxy group
  • R 5 is hydrogen or hydroxy sul Honiruokishi group
  • R 6 is hydrogen Or an acylase that catalyzes the acylation of compound IV represented by the formula (5), (7) the precursor peptide of the heteromeric peptide according to (1),
  • [12] A method of culturing the host cell according to [11] in a medium, and recovering a heteromeric peptide or a precursor of the heteromerbide peptide from the host cell and / or a culture supernatant thereof.
  • step (b) a step of recovering the reaction product in the step (a), a method for producing a reaction product produced by being catalyzed by the immobilized heteropeptide, (16) The chitin in the heteromeric peptide according to (4), the immobilized heteromeric peptide in which the cellulose-binding domain is bound to chitin and / or cellulose,
  • R 1 is Ashiru group
  • R 2 is hydroxy or Ashiruokishi group
  • R 3 is hydrogen or hydroxy group
  • R 4 is hydrogen or hydroxy group
  • R 5 is hydrogen or hydroxy sul Honiruokishi group
  • R 6 is hydrogen Or a compound of the formula (IV) or a salt thereof
  • R 2 , R ⁇ R ⁇ R 5 and R 6 represent the same groups as described above, or a method for producing a compound (III), characterized by obtaining a salt thereof.
  • a chitin / cellulose-binding domain is added to at least one of the subunits constituting the monocephalosporan acylase, and the chitin / cellulose-binding is added. Domain immobilized on chitin and / or cellulose,
  • a chitin / cellulose binding domain is added to at least one of the subunits constituting a catalytic lipopeptide acylase, and the chitin / cellulose binding domain is immobilized on chitin and / or cellulose.
  • R 2 , R 3 , R ⁇ R 5 and R 6 represent the same groups as described above).
  • the present invention relates to a heteromeric peptide in which at least one of the subunits constituting a heteromeric peptide produced by cleavage of a precursor peptide has a chitin / cellulose binding domain added thereto.
  • heteromeric peptide in the present invention means that a single-chain precursor peptide is first synthesized by transcription and translation of a gene encoding the heteromer peptide, and then the precursor peptide is cleaved by processing.
  • chitin 'cellulose binding domain refers to a peptide domain that binds to chitin and / or cellulose. Such a domain is a cellulase
  • the chitin / cellulose binding domain used in the present invention is not limited in its origin as long as it binds to chitin and Z or cellulose.
  • a partial peptide (SEQ ID NO: 22) of chitinase Al (GenBank Ac. No. M57601, J05599) derived from Bacillus acilluscirculans can be mentioned.
  • a peptide domain capable of binding to a high molecular polymer other than the chitin / cellulose binding domain.
  • Substrate binding domains are included.
  • PHA poly (hydroxyalkanoic acid) depolymerase
  • the heteromeric peptide of the present invention is a recombinant protein obtained by using a known gene recombination technique. It can be prepared as a quality. That is, a DNA coding for a chitin'cellulose binding domain is bound to a DNA coding for a subunit of the target heteropeptide so that the reading frame of the protein matches, and the subunit and the chitin are ligated. 'A fusion protein with the cellulose binding domain may be produced.
  • the position where the chitin / cellulose binding domain is fused is not particularly limited as long as the heteromeric peptide is formed and the original activity of the peptide is maintained.
  • a chitin'cellulose binding domain can be added to the N-terminus or C-terminus of a single subunit, or inserted into a subunit polypeptide.
  • any heteromeric peptide generated by cleavage of the precursor peptide can be used.
  • Such peptides include, for example, hormones, cytokines, enzymes, signaling factors, receptors, and the like.
  • Subunits also include those connected by, for example, a disulfide bond.
  • examples of such proteins include, but are not limited to, insulin, insulin receptor, NMDA (N-methyl-D-aspartate) receptor, HGF (hepatocyte growth factor), and the like.
  • the peptide in which the chitin / cellulose binding domain is added to the natural peptide may be a peptide having a natural amino acid sequence, and the natural peptide may have one or more amino acids. May be a peptide modified by substitution, deletion, addition and / or insertion of a peptide. Such modification of the amino acid sequence can be performed to improve the stability and activity of the heteromer peptide.
  • the heteromeric peptide of the present invention is preferably a protein produced by self-slicing. Self-splicing refers to autonomously cleaving the precursor peptide and generating a heteromer, and expressing the activity of the protein, without relying on the catalysis of other molecules such as protease.
  • Such proteins include intein (Intein; Pietrokovski, S., 1998, Protein Sci. 7: 64-71), N-terminal nucleophilic hydrolase; Ntn) (JA Brannigan et al., 1995, Nature 378: 416-419), and proteins such as maze.
  • intin has a yeast membrane ATPase (Chong, S. et al. 3 1998, J. Biol. Chem., 273: 10567-77; Chong, S. et al., 1996, J. Biol. Chem. 271). : 22159-68), and Pyrococcus kodakaraensis DNA polymerase (Nishioka, M.
  • N-terminal nucleophilic hydrolases include, for example, ⁇ , ⁇ ; bacterial glycosylasparaginase (Liu, Y. et al., 1998, J. Biol. Chem. 273). : 9688-94), Proteasom
  • aminoacylase is an enzyme that catalyzes a reaction that hydrolyzes an acylamino group into an amino group.
  • aminoacylase includes, for example, penicillin acylase (Oh, SJ et al., 1987, Gene 56: 87-97; Verhaert, RM et al., 1997, Appl. Environ. Microbiol. 63: 3412-8 ), Cephalosporan acylase
  • ⁇ - (4-carboxybutaneamide) -cephalosporanate acylase (abbreviated as “GL-7ACA acylase”) is an enzyme with high industrial utility as described above (for example, Ishii, Y. et al., 1995, Eur. J. Biochem. 230: 773-8; Saito, Y. et al., 1996, Annals. NY Acad. Sci. 782: 226-240; see Tsuzuki Katsuaki et al., 1989, Nippon Nogeikagaku Kaishi 63: 1847-1853).
  • GL-7ACA acylase for example, a protein derived from Pseudo marnas as mendocina) C427 strain specified by FERM BP-3425 is preferably used (see Japanese Patent Application Laid-Open No. Hei 7-313161).
  • heteromeric peptide of the present invention in which a chitin'cell-mouth binding domain is added to GL-7ACA acylase derived from the C427 strain include, for example, the amino acid set forth in SEQ ID NO: 17, 19, or 21.
  • Heteromeric peptides generated from a precursor peptide consisting of a sequence are preferably used, but are not limited thereto.
  • V22 derived from the genus Pseudomonas (Aramori, I. et al., 1991, J. Fermentation Bioengineering 72: 232-243), and A14 (Aramori, I. et al., 1992, J. Fermentation Bioengineering 73: 185- 192) can also be used o
  • the cyclic lipopeptide acylase is not particularly limited as long as it has an activity of deacylating the acylamino group of the cyclic lipopeptide.
  • the term “cyclic lipopeptide” refers to a compound having a polypeptide ring and having an amino group as a side chain on the ring. The compound may further have another side chain.
  • Examples of the cyclic lipopeptide include a compound represented by the structural formula (IV). This compound includes a FR901379 substance represented by the following structural formula [IVa] (described in JP-A-3-184921).
  • cyclic lipopeptide acylase examples include, for example, a cyclic lipopeptide acylase derived from the genus Sirep topees. Cyclic lipopeptide acylases from the genus are described, for example, in International Publication No. W097 / 32975 and International Patent Application No. PCT / JP00 / 04285.
  • the heteromeric peptide of the present invention in which a chitin / cellulose binding domain is added to a cyclic lipopeptide acylase derived from Streptooyces sp. NO. 6907 (see International Publication No.
  • Heteromeric peptides generated from the precursor peptide having the amino acid sequence described in No. 28 are preferably used, but are not limited thereto.
  • the precursor peptide of the present invention can be a peptide having any amino acid sequence as long as it has chitin'cellulose binding activity and can form a heteromer having the intended activity.
  • the peptide described in the following (b) or (c) can also be used as a precursor peptide in the present invention.
  • a peptide capable of constructing a heteromer having the same includes, for example, site-directed mutagenesis. More specifically, the present invention provides any of the nucleotide sequences described in SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 27 by using the method, and Can be obtained.
  • the gene encoding the precursor peptide of the present invention includes all of the DNA substantially consisting of the nucleotide sequences shown in SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 27 Or include a part.
  • the gene encoding the precursor peptide of the present invention includes, in addition to the DNA having the above specific nucleotide sequence, a DNA having a nucleotide sequence capable of hybridizing with the DNA having the above specific nucleotide sequence under stringent conditions. including.
  • a condition under which a DNA having a homology of about 60% or more in base sequence can hybridize is referred to as a stringent condition.
  • the gene encoding the precursor peptide of the present invention has at least (1) 60% identity, (2) 70% identity, and (3) 80% identity in the above specific nucleotide sequence.
  • (4) Includes a gene consisting of a nucleotide sequence having 90% identity or (5) 95% identity.
  • the precursor peptide encoded by the gene includes a precursor peptide having chitin'cellulose binding activity and capable of constructing a heteromer having aminoacylase activity.
  • the present invention also relates to a precursor peptide of the above-mentioned heteromeric peptide of the present invention.
  • the present invention also relates to a DM encoding the precursor peptide, an expression vector containing the DNA, and a host cell transformed with the vector.
  • the precursor peptide can be prepared by culturing a host cell transformed with an expression vector containing DM encoding the same in a medium, and collecting the precursor peptide from the culture.
  • the source of the DNA encoding the precursor peptide may be cDNA or It may be nome DNA or synthetic DNA.
  • the DNA encoding the precursor peptide of the present invention includes not only a DNA containing a nucleotide sequence of a natural gene, but also a DNA containing an arbitrary nucleotide sequence based on codon degeneracy.
  • the DNA encoding the chitin'cellulose binding domain is added to the site where the chitin'cellulose binding domain is to be added using known genetic engineering techniques. What is necessary is just to insert so that frames may match. There is no restriction on the position where the chitin / cellulose binding domain is inserted, as long as the heterologous peptide is generated by the expression of the precursor peptide and the original enzyme activity is maintained, and the N-terminal, C-terminal, or intermediate position of each subunit Can be inserted.
  • the chitin'cellulose binding domain can be inserted into at least one optional subunit that makes up the heteromeric peptide. It can also be inserted into multiple subunits.
  • insertion of a chitin'cellulose binding domain at the N-terminus, C-terminus or in the middle of a spleen //? Subunit forms a heteromeric peptide and maintains enzymatic activity .
  • Specific examples of the DNA encoding the precursor peptide of the heteromeric peptide include a DNA having the nucleotide sequence of SEQ ID NO: 16, 18, or 20.
  • a DM peptide encoding a precursor of a heteromeric peptide in which a chitin'cellulose binding domain is inserted at the C-terminus of a large subunit of a cyclic lipopeptide acylase derived from Streptomyces sp. NO.
  • Specific examples include, for example, a DNA having the base sequence of SEQ ID NO: 27.
  • the position at which the chitin / cellulose binding domain is inserted is not limited to this, and it can be inserted at the N-terminus, C-terminus, or middle of any subunit.
  • the host cell is not particularly limited, but includes, for example, bacteria.
  • Bacteria include strains belonging to the genus Escherichia (eg, E. coli JM109 ATCC 53323, E. coli HB101 ATCC 33694, E.coii MN102, E.coi / HB101-16 FERM BP-1872, E. coli 294). ATCC 31446) and strains belonging to the genus Bacillus (such as Bacillus subtilis).
  • actinomycetes for example, strains belonging to the genus Streptomyces (Streptomyces), such as Streptomyces lividans, may be mentioned.
  • a strain belonging to the genus Escherichia specifically, E.co/j'HBlOl or E. coli JM109 is preferably used.
  • E. coli for example, NEB ER2566, etc.
  • the DNA encoding T7 RNA polymerase has been integrated into the genome, etc. It is suitable.
  • the expression vector When bacteria, especially E.co, are used as host cells, the expression vector generally encodes at least a promoter--operator region, initiation codon, and amino acid sequence of a precursor of the heteromeric peptide of the present invention. It is composed of DNA, stop codon, one minute and one region, and a replicable unit.
  • the operator-one region contains a promoter, an operator, and a Shine-Dalgarno (SD) sequence (eg, AAGG).
  • the promoter / operator region is a promoter / operator / region which is conventionally used (eg, a PL-promoter of E. co7 /, a trp-promoter, or a T7 promoter). May be included.
  • Suitable initiation codons include methionine codon (ATG).
  • the DNA encoding the precursor peptide of the present invention is not particularly limited as long as it can encode the amino acid sequence of the precursor peptide of the present invention.
  • the DNA encoding the precursor peptide of the present invention can be prepared by a conventional method.
  • a DM synthesizer may be used to synthesize some or all of the DNA and / or to insert the DNA into a suitable vector (such as a plasmid) obtained from a transformant (eg, E. coli).
  • a suitable vector such as a plasmid
  • the complete DNA sequence encoding the precursor of the type heteromeric peptide can be obtained in a suitable manner, for example, by a suitable enzyme (eg, restriction enzyme, alkaline phosphatase, polynucleotide kinase, DNA ligase, DNA polymerase, etc.).
  • a suitable enzyme eg, restriction enzyme, alkaline phosphatase, polynucleotide kinase, DNA ligase, DNA polymerase, etc.
  • stop codon examples include commonly used stop codons (eg, TAG, TGA, etc.).
  • the evening / mine / overnight region includes a natural or synthetic terminator (eg, synthetic fd phage evening / overnight).
  • a replicable unit is a DNA compound capable of replicating its entire DNA sequence in a host cell, and is a natural or artificially modified plasmid (e.g., prepared from a natural plasmid). DNA fragments) and synthetic plasmids.
  • Suitable plasmids include the plasmid pBR322 in E. coli or an artificially modified product thereof (a DNA fragment obtained by treating PBR322 with an appropriate restriction enzyme).
  • the expression vector is composed of a promoter, an initiation codon, a DNA encoding the amino acid sequence of the precursor peptide of the present invention, a termination codon, and an evening / minine region.
  • An example is pET vector (Novagen).
  • an appropriate DNA fragment eg, phosphoric acid, another restriction site, etc.
  • a conventional method eg, digestion with a restriction enzyme, ligation using T4 DNA ligase. It can also be used.
  • Transformants can be prepared by introducing the above-described expression vector into host cells. Introduction (transformation (transfection)) of an expression vector into a host cell can be performed using a conventionally known technique (for example, the Kushner method in the case of E. coli).
  • the heteromeric peptide and / or its precursor peptide of the present invention is The transformant containing the expression vector prepared as described above can be produced by culturing the transformant in a nutrient medium.
  • the nutrient medium consists of a carbon source (eg, glucose, glycerin, mannitol, fructose, lactose, etc.) and an inorganic or organic nitrogen source (eg, ammonium sulfate, ammonium chloride, casein hydrolyzate, yeast extract, polypropylene). Tonnes, pact trypton, beef extract, etc.).
  • a carbon source eg, glucose, glycerin, mannitol, fructose, lactose, etc.
  • an inorganic or organic nitrogen source eg, ammonium sulfate, ammonium chloride, casein hydrolyzate, yeast extract, polypropylene. Tonnes, pact trypton, beef extract, etc.
  • other nutrient sources eg, inorganic salts (eg, sodium or potassium diphosphate, dipotassium hydrogen phosphate, magnesium chloride, magnesium sulfate, calcium chloride), vitamins (eg, vitamins) B
  • Culturing of the transformant is performed by a method known in the art.
  • Culture conditions such as temperature, medium pH and culture time, are selected to maximize the recovery of the desired hetero-peptide or its precursor peptide, usually between pH 5.5 and 8.5 (preferably pH 7 to 7.5) and 5 to 40 ° C (preferably 10 to 30 ° C) for 5 to 50 hours.
  • pH 5.5 and 8.5 preferably pH 7 to 7.5
  • 5 to 40 ° C preferably 10 to 30 ° C
  • these conditions may vary depending on the transformant.
  • the transformed host cell is cultured in a medium, and the heteromeric peptide or the precursor of the heteromeric peptide is recovered from the host cell and / or a culture supernatant thereof, thereby obtaining the heteromeric protein of the present invention.
  • Peptides or precursor peptides of the heteromeric peptides can be produced.
  • E. coli is used as a host, the telomeric peptide of the present invention or its precursor peptide is usually present in the periplasm or cytoplasm of a cultured transformant. Therefore, these peptides can be obtained, for example, by the following method.
  • cells are collected by a conventional method such as filtration and centrifugation, and the cell wall and / or cell membrane of the cells are treated with, for example, ultrasound and / or lysozyme to obtain cell debris.
  • the obtained cell debris is dissolved in an appropriate aqueous solution (eg, 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 0.1 mM EDTA, 0.1% Triton X-100).
  • an appropriate aqueous solution eg, 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 0.1 mM EDTA, 0.1% Triton X-100.
  • the heteromeric peptide of the present invention or its precursor peptide is isolated and purified according to a commonly used method.
  • isolation and purification methods include dialysis, gel filtration, affinity column chromatography using a monoclonal antibody against the heteromeric peptide or its precursor peptide of the present invention, and column chromatography on a suitable adsorbent.
  • high performance liquid chromatography is exemplified.
  • the heteromeric peptide immobilized on chitin and / or cellulose is obtained.
  • the cell lysate can be directly mixed with a chitin or cellulose carrier in an appropriate buffer and purified by affinity for chitin or cellulose.
  • the chitin / cellulose carrier may be any crystalline chitin / cellulose, and specific examples include bacteriocellulose, cotton fiber, and chitin'cellulose molded beads.
  • the heteropeptide or the precursor peptide of the heteropeptide of the present invention is fixed by mixing it with a chitin 'cellulose carrier.
  • These carriers are not limited to those composed of a single material, but may be a combination of multiple chitin / cellulose materials or a combination of other materials and a chitin / cellulose carrier. .
  • a high molecular weight polymer binding domain other than the chitin / cellulose binding domain it is possible to use a high molecular weight polymer formed into various forms.
  • the immobilized heteromeric peptide bound to chitin and / or cellulose prepared in this manner can be used for various purposes.
  • the method therefor comprises: (a) a step of bringing a substrate of the heteromeric peptide into contact with the immobilized heteropeptide of the present invention; and (b) a step of collecting a reaction product in the step (a).
  • the combination of the immobilized heteropeptide and its substrate is not particularly limited.
  • a DNA polymerase can be immobilized, and DNA and nucleotides can act on the immobilized heteropolymer to synthesize DNA.
  • various acylases including GL-7ACA acylase can be immobilized to synthesize antibiotic derivatives.
  • Applications other than the production of a substance include, for example, purification of a compound that binds to a heteromeric peptide.
  • the functional analysis of the immobilized heteromeric peptide can also be performed using the peptide.
  • an immobilized heteromeric peptide may be used as a biosensor.
  • the production of a useful substance using the immobilized heteromeric peptide of the present invention is carried out, for example, by using immobilized 7-?-(4-carboxybutanamide) -monocephalosporanase as a compound represented by the formula:
  • R 2 is carboxyalkanol having 3 to 8 carbon atoms or D-glutamyl.
  • the preparation method of cephalosporanic acylase and the immobilization of the acylase can be performed, for example, according to the method described in Examples. It can be carried out.
  • the carboxyalkanol having 3 to 8 carbon atoms in the present invention include those having a straight-chain or branched acyl group, and specifically include carboxyacetyl, carboxypropionyl, and carboxybutyl. Examples thereof include ril, carboxyisobutyryl, carboxyvaleryl, carboxyisovaleryl, carboxybivaloyl, carboxyhexanoyl, and carboxyhepnoyl.
  • Suitable compounds (I) and salts of compound (II) include alkali metal salts (eg, sodium salt, potassium salt, lithium salt).
  • the above compound (I) can be produced in an aqueous medium such as water or a buffer. That is, the production of compound (I) is usually carried out by suspending the immobilized enzyme in an aqueous medium such as water or a buffer containing compound (II).
  • the production of compound (I) can be carried out by appropriately selecting a suitable pH, concentration of compound (II), reaction time and reaction temperature according to the properties of the immobilized enzyme to be used.
  • the pH is usually 6 to 10, preferably 7 to 9
  • the reaction temperature is usually 5 to 40 ° C, preferably 5 to 37 ° C
  • the reaction time is usually 0.5 to 50 hours.
  • the concentration of the compound (II) as a substrate can be suitably selected in the range of 1 to 100 mg Iml.
  • the compound (I) thus produced is purified and isolated from the above reaction mixture by a conventional method.
  • the production of a useful substance using the immobilized heteromeric peptide of the present invention can be performed, for example, by adding an immobilized cyclic lipopeptide acylase to a compound represented by the following formula:
  • R 1 is Ashiru group
  • R 2 is hydroxy or Ashiruokishi group
  • R 3 is hydrogen or hydroxy group
  • R 4 is hydrogen or hydroxy group
  • R 5 is hydrogen or hydroxy sul Honiruokishi group
  • R 6 is hydrogen Or a compound represented by the formula (IV) or a salt thereof:
  • the method for preparing a cyclic lipobeptide acylase to which a chitin / cellulose binding domain has been added, and the immobilization of the acylase can be performed, for example, in accordance with the method described in Examples. In addition, it can be carried out according to the method described in International Publication No. W097 / 32975 and International Patent Application No. PCT / JP00 / 04285.
  • Suitable salts of compound (IV) and compound (III) include conventional non-toxic mono- or di-salts, such as metal salts such as alkali metal salts (eg, sodium salt, potassium salt, etc.), Alkaline earth metal salts (eg, calcium salt, magnesium salt, etc.), ammonium salts, salts with organic bases (eg, trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, ⁇ , ⁇ ) Organic acid addition salts (eg, formate, acetate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, toluenesulfonate, etc.), inorganic Acid addition salts (eg, hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, etc.), amino acids (eg, arginine, alpha Salts with sparginic acid, gluta
  • the production of compound (II) can be carried out in an aqueous medium such as water or a buffer. That is, the production of compound (III) is usually carried out by suspending the immobilized enzyme in an aqueous medium such as water or a buffer containing compound (IV).
  • the production of compound (III) can be carried out by appropriately selecting ⁇ , the concentration of compound (IV), the reaction time and the reaction temperature according to the properties of the immobilized enzyme to be used.
  • ⁇ is usually 5 to 10, preferably ⁇ 6 to 9
  • the reaction temperature is usually 10 to 70 ° C, preferably 30 to 50 ° C
  • the reaction time is usually 0.5 to 50 hours. .
  • the concentration of compound (IV) as a substrate can be suitably selected in the range of 1 to 100 mg / ml.
  • Compound (III) produced in this manner is as described above.
  • the reaction mixture is purified and isolated from the reaction mixture by appropriately combining conventional methods such as concentration under reduced pressure, lyophilization, extraction, pH adjustment, adsorption resin, ion exchange resin, crystallization, and recrystallization.
  • the chitin or cellose carrier subjected to the immobilization reaction in the present invention can be used repeatedly by eluting and regenerating the heteromerbide whose activity has been reduced and newly immobilizing the heteromerbide. It is.
  • Eluents include protein denaturants (eg, guanidine hydrochloride solution, urea solution, etc.), surfactants (eg, sodium dodecyl sulfate solution, etc.), and low polar solvents (eg, demineralized water, depending on the properties of the chitin / cellulose binding domain). , Ethylene glycol solution, etc.), etc., and the re-immobilization of the heteropeptide can be performed in the same manner as when it is not used.
  • protein denaturants eg, guanidine hydrochloride solution, urea solution, etc.
  • surfactants eg, sodium dodecyl sulfate solution, etc.
  • low polar solvents eg, demineralized water, depending on the properties of the chitin / cellulose binding domain.
  • Ethylene glycol solution, etc. etc.
  • FIG. 1 is a diagram showing the structure of an expression plasmid of a peptide (C427 GL-7ACA acylase) fused with a chitin cell-cell binding domain.
  • CBD stands for chitin or cell-site binding domain, spikes and /? Stand for subunits and /? Subunits, respectively, and SP stands for spacer peptide.
  • T7 represents the T7 promoter.
  • FIG. 9 is a photograph showing the results of SDS-PAGE (A) and Western analysis (B) of an E. coli extract using an anti-C427 GL-7ACA acylase polyclonal antibody.
  • FIG. 3 is a photograph showing an SDS-PAGE of a chitin bead extract of a peptide (C427 GL-7ACA acylase) fused with a chitin / cellulose binding domain expressed in E. coli.
  • FIG. 4 is a diagram showing the structure of a plasmid pUAYCBD encoding a cyclic lipopeptide acylase (TA6907 acylase) fused with a chitin / cellulose binding domain.
  • FIG. 5 is a diagram showing the structure of the actinomycete expression plasmid pJAYCBD encoding a cyclic lipopeptide acylase (TA6907 acylase) fused with a chitin / cellulose binding domain.
  • PYS9107K plasmid DNA (JP-A-7-313161) was digested with StuI and ClaI and cut into three fragments, and the cut ends were blunt-ended using Klenow fragment. This was electrophoresed on a 1% agarose gel and 2170 bp containing the ORF of C427 GL-7ACA acylase (JP-A-7-313161; Y. Ishii et al., 1994, J. Fermentation Bioengineering 77: 591-597). The fragment was collected. Separately, pUC19 plasmid DNA was digested with Kpnl, blunt-ended similarly, and fractionated by 1% agarose gel electrophoresis.
  • Both DNA fragments were ligated with T4 DNA ligase and transformed into E. coli DH5 by the calcium method. Screening of ampicillin-resistant bacteria was performed based on the fact that a plasmid DNA purified from E. coli was digested with BamHI and a fragment of about 600 bp was cut out, and the plasmid was inverted in the direction opposite to the pUC19 5-galactosidase gene.
  • C427 A plasmid incorporating the GL-7ACA acylase gene was selected and designated as PUC427.
  • C427 GL-7ACA acylase A plasmid was constructed that ligated and expressed CBD at the 5 'and 5' ends of the DNA encoding the heavy chain.
  • the heavy chain and the CBD were linked at the Kpn I site.
  • pTYB2 From the CBD coding region of pTYB2, an expression plasmid of the IMPACT TM T7 system (manufactured by New England BioLabs) used for peptide purification, a PCR reaction using primers 3 and 4 resulted in an approximately 150 bp The fragment was amplified.
  • pTYB2 carries a CBD derived from the 'sils' circulans chitinase A1 (T. Watanabe et al., 1994, J. Bacterid. 176: 4465-4472; GenBank Accession No. M57601, J05599).
  • C427 GL-7ACA acylase Approximately 600 bp fragment was amplified by PCR using primers 5 and 2 with pUC427 plasmid as a template to introduce a KpnI site at the 5 'end of DNA encoding the heavy chain. did.
  • the amplified CBD fragment was digested with NdeI and KpnI, and the 5 ′ terminal fragment of the C427 GL-7ACA acylase gene was digested with KpnI and BamHI. These were incorporated into £ 188427 plasmid digested with Nde I, 8 & 111 111 to give pETN-427 plasmid ( Figure 1).
  • SEQ ID NO: 16 The nucleotide sequence of the coding region of the fusion protein of pETN-427 plasmid is shown in SEQ ID NO: 16, and the amino acid sequence encoded by this nucleotide sequence is shown in SEQ ID NO: 17.
  • SEQ ID NO: 16 1st to 165th position (arrangement (Column number: 1st to 55th in 17) corresponds to CBD.
  • a plasmid was constructed to ligate and express CBD at the third and third ends of the DNA encoding the C427 GL-7ACA acylase chain.
  • the chain and CBD were linked at the Kpn I site.
  • the amplified CBD fragment was digested with SacI and KpnI, and the 3 ′ end fragment of the C427 GL-7ACA acylase gene was digested with KpnI and MluI, respectively. These were incorporated into pUC427 plasmid digested with SacI and Mlul to obtain pUCC-427 plasmid. Furthermore, this plasmid was digested with SacI and NdeI, and incorporated into pET24a plasmid digested with the same restriction enzymes to obtain pETC-427 (FIG. 1).
  • the nucleotide sequence of the coding region of the fusion protein of pETC / 5427 plasmid is shown in SEQ ID NO: 18, and the amino acid sequence encoded by this nucleotide sequence is shown in SEQ ID NO: 19. Positions 2077 to 2238 of SEQ ID NO: 18 (positions 693 to 746 of SEQ ID NO: 19) correspond to CBD.
  • a plasmid was constructed to ligate and express CBD 3 amino acids (9 bp) upstream from the 3 end of the DNA encoding the C427 GL-7ACA acylase heavy chain. Both ends of the CBD were closed at the Kpn I site.
  • the fragment was amplified.
  • the amplified CBD fragment was digested with KpnI and incorporated into pET427C «Kp plasmid digested with ⁇ I to obtain ETC-427 plasmid (Fig. L).
  • the nucleotide sequence of the coding region of the fusion protein of pETC-427 plasmid is shown in SEQ ID NO: 20, and the amino acid sequence encoded by this nucleotide sequence is shown in SEQ ID NO: 21.
  • Positions 484 to 651 of SEQ ID NO: 20 positions 162 to 217 of SEQ ID NO: 21) correspond to CBD.
  • the nucleotide sequence of the PCR amplified portion was confirmed by a fluorescent labeling cycle sequence method using the primers shown below.
  • Primer 1 (antisense)? (5rev): 5'-CGCCTCGTAGTAGGTGAAGTAG-3 '(SEQ ID NO: 2)
  • Primer 1 (antisense) CBDKp3 5'-CCTTCCTGGGTACCTTGAAGCTGCCACAAG-3 '(SEQ ID NO: 4)
  • Primer 5 (sense) 427Kp5: 5, -GCGTCGCCGGTACCCTGGCCGAGCCGA-3 5 (SEQ ID NO: 5)
  • Primer 1 (sense) Mlu (upver2): 5'-ATGAGCTACGGCAATTCTCG-3 '(SEQ ID NO: 8)
  • Primer 9 (antisense) 427Kp3: 5, -GGTCAGGCGGTMCCTGGCTTGMGTT-3, (SEQ ID NO: 9)
  • Primer 10 (antisense) 427insKp3: 5 in GGCGGGTCGGTACCGCCCAGGGTGCGGCCG GGCGACGCGA-3 '(SEQ ID NO: 10)
  • Primer 11 (sense) 427insKp5: 5'-GCCGCACCGGTACCGAGGGCGACCCGCCGGACCTGG-3, (SEQ ID NO: 11)
  • Primer 12 antisense Nco (under3): 5, -CCGTGATCATGTCGAAATACTGCTCG-3, (SEQ ID NO: 12)
  • Primer 13 sense) T7promoter: 5'-TAATACGACTCACTATAGGG-3 '(SEQ ID NO: 13)
  • Each of the prepared plasmids was transformed into the host E. coli ER2566 strain by the calcium method.
  • One loopful of each transformed strain was inoculated into 1 ⁇ medium in 31 ⁇ / 15 test tubes and cultured at 37 ° C for about 15 hours. Inoculate 1 mL of this culture into a 100 mL I 500 mL flask of LB medium at 30 ° C. . Was cultured until the concentration became 0.5 to 0.6, and IPTG was added thereto so that the concentration became 1 mM. Addition of IPTG induces the expression of the integrated gene.
  • the culture temperature was set to 20 to 25 ° C, and the cells were further cultured for 20 to 40 hours, and 0.2 to 0.5 U / mL—enzyme activity of the culture was expressed. Per 1 mol of 7ACA).
  • the cells were collected by centrifuging the culture at 4,000 rpm for 10 minutes at 5 ° C, and 5 mL of 20 mM Tris-HCl buffer pH 8.5, 500 mM NaC 0.1% Triton X-100 Again.
  • the suspension was crushed three times with an ultrasonic crusher under ice cooling for 1 minute, and then centrifuged at 12,000 rpm at 5 ° C for 10 minutes to obtain a supernatant fraction as an enzyme solubilizing solution.
  • adsorption buffer 20 mM Tris-HCl buffer pH 8.5, 500 mM NaCl, 0.1% Triton X-100 (New England) BioLabs) was added with a solubilized enzyme solution such that the enzyme activity was 0.5 to 5 U / ml. This was mixed by shaking at 5 ° C for 5 hours or more, and the enzyme was adsorbed on chitin beads to obtain enzyme-immobilized beads.
  • Glucuryl-7ACA (substrate) powder is dissolved at a concentration of lOmg / mL in 0.15 MTris-HCl buffer (pH 8.7), and the enzyme-immobilized bead solution is activated to about 1-3 U / mL. It was prepared as follows. In a small test tube, O.lmL of the enzyme-immobilized bead solution was added and mixed with 0.9mL of the substrate solution pre-incubated at 37 ° C for about 5 minutes, and reacted at 37 ° C for 5 minutes. After a lapse of time, 1 mL of 4% acetic acid was added to stop the reaction, and the mixture was diluted three times with ion-exchanged water, and then the 7ACA concentration was quantified by HPLC under the following conditions.
  • the enzyme activity was measured by the activity calculation formula “7ACA concentration ( ⁇ g / ml) ⁇ 6 10/5 / 272.4 ⁇ dilution factor at the time of preparing the enzyme solution”.
  • “6” is the dilution factor after the reaction
  • “10” is the dilution factor of the enzyme solution during the reaction
  • "5" is the reaction time (min.)
  • “272.4” is the 7ACA molecular weight.
  • the immobilized beads were directly treated with SDS-PACE sample buffer and analyzed by SDS-PAGE. As a result, it was confirmed that the beads were highly purified (Fig. 3).
  • the activity of // lactamase which shows the catalytic activity of decomposing the substrate and the product, which is commonly produced by the host Escherichia coli.
  • the solubilized enzyme extracted with 1 mL of 20 mM Tris-HCl buffer pH 8.5, 500 mM NaC 0.1% Triton X-100 (adsorption buffer) The liquid was used as a culture solution sample. Using 1 mL of this solubilized enzyme solution, it was immobilized on chitin beads so that GL-7ACA acylase activity became 0.5 U / mg-dry beads.
  • the 7ACA concentration was quantified by HPLC in the same manner as above, and from the amount of decomposed 7ACA, the activity calculation formula “(Blank 7ACA concentration ( ⁇ g / ml) —sample 7ACA concentration (g / ml)) / 120 / 272.480 x 10/30 "to determine the enzyme activity.
  • “120” is the reaction time (min-)
  • “272.4” is the 7ACA molecular weight
  • "80” is the dilution factor after the reaction
  • “10” is the sample dilution factor during the reaction
  • “30” is the sample preparation for the culture solution. Represents the concentration factor.
  • Streptomices sp. No. 6907 strain (FERM BP-5809; W097 / 32975); this strain is the Institute of Biotechnology, Industrial Technology Institute of the Ministry of International Trade and Industry (1-1-3 Tsukuba East, Ibaraki, Japan) , FR901379 acylase (see W097 / 32975) derived from the original deposit date of March 8, 1996, international deposit date (transferred February 3, 1997); Streptomices in the present invention.
  • This acylase derived from sp. No. 6907 strain is also referred to as “TA6907 acylase”). I do. TA6907 acylase and CBD are linked at the Kpn I site.
  • IMPACT TM T7 system (New England) used for peptide purification as CBD From the CBD coding region of pTYB2, an expression plasmid of Biolabs, a fragment of about 150 bp was obtained by PCR using Primer 14 (SEQ ID NO: 23) and Primer 15 (SEQ ID NO: 24). To amplify. The amplified fragment has a Kpn I site at the 5 'end and a stop codon and a Bajn HI site at the 3' end.
  • SEQ ID NO: 27, position 3337 1 to position 3526 correspond to CBD. Further, this plasmid was digested with Sac I and Bajn HI, and the excised fragment of about 3000 bp was incorporated into an actinomycete vector PIJ702 (see W097 / 32975) digested with Sac I and Bgl II. (Fig. 5).
  • Streptomyces lividans 1326 J. General Microbiology 1983, 129, 2703-2713 according to the method described in Transform with pJAYCBD.
  • One of the obtained transformants is selected and designated as Streptomyces lividans 1326 / pJAYCBD.
  • Primer 1 sense
  • CBDKp5 5'-AAGCCAGGTACCACGACAAATCC-3 '(SEQ ID NO: 23)
  • Primer_2 (antisense) CBDBam3: 5, -MTTCGGGATCCCTATTGAAGCTGCC-3, (SEQ ID NO: 24)
  • Primer 4 (antisense) AcyKp3: 5, -CCGCCCACCGGTACCCCGCCGCTCGTGCAC-3 '(SEQ ID NO: 26)
  • FR901379 acylase activity of the culture Incubate at 30 ° C for 3 days (260 rpm), measure the FR901379 acylase activity of the culture, and confirm the activity to produce FR179642 (desacylated FR901379 substance) (both refer to W097 / 32975) .
  • the acylase activity can be measured as follows.
  • FR901379 100mg / ml FR901379 (see W097 / 32975) 0.1 ml of aqueous solution, phosphate buffer (pH 6.0) 0.1 ml of methanol 0.1 ml, distilled water 0.6 ml, culture solution or immobilized enzyme suspension Add 0.1 ml and react at 37 ° C (125 rpm). After 15 minutes, the reaction is terminated by adding 1 ml of 4% acetic acid and 2 ml of distilled water. Then, the generated FR179642 (desacylated FR901379 substance) is quantified using high performance liquid chromatography (HPLC) under the following conditions.
  • HPLC high performance liquid chromatography
  • Enzyme-immobilized beads are recovered by centrifugation at 3000 rpm for 10 minutes at 5 ° C, and resuspended in washing buffer (20 mM Tris-HC1 buffer pH 8.5, 500 mM NaC 0.1% Triton X 100). Wash by re-centrifugation twice.
  • CBD fusion acylase shows high adsorption activity to chitin beads. Purification with high purity can be confirmed by directly treating the immobilized beads with SDS-PAGE sample buffer and performing SDS-PAGE analysis.
  • Industrial applicability According to the present invention, a heteropeptide fused with a chitin / cellulose binding domain is provided. According to the present invention, it is possible to immobilize an enzyme which is industrially useful and has a specific processing mechanism while maintaining its activity. Chitin and cellulose are industrially usable carriers, and the heteromeric peptide of the present invention using a chitin-cellulose binding domain exhibiting an adsorption ability is suitable for industrial use.
  • Escherichia coli contains an enzyme, lactamase, which degrades a compound having a cephaloskeleton, and removal of this enzyme was indispensable in the conventional immobilized enzyme method. Can be removed.
  • the product yield was reduced by the adsorption of the substrate and the product on the carrier, but the immobilization method of the present invention does not adsorb the substrate and the product, It is possible to prepare an immobilized heteromeric peptide in high yield.

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Abstract

L'invention concerne une méthode permettant d'immobiliser des peptides hétéromères comportant plusieurs sous-unités formées par découpage de peptides précurseurs, sur un support de chitine ou de cellulose. Ladite méthode consiste à exprimer une protéine possédant un domaine de liaison chitine-cellulose (CBD), fusionné à ladite protéine par une technique de manipulation génétique. L'invention concerne également les peptides hétéromères fusionnés à une domaine de liaison chitine-cellulose, et une méthode permettant d'immobiliser ces peptides hétéromères. Lesdits peptides hétéromères pourvus d'un domaine de liaison chitine-cellulose sont utilisés dans des techniques d'immobilisation d'enzymes catalytiques visant à produire des substances utiles, etc. Cette méthode d'immobilisation des peptides hétéromères peut s'appliquer à des utilisations industrielles.
PCT/JP2000/007275 1999-10-22 2000-10-19 Immobilisation de peptides heteromeres par genie genetique Ceased WO2001031038A1 (fr)

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Cited By (6)

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Publication number Priority date Publication date Assignee Title
EP1197557A4 (fr) * 1999-07-02 2002-09-11 Fujisawa Pharmaceutical Co Gene codant pour une acylase des lipopeptides cycliques et expression dudit gene
EP0885957A4 (fr) * 1996-03-08 2004-06-09 Fujisawa Pharmaceutical Co Processus de deacylation de lipopeptides cycliques
US8053215B2 (en) 2001-09-15 2011-11-08 Ahram Biosystems, Inc. Method and apparatus for amplification of nucleic acid sequences by using thermal convection
US9346055B2 (en) 2001-10-30 2016-05-24 Ahram Biosystems, Inc. Method and apparatus for amplification of nucleic acid sequences using immobilized DNA polymerase
US9573133B2 (en) 2010-01-12 2017-02-21 Ahram Biosystems, Inc. Two-stage thermal convection apparatus and uses thereof
US9573134B2 (en) 2010-01-12 2017-02-21 Ahram Biosystems, Inc. Three-stage thermal convection apparatus and uses thereof

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WO1994024158A1 (fr) * 1993-04-14 1994-10-27 The Regents Of The University Of California Domaine de liaison de cellulose
JPH07313161A (ja) * 1994-05-24 1995-12-05 Fujisawa Pharmaceut Co Ltd ７−β−（４−カルボキシブタンアミド）−セファロスポラン酸アシラーゼ及びその製造方法
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SHAORONG CHONG ET AL.: "Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element", GENE, vol. 192, no. 2, 19 June 1997 (1997-06-19), pages 271 - 281, XP002936068 *
TAKESHI WATANABE ET AL.: "The roles of the C-terminal domain and type III domains of chitinase A1 from bacillus circulans WL-12 in chitin degradation", JOURNAL OF BACTERIOLOGY, vol. 176, no. 15, 1 August 1994 (1994-08-01), pages 4465 - 4472, XP002936066 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0885957A4 (fr) * 1996-03-08 2004-06-09 Fujisawa Pharmaceutical Co Processus de deacylation de lipopeptides cycliques
EP2172546A1 (fr) * 1996-03-08 2010-04-07 Astellas Pharma Inc. Processus de déacylation de lipopeptides cycliques
EP1197557A4 (fr) * 1999-07-02 2002-09-11 Fujisawa Pharmaceutical Co Gene codant pour une acylase des lipopeptides cycliques et expression dudit gene
US8053215B2 (en) 2001-09-15 2011-11-08 Ahram Biosystems, Inc. Method and apparatus for amplification of nucleic acid sequences by using thermal convection
US9765376B2 (en) 2001-09-15 2017-09-19 Ahram Biosystems, Inc. Method and apparatus for amplification of nucleic acid sequences by using thermal convection
US9346055B2 (en) 2001-10-30 2016-05-24 Ahram Biosystems, Inc. Method and apparatus for amplification of nucleic acid sequences using immobilized DNA polymerase
US9573133B2 (en) 2010-01-12 2017-02-21 Ahram Biosystems, Inc. Two-stage thermal convection apparatus and uses thereof
US9573134B2 (en) 2010-01-12 2017-02-21 Ahram Biosystems, Inc. Three-stage thermal convection apparatus and uses thereof
US10086374B2 (en) 2010-01-12 2018-10-02 Ahram Biosystems, Inc. Three-stage thermal convection apparatus and uses thereof
US10086375B2 (en) 2010-01-12 2018-10-02 Ahram Biosystems, Inc. Two-stage thermal convection apparatus and uses thereof

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