WO2000061760A1 - Domaines de fixation glucidiques de piromyces equi - Google Patents

Domaines de fixation glucidiques de piromyces equi Download PDF

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Publication number
WO2000061760A1
WO2000061760A1 PCT/GB2000/001317 GB0001317W WO0061760A1 WO 2000061760 A1 WO2000061760 A1 WO 2000061760A1 GB 0001317 W GB0001317 W GB 0001317W WO 0061760 A1 WO0061760 A1 WO 0061760A1
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protein
derivative
carbohydrate
affinity
plant
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PCT/GB2000/001317
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English (en)
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Alexander Carl Freelove
Peter John White
Geoffrey Peter Hazlewood
Harry John Gilbert
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Babraham Institute
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Priority to AU38289/00A priority Critical patent/AU3828900A/en
Publication of WO2000061760A1 publication Critical patent/WO2000061760A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • 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
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to novel carbohydrate binding domains, to molecules containing the domains, to nucleic acid molecules encoding the novel domains and the molecules containing them, and to methods for their production.
  • the invention also relates to affinity purification methods, methods for enhancing the catalytic activity of carbohydrate inhibited enzymes, methods for protecting carbohydrates or carbohydrate moieties from enzymatic or chemical reagents, and to use of molecules containing the novel binding protein in such methods .
  • the invention further relates to methods for enhancing plant cell growth, to methods for enhancing the solubility of expressed proteins, to methods for selectively targeting a reagent to a substrate and to use of molecules containing the novel domains in such methods .
  • Selective targeting of molecules containing the novel domains can be used in, for example, herbicide or pesticide treatment of crops, prevention or amelioration of bio-deterioration of harvested food crops or textiles, and in food analysis.
  • the cellulase/hemicellulase system of the anaerobic fungus Piromyces equi constitutes a multi-enzyme complex, which binds strongly to cellulose and contains endoglucanase, xylanase, mannanase and ⁇ -glucosidase enzymes. Formation of the complex appears to be mediated by a 40 residue reiterated domain, known as a "docking" domain, which is highly conserved in enzymes that are part of the complex. It is thought that these "docking" domains are involved in protein-protein interactions between the catalytic subunits in which they are present and a putative scaffolding protein. No domains responsible for binding to cellulose/hemicellulose have been found on the catalytic subunits and the mechanism of attachment of the enzyme complex to its natural substrate remains unclear.
  • the protein has no detectable enzyme activity or any significant homology to known proteins .
  • the protein comprises 3 docking domains and two novel repeat (Rpt) domains (Rptl and Rpt2, shown in Figure 1) separated by a T- rich linker. Each Rpt domain is approximately 144 amino acids in length (16Kd) .
  • An RptDom domain is made up of the Rptl and Rpt2 domains together with the T-rich linker between them.
  • the novel repeated domain (RptDom) as well as the second of the two repeats (Rpt2) have been successfully expressed in E. coli and purified to homogeneity.
  • carbohydrate binding protein in substantially isolated form obtainable from Piromyces equi which binds to galactomannan, beta glucan, arabinoxylan, and cellulose.
  • cellulose used herein includes soluble substituted forms of cellulose such as hydroxyethyl cellulose, methyl cellulose and carboxy methyl cellulose.
  • a carbohydrate binding protein in substantially isolated form obtainable from Piromyces equi which binds to at least one of locust bean galactomannan, carob galactomannan, barley beta glucan, hydroxyethyl cellulose or rye arabinoxylan with higher affinity than it binds to at least one of 4-o-methyl- D-glucurono-D-xylan, oat spelt xylan, wheat arabinoxylan, or birchwood xylan.
  • a carbohydrate binding protein in substantially isolated form obtainable from Piromyces equi which selectively discriminates in its binding activity between xylans from different plant species .
  • the carbohydrate binding protein binds with higher affinity to oat spelt xylan than birchwood xylan.
  • carbohydrate binding protein in substantially isolated form obtainable from Piromyces equi which selectively discriminates in its binding activity between arabinoxylans from different plant species .
  • the carbohydrate binding protein binds with higher affinity to rye arabinoxylan than wheat arabinoxylan.
  • a carbohydrate binding protein in substantially isolated form obtainable from Piromyces equi which selectively discriminates in its binding activity between galactomannans from different plant species.
  • the carbohydrate binding protein binds with higher affinity to locust bean galactomannan than carob galactomannan.
  • carbohydrate binding protein in substantially isolated form obtainable from Piromyces equi comprising one or more Rpt domains .
  • the carbohydrate binding protein may further comprise one or more docking domains. Two or more Rpt domains may be arranged in tandem.
  • the carbohydrate binding protein may comprise two or more Rpt domains wherein the Rpt domains bind cooperatively to a carbohydrate.
  • the carbohydrate is locust bean galactomannan, carob galactomannan, barley beta glucan, hydroxyethyl cellulose, rye arabinoxylan, 4-o-methyl-D- glucurono-D-xylan, oat spelt xylan, or wheat arabinoxylan.
  • a carbohydrate binding protein in substantially isolated form obtainable from Piromyces equi which comprises the amino acid sequence of SEQ ID NO:l, or a derivative thereof which retains carbohydrate binding activity.
  • a carbohydrate binding protein in substantially isolated form obtainable from Piromyces equi which comprises the amino acid sequence of SEQ ID NO: 2, or a derivative thereof which retains carbohydrate binding activity.
  • a carbohydrate binding protein in substantially isolated form obtainable from Piromyces equi which comprises the amino acid sequence of SEQ ID NO: 3, or a derivative thereof which retains carbohydrate binding activity.
  • a carbohydrate binding protein in substantially isolated form obtainable from Piromyces equi which comprises the amino acid sequence of SEQ ID NO: 4, or a derivative thereof which retains carbohydrate binding activity.
  • derivative used herein means any derivative of the protein and includes, for example, a protein with a sequence which differs from the sequence of the protein by virtue of an amino acid substitution, deletion, or addition, protease truncation or post-translational modification.
  • nucleic acid molecule in substantially isolated form which encodes a protein or derivative according to the invention.
  • nucleic acid molecule in substantially isolated form comprising a sequence which corresponds to, or is genetically redundant to, the sequence of SEQ ID NO: 5.
  • nucleic acid molecule in substantially isolated form comprising a sequence which corresponds to, or is genetically redundant to, the sequence of SEQ ID NO: 6.
  • nucleic acid molecule in substantially isolated form comprising a sequence which corresponds to, or is genetically redundant to, the sequence of SEQ ID NO: 7.
  • nucleic acid molecule in substantially isolated form comprising a sequence which corresponds to, or is genetically redundant to, the sequence of SEQ ID NO: 8.
  • the vector may be an expression vector in which the nucleic acid molecule is operably linked to sequences capable of directing expression of the protein encoded by the nucleic acid molecule.
  • the expression vector may, for example, be derived from pET23 (Novagen) .
  • pET23a is shown in Figure 1.
  • a method for preparing a protein or derivative of the invention which comprises culturing a host cell comprising DNA encoding the protein or derivative under conditions permitting expression of the protein or derivative from the DNA in the host cell; and recovering the protein or derivative from the host cell.
  • Methods for preparing a protein or derivative of the invention may further comprise transforming the host cell with the DNA encoding the protein or derivative.
  • Methods for preparing a protein or derivative of the invention may further comprise preparing a replicable expression vector comprising the DNA encoding the protein or derivative which is capable of directing expression of the protein or derivative in the host cell.
  • proteins or derivatives according to the invention can be used for bio-separations, for example in affinity chromatography.
  • the naturally occurring configuration of two binding domains existing in tandem is especially advantageous .
  • Affinity for the carbohydrate cellulose, in particular acid-washed cellulose, is enhanced when the two domains are expressed together. Since each domain can function independently then it is possible to modulate the affinity of the fusion for the affinity matrix.
  • Proteins or derivatives according to the invention have lectin-like properties which could be exploited for purification of glycoproteins or whole cells and the characterization of sugar chains associated with them.
  • a method for substantially purifying a protein according to the invention from a mixture of that protein with impurities which comprises : contacting the mixture with an affinity matrix having relatively high affinity for the protein and relatively low affinity for the impurities to bind the protein to the affinity matrix; washing the affinity matrix to substantially separate the impurities from the protein; and eluting the protein from the affinity matrix.
  • a method for substantially purifying a fusion protein according to the invention from a mixture of that fusion protein with impurities which comprises : contacting the mixture with an affinity matrix having relatively high affinity for the first protein or derivative of the fusion protein and relatively low affinity for the impurities and the second protein of the fusion protein to bind the fusion protein to the affinity matrix; washing the affinity matrix to substantially separate the impurities from the fusion protein; and eluting the fusion protein from the affinity matrix.
  • a method for substantially purifying a glycoprotein having one or more carbohydrate moieties from a mixture of that glycoprotein with impurities which comprises : contacting a protein according to the invention, having a relatively high affinity for one or more of the carbohydrate moieties of the glycoprotein and a relatively low affinity for the impurities, with an affinity matrix having a relatively high affinity for the protein and a relatively low affinity for the glycoprotein and the impurities to bind the protein to the affinity matrix; washing the affinity matrix to substantially remove unbound protein; contacting the mixture with the protein bound affinity matrix to bind the glycoprotein to the protein bound to the affinity matrix; washing the glycoprotein and protein bound affinity matrix to substantially remove the impurities; and eluting the glycoprotein from the protein.
  • Methods according to the invention for substantially purifying a glycoprotein may further comprise eluting the protein from the affinity matrix before the glycoprotein is eluted from the protein.
  • a method for substantially purifying a cell expressing a glycoprotein on its surface from a mixture of that cell with impurities which comprises : contacting a protein according to the invention, having a relatively high affinity for one or more of the carbohydrate moieties of the glycoprotein and a relatively low affinity for the impurities, with an affinity matrix having a relatively high affinity for the protein and a relatively low affinity for the glycoprotein and the impurities to bind the protein to the affinity matrix; washing the affinity matrix to substantially remove unbound protein; contacting the mixture with the protein bound affinity matrix to bind the cell to the protein bound to the affinity matrix; washing the cell and protein bound affinity matrix to substantially remove the impurities; and eluting the cell from the protein.
  • the affinity matrix may comprise cellulose, preferably acid- washed cellulose.
  • a protein according to the invention in affinity purification of a carbohydrate, a glycoprotein, or a cell expressing a glycoprotein on its surface, which selectively binds to the protein.
  • an affinity matrix for use in methods according to the invention which comprises a protein according to the invention.
  • fusion proteins can be provided which comprise a protein or derivative of the invention fused to a protein having a different activity.
  • fusion proteins comprising a protein or derivative according to the invention have uses for biocatalysis .
  • a protein or derivative of the invention can be fused to a glycosyl hydrolase for which there is no known binding domain, for example a mannanase or a ⁇ -glucanase. This can help to increase the specific activity of the glycosyl hydrolase for its natural substrate (or a synthetic substrate) .
  • fusion protein comprising a first protein or derivative of the invention fused to a second protein.
  • the second protein may be an enzyme, wherein at least part of the substrate of the enzyme or of a molecule or other moiety bound to the substrate can be bound by the first protein or derivative. This allows the enzyme to be targeted to its substrate. It will be appreciated that the different affinities of proteins or derivatives of the invention for different carbohydrates may be exploited to preferentially target the enzyme to a particular substrate.
  • the second protein may have glycosyl hydrolase activity, for example mannanase or ⁇ -glucanase activity, or glycosyl transferase activity.
  • proteins or derivatives of the invention can bind and sequester carbohydrates and thereby enhance the catalytic activity of enzymes which are inhibited by carbohydrates.
  • glycosyl hydrolase enzymes are product inhibited.
  • Proteins or derivatives of the invention can sequester the carbohydrate products of such enzymes as they are formed and thereby reduce product inhibition.
  • a method for preventing or relieving inhibition of an enzyme by a carbohydrate inhibitor of the enzyme which comprises contacting a mixture of the enzyme and the carbohydrate inhibitor with a protein or derivative of the invention which can bind and sequester the carbohydrate inhibitor. Relief of inhibition may be partial or total.
  • a substrate of the enzyme is also a carbohydrate
  • the protein or derivative should bind preferentially to the carbohydrate inhibitor instead of the substrate.
  • a protein or derivative of the invention to prevent or relieve inhibition of an enzyme by a carbohydrate inhibitor of the enzyme .
  • the carbohydrate inhibitor may be a product inhibitor of the enzyme .
  • binding of proteins or derivatives of the invention to carbohydrates or carbohydrate moieties can protect them from reaction with enzymatic or chemical reagents. This can be of particular use to protect carbohydrate side chains of compounds which are part of a synthetic pathway from undesired reaction with enzymes involved in the pathway.
  • a method for protecting a carbohydrate or carbohydrate moiety from reaction with an enzymatic or chemical reagent which comprises contacting the carbohydrate or carbohydrate moiety with a protein or derivative of the invention capable of binding the carbohydrate or carbohydrate moiety.
  • the carbohydrate moiety may be a side chain group of a polysaccharide or a glycoprotein.
  • the carbohydrate moiety may be a side chain group of a substrate of the enzymatic reagent or of a product of a reaction of the enzymatic reagent with its substrate.
  • the enzymatic reagent may be a glycosyl hydrolase, a glycosyl synthetase, or a glycosyl transferase.
  • proteins or derivatives of the invention can be used as targeting means to target an agent to a desired site.
  • a herbicide or pesticide which is coupled to a protein or derivative of the invention could be targeted to a particular plant or plant cell using the affinity of the protein or derivative for a carbohydrate of the plant or plant cell.
  • an agent can be selectively targeted to rye rather than wheat by coupling the agent to a protein or derivative of the invention which binds to rye arabinoxylan with higher affinity than wheat arabinoxylan.
  • Selective targeting could also control post- harvest bio-deterioration of food crops or textiles [How?] .
  • a target molecule for targeting an agent to a site having a carbohydrate moiety which comprises a protein or derivative of the invention coupled to the agent, wherein the protein or derivative can bind to the carbohydrate moiety.
  • a selective target molecule for preferentially targeting an agent to a subset of sites out of a set of sites, each site having a carbohydrate moiety which comprises a protein or derivative of the invention coupled to the agent, wherein the protein or derivative can bind to the carbohydrate moiety of each site of the subset with higher affinity than to the carbohydrate moiety of each of the remaining sites of the set.
  • a method for targeting an agent to a site having a carbohydrate moiety which comprises contacting the site with a protein or derivative of the invention coupled to the agent, wherein the protein or derivative can bind to the carbohydrate moiety.
  • a method for preferentially targeting an agent to a subset of sites out of a set of sites, each site having a carbohydrate moiety which comprises contacting a protein or derivative of the invention coupled to the agent with each site of the set, wherein the protein or derivative can bind to the carbohydrate moiety of each site of the subset with higher affinity than to the carbohydrate moiety of each of the remaining sites of the set.
  • the carbohydrate moiety may comprise an arabinoxylan, a galactomannan, a xylan, a ⁇ -glucan, or a derivative thereof.
  • Each site of the subset may comprise a rye arabinoxylan or derivative thereof and each site of the remaining sites of the set may comprise a wheat arabinoxylan or derivative thereof.
  • Each site of the subset may comprise a locust bean galactomannan or derivative thereof and each site of the remaining sites of the set may comprise a carob galactomannan or derivative thereof.
  • Each site of the subset may comprise an oat spelt xylan or derivative thereof and each site of the remaining sites of the set may comprise a birchwood xylan or derivative thereof.
  • Each site may be a plant.
  • the plants of the subset may be a different species to the remaining plants of the set.
  • Each site may be a plant cell.
  • the plant cells of the subset may be a different species or cell type to the remaining plant cells of the set.
  • the agent may be a herbicide or a pesticide.
  • the agent may be an antifungal agent such as Agrocybin or trichothecin.
  • each site is a plant.
  • the plants of the subset of sites may be a different species to the plants of the remaining sites of the set, and the carbohydrate moiety may be specific to the species of the subset.
  • the agent can then be preferentially targeted to the plants of the subset using a target molecule of the invention.
  • the agent is a herbicide, a pesticide or an antifungal agent.
  • herbicides, pesticides or antifungal agents can be preferentially targeted to a particular species of plant.
  • the protein or derivative of the invention can bind to rye arabinoxylan with higher affinity than to wheat arabinoxylan. Consequently, a target molecule of the invention can be used to selectively target the agent to rye instead of wheat. If the carbohydrate moiety comprises a galactomannan the protein or derivative of the invention can bind to locust bean galactomannan with higher affinity than carob galactomannan to selectively target the agent to locust bean instead of carob.
  • the protein or derivative of the invention can bind to oat spelt xylan with higher affinity than birchwood xylan to selectively target the agent to oat spelt instead of birchwood.
  • Target molecules of the invention can also be used to target, or selectively target, agents to plant cells or textiles.
  • a target molecule of the invention in a method for targeting or preferentially targeting an agent to a site.
  • a target molecule of the invention in the herbicide, pesticide, or antifungal treatment of a crop.
  • a target molecule of the invention as a selective herbicide, pesticide, or antifungal agent, for example as a herbicide or pesticide for rye instead of wheat.
  • a target molecule of the invention in the prevention or amelioration of bio-deterioration of a harvested food crop or a textile.
  • proteins or derivatives of the invention can be used to test whether food has a particular type of carbohydrate present. This has application in testing for the presence of a genetically modified component in a food.
  • a protein or derivative of the invention is coupled to a detectable reporter and contacted with the food under conditions permitting binding of the protein or derivative to carbohydrate in the food.
  • Suitable reporters include a fluorescent or luminescent reporter, or a polypeptide (such as an antibody) or enzyme (such as firefly luciferase) which can be detected by assay.
  • a protein or derivative of the invention in testing food for the presence of a carbohydrate which can be bound by the protein or derivative .
  • a protein or derivative of the invention may enhance the growth of a plant cell, thereby increasing its cell volume, surface area and wet or dry weight.
  • a method for enhancing plant cell growth which comprises providing a plant cell with an amount of a protein or derivative of the invention sufficient to enhance the growth of the plant cell compared to the growth of an equivalent plant cell which has not been provided with the protein or derivative.
  • the plant cell may be m vivo or in cell culture.
  • the plant cell may be provided with the protein or derivative by adding it to, or expressing it in, the plant cell.
  • the protein or derivative may be expressed n the plant cell from a vector comprising genetic material encoding the protein or derivative or from genetic material which has been stably integrated into a natural or artificial chromosome of the plant cell.
  • a genetically engineered plant cell which comprises foreign nucleic acid encoding a protein or derivative of the invention operably linked to regulatory sequences capable of directing expression of the protein the plant cell, wherein expression of the protein or derivative causes the growth characteristics of the plant cell to be enhanced compared to an equivalent plant cell in which the protein or derivative is not expressed.
  • a genetically engineered plant which comprises foreign nucleic acid encoding a protein or derivative of the invention operably linked to regulatory sequences capable of directing expression of the protein m the plant, wherein expression of the protein or derivative causes the growth characteristics of the plant to be enhanced compared to an equivalent plant in which the protein or derivative is not expressed.
  • the foreign nucleic acid is stably integrated in the plant cell or plant.
  • Expression of the protein or derivative may be regulatable so that its expression may be increased or decreased at a desired time or so that the level of expression of the protein or derivative is higher or lower in a particular cell type than in other cell types .
  • a method for producing a genetically engineered plant cell with enhanced growth characteristics which comprises transforming a plant cell with nucleic ac d encoding a protein or derivative of the invention operably linked to regulatory sequences capable of directing expression of the protein or derivative in the genetically engineered plant cell and culturmg the plant cell under conditions for expression of the protein or derivative.
  • Methods for producing the genetically engineered plant cell may further comprise selecting for the transformed plant cell.
  • Culturmg the plant cell to produce a plant comprising the foreign nucleic acid.
  • foreign nucleic acid means nucleic acid not present in the natural nucleic acid content of the plant cell.
  • Methods for producing plants with enhanced growth characteristics may further comprise transforming the plant cell with the foreign nucleic acid.
  • protoplasts obtained from undifferentiated cells callus tissue can be transformed with foreign nucleic acid using Agrobacterium, biolistic or plant virus based transformation protocols as described in:
  • proteins or derivatives of the invention enhance plant cell growth by increasing the enzymatic production of plant cellulose and hemicellulose and inhibiting the crystallisation of plant cell wall structural polysaccharides such as cellulose and galactomannan.
  • the second polypeptide may be co-expressed as a separate polypeptide to the first polypeptide or, preferably, as a fusion protein with the first polypeptide.
  • the expression system may be any type of expression system such as a bacterial, yeast, plant, insect, or mammalian expression system, but is preferably an E. coli expression system.
  • the first polypeptide is preferably a multidomain protein such as firefly luciferase.
  • polypeptide comprising a sequence corresponding to the sequence of SEQ ID NO: 3, or a derivative thereof which retains carbohydrate binding activity, to enhance the soluble amount of another polypeptide expressed in an expression system.
  • Figure 1 shows a map of the pET-23 vector of Novagen
  • Figure 2 shows the molecular architecture of a carbohydrate binding protein according to an embodiment of the invention
  • Figure 3 shows an amino acid sequence (SEQ ID NO:l) of the embodiment of the invention shown in Figure 2 and the DNA sequence (SEQ ID NO: 5) of an entire cDNA clone encoding the amino acid sequence of SEQ ID NO:l;
  • Figure 4 shows the amino acid and DNA sequences of SEQ ID NOS: 2-4,6-8;
  • Figure 5 shows a binding isotherm for the Rpt2 domain of the embodiment of the invention shown in Figure 2;
  • Figure 6 shows affinity electrophoresis gels resulting from experiments to screen the RptDom and Rpt2 domains of the embodiment of the invention shown in Figure 2 for carbohydrate binding activity by affinity electrophoresis;
  • Figure 7 shows the organisation of open reading frames of the pET-23a-based expression vector;
  • Figure 8 shows the 5' and 3' regions of the fusion constructs of the Rpt2 and RptDom domains with firefly luciferase;
  • Figure 9 shows the organisation of domains in the fusion proteins RptDom-luciferase and Rpt2-luciferase-
  • Figure 10 shows an SDS-PAGE gel of material from each step of purification of the RPt2-luciferase fusion protein with an acid swollen cellulose affinity matrix; and
  • Figure 11 shows an SDS-PAGE gel of crude cell free E. coli extracts showing the soluble expression of Rpt2-luciferase
  • the carbohydrate binding protein shown in figure 2 has no detectable enzyme activity and no significant homology to known proteins.
  • Each Rpt repeat is approximately 144 amino acids in length (16Kd) .
  • the RptDom domain is made up of the Rptl and Rpt2 domains together with the T-rich linker between them.
  • RptDom and Rpt2 were screened for carbohydrate-binding activity against soluble carbohydrates by affinity electrophoresis (AE) ( Figure 6) .
  • the dissociation constant of the interaction may be calculated from the change of mobility (of the binding domains) as a function of carbohydrate concentrations .
  • Several carbohydrates were tested by this method and the f for RptDom and Rpt2 calculated (Table 1) .
  • Table 1 Affinities of recombinant RptDom and Rpt2 for various soluble ligands .
  • ND no binding detectable Neither RptDom nor Rpt2 bind to potato galactan, larchwood arabinogalactan or mannan from S. cerevisiae .
  • the level of cooperativity is highest for locust bean galactomannan (around 30-fold increase) .
  • Cooperativity for rye arabinoxylan is 21-fold
  • barley beta glucan is 13-fold
  • HEC is 7-fold
  • carab galactomannan is 6-fold.
  • Binding to birchwood xylan is negligible although binding to oat spelt xylan is clearly detectable.
  • a cDNA encoding the amino acid sequence of SEQ ID No. 1 was cloned in to pBluescript.
  • the sequence of the cDNA (SEQ ID No. 5) is shown in Figure 3.
  • cDNA encoding the RptDom domain was then cloned as a PCR fragment into the expression plasmid pQE-30 (Qiagen) .
  • This plasmid was the template for further PCR amplifications to create fusion proteins between the Rpt Dom or Rpt 2 domains and firefly luciferase in the translation vector pET23a (Novagen) (Fig. 5).
  • Rpt Dom starts with nucleotide (nt) 520 and ends with nt 1434 whilst Rpt (2) starts with nt 1006 and ends with nt 1434 (numbering from gene sequence in Fig. 3) .
  • the amplified fragments contain a 5' Nde I and a 3' BamH I restriction site to allow cloning into the polylinker of pET23a (Fig. 7) .
  • the cDNA for firefly luciferase was then added to these constructs as an in-frame translational fusion containing a 5' BamH I and 3' Sal I restriction site (Figs 7 & 8) .
  • the fusion proteins were then tested to check the usefulness of the repeat domains as affinity tages in expression and purification applications.
  • a 40 ml culture of E. coli BL21(DE3) cells expressing either firefly luciferase or the Rpt 2-luciferase fusion protein was harvested by centrifugation. The resulting cell pellets were then resuspended in 4ml of B-PER bacterial protein extraction reagent (Pierce & Warriner) containing lOOmM ammonium sulphate and ImM dithiothreitol . The homogeneous extracts were shaken gently at room temperature for 10 minutes and then centrifuged at 18 000 rpm in a Beckman JA20 rotor at 4C for 20 minutes. The cell free extract from each extraction was then carefully removed and used in the binding experiment .
  • B-PER bacterial protein extraction reagent Pulierce & Warriner
  • Step 1 Acid swollen cellulose (20mg/ml) was added to 1ml of the cell free extracts in a 2ml eppendorf tube to a final concentration of 0.95, 1.82 and 3.33mg/ml. The extracts were then incubated on ice with occasional mixing for 30 minutes.
  • Step 2 The cell free extract/cellulose mixtures were then centrifuged at 14 000 rpm in an eppendorf centrifuge for 5 minutes at room temperature .
  • Step 3 Luciferase activity present in equivalent aliquots of the supernatants was then measured using a luminometer and expressed as a percentage of the activity present in the cell free extracts before the addition of cellulose (Table 2) . Firefly luciferase did not bind to acid swollen cellulose.
  • a 400ml culture of E. coli BL21 (DE3) cells expressing the Rpt 2-luciferase fusion was harvested by centrifugation. The resulting cell pellet was then resuspended in 35ml of B-per reagent (Pierce & Warriner) containing lOOmM ammonium sulphate and ImM dithiothreitol. The homogeneous extract was shaken gently at room temperature for 10 minutes and then centrifuged at 18 000 rpm in a Beckman JA20 rotor at 4C for 20 minutes. The cell free extract from the extraction was then carefully removed and used in the purification protocol.
  • B-per reagent Pulierce & Warriner
  • Step 1 Acid swollen cellulose was added to the cell free extract (5.2 ml of a 20mg/ml suspension) mixed and then divided equally into two 50ml centrifuge tubes. The solutions were left on ice for 20 minutes with occasional mixing .
  • Step 2 The cell free extract/cellulose mixtures were then centrifuged at 15 000 rpm in a Beckman JA20 rotor at 4C for 20 minutes.
  • Step 3 The supernatants were removed and each cellulose pellet was resuspended in 5.2ml of wash buffer; 25mM potassium phosphate, pH 7.0 containing 0.1% (w/v) Triton X- 100. The resuspended pellets were left on ice for 5 minutes and then centrifuged at 15 000 rpm in a Beckman JA20 rotor at 4C for 6 minutes. The supernatants were then carefully decanted and the cellulose pellets washed once more.
  • Step 3 Elution of the Rpt 2-luciferase fusion was achieved by resuspending the pellets from the previous step in 7ml of elution buffer; 250mM potassium phosphate pH 7.0, 0.1% (w/v) Triton X-100 containing lmg/ml low viscosity carob galactomannan (Megazyme) .
  • the resuspended pellets were kept on ice for 15 minutes before centrifugation at 15 000 rpm in a Beckman JA20 rotor at 4C for 6 minutes. The supernatants were collected and the elution procedure was repeated twice resulting in a total of 28ml of supernatant.
  • Step4 The eluted material was then dialysed against 1L of 50mM Tris-Tricine pH 7.8 containing lmM DTT .
  • Luciferase activity was monitored in all the unbound, wash and elution fractions and expressed as a percentage of the total luciferase activity present in the cell free extract prior to purification (Table 4) .
  • the results described here show a novel sequence and structure and a previously undescribed binding domain group. Cooperative interaction between the two binding domains in binding with soluble carbohydrates is shown.
  • the novel protein shown in Figure 3 is the only known example of a cellulosomal-like subunit which lacks a catalytic domain.
  • the novel binding domain described is the only known example of a carbohydrate binding domain which can bind the plant polysaccharides galactomannan, beta glucan and arabinoxylan and can selectively discriminate bewteen xylans from different plant sources.
  • the pET-23a vector of Novagen The cloning/expression region represented by the solid black arrow has : Bgl II (334), Xba I (276), Nde I (238), Nhe I (231), BamH I (198), Eco RI (192), Sac I (190), Hmc II (181), Sal I (179), Hmd III (173), Eag I (166), Not I (166), Xho I (158) , Bpu I (102) , Sty I (57) ; T7 promoter 303-319; T7 transcription start 302; T7.Tag coding sequence 207-239, His. Tag coding sequence 140-157; T7 terminator 26-72
  • RPT 1 and RPT 2n have 34% identity and 64% similarity
  • Figure 3 Nucleotide sequence (SEQ ID NO: 5) and deduced ammo acid sequence (SEQ ID NO:l) of the entire cDNA clone containing the carbohydrate binding domains Repeat 1 and 2.
  • the domains are (the letters and numbering refer to the deduced ammo acid sequence) :
  • the arrow indicates protein of apparent molecular MW 77 Kda

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Abstract

L'invention concerne de nouveaux domaines de fixation glucidiques se fixant au galactomannane, au béta glucane, à l'arabinoxylane et à la cellulose. Ces domaines peuvent, dans leur activité de fixation, faire une distinction sélective entre les xylanes, les arabinoxylanes et les galactomannanes de différentes espèces végétales, et peuvent s'utiliser pour cibler de manière sélective des agents d'espèces végétales particulières.
PCT/GB2000/001317 1999-04-09 2000-04-07 Domaines de fixation glucidiques de piromyces equi WO2000061760A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994000578A1 (fr) * 1992-06-24 1994-01-06 Commonwealth Scientific And Industrial Research Organisation Cellulases recombinees
WO1998014597A1 (fr) * 1996-10-04 1998-04-09 University Of Georgia Research Foundation Inc. Cellulases d'orpinomyces et sequences codantes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994000578A1 (fr) * 1992-06-24 1994-01-06 Commonwealth Scientific And Industrial Research Organisation Cellulases recombinees
WO1998014597A1 (fr) * 1996-10-04 1998-04-09 University Of Georgia Research Foundation Inc. Cellulases d'orpinomyces et sequences codantes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ALI B R S ET AL: "Cellulases and hemicellulases of the anaerobic fungus Piromyces constitute a multiprotein cellulose-binding complex and are encoded by multigene families.", FEMS MICROBIOLOGY LETTERS 1995 DEP. OF CELLULAR PHYSIOL., BABRAHAM INST., BABRAHAM, CAMBRIDGE CB2 4AT, UK, vol. 125, no. 1, pages 15 - 22, XP000938439 *
HALSTEAD JONATHAN R ET AL: "A family 26 mannanase produced by Clostridium thermocellum as a component of the cellulosome contains a domain which is conserved in mannanases from anaerobic fungi.", MICROBIOLOGY (READING), vol. 145, no. 11, November 1999 (1999-11-01), pages 3101 - 3108, XP002145324, ISSN: 1350-0872 *
TOMME P ET AL: "Characterization and affinity applications of cellulose-binding domains", JOURNAL OF CHROMATOGRAPHY B: BIOMEDICAL SCIENCES & APPLICATIONS,NL,ELSEVIER SCIENCE PUBLISHERS, vol. 715, no. 1, 11 September 1998 (1998-09-11), pages 283 - 296, XP004147002, ISSN: 0378-4347 *

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