Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
  • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
  • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
  • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B15/00—Preparation of other cellulose derivatives or modified cellulose, e.g. complexes
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B15/00—Preparation of other cellulose derivatives or modified cellulose, e.g. complexes
  • C08B15/10—Crosslinking of cellulose
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B31/00—Preparation of derivatives of starch
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; 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)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
  • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• • D—TEXTILES; PAPER
  • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
  • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
  • D06M15/00—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment
  • D06M15/01—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment with natural macromolecular compounds or derivatives thereof
  • D06M15/15—Proteins or derivatives thereof
• • D—TEXTILES; PAPER
  • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
  • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
  • D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
  • D06M16/003—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic with enzymes or microorganisms
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
  • D21C9/00—After-treatment of cellulose pulp, e.g. of wood pulp, or cotton linters ; Treatment of dilute or dewatered pulp or process improvement taking place after obtaining the raw cellulosic material and not provided for elsewhere
  • D21C9/001—Modification of pulp properties
  • D21C9/002—Modification of pulp properties by chemical means; preparation of dewatered pulp, e.g. in sheet or bulk form, containing special additives
  • D21C9/005—Modification of pulp properties by chemical means; preparation of dewatered pulp, e.g. in sheet or bulk form, containing special additives organic compounds
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
  • D21H17/00—Non-fibrous material added to the pulp, characterised by its constitution; Paper-impregnating material characterised by its constitution
  • D21H17/005—Microorganisms or enzymes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the present invention relates to methods and compositions for altering the structural, chemical, physical and mechanical properties of polysaccharide materials using biological crosslinking agents based on multimeric structures of polysaccharide binding domains fused or linked to a biological or chemical entity and the resulting biological compositions.
• the invention is exemplified by the use of a cellulose binding domain (CBD) fusion ⁇ protein containing two cellulose binding domains, a cellulose binding domain-Protein A-Ab complex or a S-peptide-cellulose binding domain-S-protein fusion to enhance mechanical properties, such as wet strength, of tissue paper, filter paper and cotton yarn.
• CBD cellulose binding domain
• Polysaccharides are ubiquitous, stable structural components found in nature. Many organisms use polysaccharides as structural material inside and outside of their cells to provide 3 -dimensional shape and surface structure. The structural integrity of polysaccharides from natural sources is often retained after the isolation of the polysaccharide, allowing it to be used for a variety of commercial purposes. Owing to their desirable physical characteristics polysaccharides have also been produced by synthetic methods for commercial purposes. In either case, polysaccharides such as celluloses from either synthetic or non-synthetic sources comprise the raw material for a variety of commercially important products such as paper pulp, and textile fibers.
• the paper manufacturing process conventionally includes four main steps: forming an aqueous suspension of cellulosic fibers, commonly known as pulp; adding various processing and paper enhancing materials, such as strengthening and/or sizing materials to the pulp slurry; sheeting the paper by pouring the resulting suspension over forming fabric which filters out most of the water and drying the fibers to form a desired cellulosic web; and post-treating the web after an initial drying of the paper to provide various desired characteristics to the resulting paper, including surface application of sizing materials to increase the dry strength of the paper.
• processing and paper enhancing materials such as strengthening and/or sizing materials
• wet-end additives include retention aids to retain fines and fillers, for example, alum, polyethylene imine, cationic starches and the like; drainage aids, such as polyethylene imine; defoamers; and pitch or additives such as microfibers and adsorbent fillers.
• Other wet- end additives include polymers such as, cationic polyarylamides and poly(amide amine/epichlorohydrin) which are added to improve wet strength as well as dry strength of the paper. Starch, guar gums, and polyacrylamides are also added to yield dry strength improvements.
• Sizing agents are occasionally added to impart hydrophobic character to the hydrophilic cellulosic fibers. These agents are used in the manufacture of paper for liquid containers, for example, milk or juice, paper cups and surfaces printed by aqueous inks where it is desired to prevent the ink from spreading. Such sizing agents include rosin sizes derived from pine trees, wax emulsions and, more recently, cellulose-reactive sizes.
• sizing agents include rosin sizes derived from pine trees, wax emulsions and, more recently, cellulose-reactive sizes.
• the application of additives to paper after an initial drying of the sheet by spraying, capillary sorption, immersion, roll-coating and the like, is often referred to as a dry-end addition. Poly(vinyl alcohol), acrylic or vinyl acetate emulsions, starches, sizing agents, polyurethanes, and SBR latex are commonly added at the dry end.
• corrugating medium the middle fluting paper used in corrugated containers.
• Starch makes up 2-5% of the total weight of fluting paper.
• Various techniques have been used to improve the wet strength of corrugating medium, including the use of chemical crosslinkers, such as formaldehyde resins, or the application of hydrophobic materials, such as wax.
• chemical crosslinkers such as formaldehyde resins
• hydrophobic materials such as wax.
• Other techniques used have employed more expensive raw materials such as semichemical pulp in order to increase the weight and strength of the paper per square meter. This latter approach leads to increased cost of both starting materials and the manufacturing process itself.
• the processing of cellulosic material as for example cotton fiber into a textile fabric, like paper making, also involves several steps: spinning of the fiber into a yam; construction of woven or knit fabric from the yam and subsequent preparation, dyeing and finishing operations.
• Woven goods are the prevalent forms of textile fabric construction.
• the yams generally are sized in a size box, then the water is removed on steam cans and the yams formed into a sheet which is run across bust rods to break the sheet back into individual yams.
• the yams are then woven, which is done by weaving a filling yam between a series of warp yarns.
• the sub-steps involved in preparation are desizing, scouring and bleaching.
• a one-step combined scour/bleach process is also used in the industry.
• Various compounds are used as sizing agents for warp yams to prevent breakage of the yam during weaving.
• a good yarn sizing agent is one which forms a film with sufficient strength to provide protection to the yam being sized but is not so strong that the yarn will break under the size film.
• the sizing agents are placed on the warp yarns prior to weaving to provide strength and to protect the yarns from abrasion.
• Traditional sizing agents for cotton-containing yams have generally included film formers such as starch, derivatives of starch, polyvinyl alcohol, polyester resins, waxes, acrylic polymers and copolymers and their salts, wetting agents, antistatic agents, and combinations thereof.
• thermosetting resin systems either postcured or precured, result in embrittlement and reduction of mobility of the microstructural units of cellulosic fibers to such an extent that abrasion resistance, breaking strength, and tearing strength often are seriously impaired. Abrasion resistance is often reduced by 75-85%, breaking strength by 50-60%, and tearing strength by about 50%. Furthermore, if the cellulosic fiber-containing ya is sized by the conventional methods described above, it is difficult to completely desize the sized yam. Even if the sized yam is completely desized, the desizing process is complicated or expensive.
• U.S. PAT. NO. 5,137,819 to Kilbum et al. discloses the preparation of fusion proteins which include a cellulase substrate binding region and their use in immobilization and purification of polypeptides.
• U.S. PAT. NO. 5,928,917 to Kilbum et al. discloses conjugates of a non-protein chemical moiety and a polypeptide having a cellulose binding region. Polysaccharide binding proteins and conjugates are described in U.S. PAT. NO. 5,962,289 to Kilbum et al.
• U.S. PAT. NO. 5,821,358 to Gilkes et al. discloses methods and compositions for the modification of polysaccharide structures, for example, cotton and ramie fibers, using binding domains and/or catalytic domains from polysaccharidases.
• U.S. PAT. NO. 5,837,814 to Shoseyov et al. discloses a CBD having a high affinity for crystalline cellulose and chitin, together with fusion products of the CBD and a second protein.
• Applications for the CBD and the fusion products including: drug delivery, affinity separations, and diagnostic techniques are also disclosed. See also, U.S. PAT. NO. 5,719,044; U.S. PAT. NO. 5,496,934; and U.S.5,856,201; all to Shoseyov et al., the contents of each of which are incorporated by reference herein.
• a review of the utility of paper additives is given by B.B. Spence Encyclopedia of Polymer Science and Technology, Second Edition, Wiley- Interscience, Vol. 10, pgs. 761-786, New York (1987).
• compositions of the invention include polysaccharide binding domain (PBD) fusion proteins, PBD coupler units, PBD functional moieties and polysaccharides modified using these compositions.
• PBD polysaccharide binding domain
• a PBD coupler unit of the invention includes one, two or more PBDs, each of which is capable of independently binding to a polysaccharide, and optionally one or more linker unit coupled between the PBDs.
• the method includes the step of contacting a polysaccharide structure with a sufficient amount of a PBD fusion protein under conditions and for a time sufficient to modify one or more characteristic of a polysaccharide material comprising a polysaccharide structure.
• the methods and compositions find use in producing polysaccharide containing materials with altered mechanical, chemical, electrical and/or physical properties.
• a process of manufacturing a polysaccharide containing material having at least one desired structural, chemical, physical, electrical and/or mechanical property comprising the step of contacting polysaccharide structures of the polysaccharide containing material with a polysaccharide binding domain containing composition before, during and/or after processing the polysaccharide structures into the polysaccharide containing material, thereby manufacturing the polysaccharide containing material having the desired structural, chemical, physical, electrical and/or mechanical property.
• composition-of-matter comprising a polysaccharide containing material including polysaccharide structures; and a polysaccharide binding domain containing composition being bound to the polysaccharide structures of the polysaccharide containing material, providing the polysaccharide containing material with at least one desired structural, chemical, physical, electrical and/or mechanical property.
• contacting the polysaccharide structures of the polysaccharide containing material with the polysaccharide binding domain containing composition is effected before processing the polysaccharide structures into the polysaccharide containing material.
• contacting the polysaccharide structures of the polysaccharide containing material with the polysaccharide binding domain containing composition is effected during processing the polysaccharide structures into the polysaccharide containing material.
• contacting the polysaccharide structures of the polysaccharide containing material with the polysaccharide binding domain containing composition is effected after processing the polysaccharide structures into the polysaccharide containing material.
• the polysaccharide containing material is selected from the group consisting of a paper, a textile, a yam and a fiber.
• the structural property is selected from the group consisting of a predetermined level of cross-links between polysaccharide structures of the polysaccharide containing material, a predtermined aggregation of the polysaccharide structures of the polysaccharide containing material and a predetermined surface texture of the polysaccharide containing material.
• the chemical property is selected from the group consisting of a predetermined hydrophobicity, a predetermined hydrophylicity, a predetermined wet-ability, a predetermined chemical reactivity, a predetermined photochemical reactivity, a predetermined functionality and a predetermined surface tension.
• the physical property is selected from the group consisting of a predetermined Young's modulus, a predetermined strain at maximum load, a predetermined energy to break point, a predetermined water absorbency, a predetermined swellability and a predetermined toughness.
• the electrical property is selected from the group consisting of a predetermined surface charge and a predetermined electrical conductivity.
• the mechanical property is selected from the group consisting of a predetermined tensile strength, a predetermined resistance to shear, a predetermined abrasion resistance, a predetermined frictional coefficient, a predetermined elasticity and a predetermined wet strength.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and at least one additional polysaccharide binding domain covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and another protein covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a hydrophobic group covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a hydrophilic group covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a biological moiety covalently coupled thereto. According to still further features in the described prefe ⁇ ed embodiments the polysaccharide binding domain containing composition includes a polysaccharide binding domain and an enzyme covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and an chemical reactive group covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and an chemical photoreactive group covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a lipase covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a lacase covalently coupled thereto. According to still further features in the described prefe ⁇ ed embodiments the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a protein A-antibody covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a peptide covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a polypeptide covalently coupled thereto. According to still further features in the described prefe ⁇ ed embodiments the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a hydrocarbon or a hydrocarbon derivative covalently coupled thereto. According to still further features in the described prefe ⁇ ed embodiments the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a fatty acid derivative covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and an electrically charged moiety covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and an ionic moiety covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a silicon binding moiety covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a polymer binding moiety covalently coupled thereto. According to still further features in the described prefe ⁇ ed embodiments the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a metal covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a metallothionein-like protein covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and ferritin covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a metal binding moiety covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a bacterial siderophores covalently coupled thereto. According to still further features in the described preferred embodiments the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a metallothionein covalently coupled thereto. According to still further features in the described prefe ⁇ ed embodiments the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a thiol group covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and an aldehyde covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a maleimide covalently coupled thereto. According to still further features in the described prefe ⁇ ed embodiments the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a hydrazide covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and an epoxide covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a carbodiimide covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain and a phenylazide covalently coupled thereto.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain which is a cellulose binding domain. According to still further features in the described prefe ⁇ ed embodiments the polysaccharide binding domain containing composition includes a polysaccharide binding domain which is a starch binding domain.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain capable of binding to cellulose.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain capable of binding to starch.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain capable of binding to chitin.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain which is a glucan-binding domain, e.g., a ⁇ -l,3-glucan-binding domain.
• the polysaccharide binding domain containing composition includes a polysaccharide binding domain which includes streptococcal glucan-binding repeats.
• composition-of-matter comprising a polysaccharide containing material including polysaccharide stmctures; and a polysaccharide binding domain containing composition being bound to the polysaccharide stmctures of the polysaccharide containing material, the polysaccharide binding domain containing composition including at least two covalently coupled polysaccharide binding domains forming a polysaccharide binding domain coupler cross linking the polysaccharide structures of the polysaccharide containing material.
• composition-of-matter comprising a polysaccharide containing material including polysaccharide stmctures; and a polysaccharide binding domain containing composition being bound to the polysaccharide stmctures of the polysaccharide containing material, the polysaccharide binding domain containing composition including at least one polysaccharide binding domain and a functionalizing moiety being covalently coupled thereto, the at least one polysaccharide binding domain attaching the functionalizing moiety to the polysaccharide stmctures of the polysaccharide containing material.
• composition-of-matter comprising a polysaccharide containing material including polysaccharide structures; and a polysaccharide binding domain containing composition being bound to the polysaccharide stmctures of the polysaccharide containing material, the polysaccharide binding domain containing composition including at least one polysaccharide binding domain and a hydrophobic moiety being covalently coupled thereto, the at least one polysaccharide binding domain attaching the hydrophobic moiety to the polysaccharide stmctures of the polysaccharide containing material.
• composition-of-matter comprising a polysaccharide containing material including polysaccharide stmctures; and a polysaccharide binding domain containing composition being bound to the polysaccharide stmctures of the polysaccharide containing material, the polysaccharide binding domain containing composition including at least one polysaccharide binding domain and a hydrophilic moiety being covalently coupled thereto, the at least one polysaccharide binding domain attaching the hydrophilic moiety to the polysaccharide stmctures of the polysaccharide containing material.
• composition-of-matter comprising a polysaccharide containing material including polysaccharide stmctures; and a polysaccharide binding domain containing composition being bound to the polysaccharide stmctures of the polysaccharide containing material, the polysaccharide binding domain containing composition including at least one polysaccharide binding domain and a chemical reactive moiety being covalently coupled thereto, the at least one polysaccharide binding domain attaching the chemical reactive moiety to the polysaccharide stmctures of the polysaccharide containing material.
• composition-of-matter comprising a polysaccharide containing material including polysaccharide stmctures; and a polysaccharide binding domain containing composition being bound to the polysaccharide stmctures of the polysaccharide containing material, the polysaccharide binding domain containing composition including at least one polysaccharide binding domain and a photo-chemical reactive moiety being covalently coupled thereto, the at least one polysaccharide binding domain attaching the photo-chemical reactive moiety to the polysaccharide stmctures of the polysaccharide containing material.
• composition-of-matter comprising a polysaccharide binding domain coupler including at least two covalently coupled polysaccharide binding domains.
• nucleic acid constmct comprising a polynucleotide encoding a fusion protein including at least two polysaccharide binding domains.
• nucleic acid constmct further comprising at least one additional polynucleotide encoding at least one linker peptide coupling the at least two polysaccharide binding domains.
• a process of manufacturing a polysaccharide containing material having at least one desired structural, chemical, physical, electrical and/or mechanical property comprising the step of contacting polysaccharide stmctures of the polysaccharide containing material with a polysaccharide binding domain, during and/or after processing the polysaccharide stmctures into the polysaccharide containing material, and thereafter covalently coupling at least one moiety or group to the polysaccharide binding domain, thereby manufacturing the polysaccharide containing material having the desired structural, chemical, physical, electrical and/or mechanical property.
• the present invention successfully addresses the shortcomings of the presently known configurations by providing processes and reagents for manufacturing superior polysaccharide stmctures containing materials such as papers and textiles.
• Fig. 1 A is a schematic representation of pET-CBD plasmid.
• Figs.lB-C show the nucleotide sequence (SEQ ID NO:l) and the amino acid sequence (SEQ ID NO:2) of CBDclos.
• Fig. ID is a schematic representation of pET-CBD-180.
• Figs. 1E-G show the nucleotide sequence (SEQ ID NO:3) and the amino acid sequence (SEQ ID NO:4) of CBD- 180 along with restriction endonuclease recognition sites.
• Fig. 2 A is a schematic representation of pET-CCP-180 containing one copy of CBD- 180 and one of CBD fused in frame thereto.
• Figs. 2B-E show the nucleotide sequence (SEQ ID NO:5) and the amino acid sequence (SEQ ID NO: 6) of CCP (cellulose cross linking protein) along with restriction endonuclease recognition sites.
• Fig. 3 A is a schematic representation of pET-ProtA-CBD.
• Figs. 3B-G show the nucleotide sequence (SEQ ID NO:7) and the amino acid sequence (SEQ ID NO:8) of ProtA-CBD.
• Fig. 4 A is a schematic representation of pET29-Spep-CBD-Sprot.
• Figs. 4B-G show the nucleotide sequence (SEQ ID NO:9) and the amino acid sequence (SEQ ID NO: 10) of Spep-CBD-Sprot.
• Fig. 5A schematically represents a cellulose cross-linking protein having two domains for cellulose binding per molecule.
• Fig. 5B schematically represents the cellulose cross-linking protein of Fig. 5A, wherein one cellulose binding domain is bound to a first polymeric structural unit, and a second cellulose binding domain is bound to a second polymeric structural unit.
• Fig. 6 schematically represents a generic CBD coupler unit.
• Figs. 7A-C each schematically represent various embodiments of a CBD coupler unit.
• Fig. 7A shows a CBD coupler unit having a pair of terminal CBDs linked by a linker unit which includes a pair of starch binding domains each coupled to a CBD, and a starch moiety coupled to both starch binding domains.
• Fig. 7B shows a CBD coupler unit having a pair of terminal CBDs linked by a linker unit which includes a plurality of CBDs, each of which is coupled to an adjacent CBD via a JUN/FOS bridge.
• Fig. 7C shows a CBD coupler unit having a pair of terminal CBDs linked by a large protein moiety.
• Fig. 8 schematically represents a CBD functional moiety (CBDC) having at least one CBD and a functional moiety (FM) attached thereto.
• CBD CBD functional moiety
• Figs. 9A-C schematically represent various ways in which a CBD coupler unit can interact with, and bind to, a polymeric structural unit.
• Figs. 10A-D are bar graphs of Young's modulus, strain at maximum load, energy to break point and toughness, respectively, for control, CBD-treated, and CCP- treated paper strips.
• Fig. 11 schematically represents a yam coating apparatus used for treating the yam with test formulations.
• Figs. 12A-B are bar graphs of Young's modulus and strain at maximum load, respectively, for control, CCP-treated, ProtA-CBD treated, and Ab-ProtA-CBD treated yarn.
• Fig. 13 shows a photograph of the results of expression of S-protein-CBD-S- peptide (SCS) in E.coli.
• Protein marker (lane 1), total E.coli proteins before induction with IPTG (lane 2) total E.coli proteins after induction with IPTG (lane 3) and inclusion bodies containing the SCS protein (lane 4).
• Fig. 14 shows a Young's modulus map of the results of treating Whatman papers with CBD, CCP, or SCS. All measurements were taken at 23 °C, 65 % relative humidity.
• Fig. 15 shows the energy to break points of CBD, CCP, and SCS treated Whatman papers. All measurements were taken at 23 °C, 65 % relative humidity.
• Fig. 16 shows the results of a toughness test of CBD, CCP, and SCS treated Whatman papers. All measurements were taken at 23 °C, 65 % relative humidity.
• Fig. 17 shows the stress at maximum load of CBD, CCP, and SCS treated Whatman papers. All measurements were taken at 23 °C, 65 % relative humidity.
• Fig. 18 shows typical stress versus strain curves of preformed Whatman papers treated with CBD or CCP. All measurements were taken at 23°C, 65% relative humidity and at a constant deformation rate of 20 mm/min. Squers - Control; Circles - 2.5 mg/ml CBD; triangles - 2.5 mg/ml CCP.
• Fig. 19 shows water-absorption time of preformed Whatman papers treated with CBD or CCP at different concentrations. All measurements were taken at 23°C .
• Fig. 20 shows time-lapse photograps of water absorption on preformed
• Whatman pape treated with CCP Water droplets (20 ⁇ l each) were dripped onto CCP-treated paper, and pictures were taken every 25 ms.
• the first frame (A) was taken before the water made contact with the paper.
• Frames B to E ware taken after 2, 4, 6, and 8 minutes, respectively.
• the last frame (F) was taken on non-treated paper 25 ms after water came in contact with the paper. Water absorption was visualized using an optical contact angle meter.
• Polysaccharide stmctures such as fibers and filaments are treated with PBD- containing compositions during or after processing of the stmctures into polysaccharide containing materials, thereby cross-linking and/or functionalizing the polysaccharide stmctures and/or their products such as textiles and paper.
• the PBD- containing compositions function as versatile biological crosslinkers, the structure of which can be varied to accommodate a desired result such as increased elasticity or hydrophobicity of a polysaccharide containing end product.
• the biological entity is one or more second protein.
• the second protein can be another PBD, such as a cellulose binding domain or a starch binding domain or a functionalized PBD, such as a PBD bound to a hydrophobic group or an enzyme, particularly an enzyme which can improve and/or accelerate processing of the polysaccharide stmctures and/or the characteristics of the polysaccharide containing end product such as a lipase or a laccase.
• the second protein can be a non-PBD protein such as a protein A-antibody complex.
• a polysaccharide structure is contacted during or after processing into a polysaccharide material with a sufficient amount of a biological crosslinker under conditions and for a time sufficient to modify one or more characteristic of a polysaccharide material containing the polysaccharide structure.
• the PBD fusion proteins can be used to aggregate polysaccharide stmctures and/or crosslink polysaccharide structures such as polysaccharide fibers and filaments so as to increase the wet strength of the stmctures themselves during processing and/or the polysaccharide containing material produced.
• the treatment with PBD fusion proteins is in lieu of the traditional sizing step.
• PBDs comprising functional moieties can be used to functionalize a polysaccharide containing material, such as a cellulose matrix, with for example a hydrophobic moiety such as a fatty acid derivative or a hydrophobic amino acid sequence to decrease the wetability of a polysaccharide containing material such as paper.
• the subject invention offers several advantages over existing methods of treating polysaccharide stmctures as for example are used in commercial paper and textile processes.
• a suitable polysaccharide containing material such as cellulose fibers
• a biological crosslinker By treating a suitable polysaccharide containing material, such as cellulose fibers, with a biological crosslinker, a product with improved mechanical properties (for example, increased strength and durability) as compared with untreated materials and/or materials treated using enhancing materials other than PBD fusion proteins can be obtained.
• the PBD reagent can be applied in either the forming stage or before or after the sizing stage to increase the wet strength of the paper. If applied in the forming stage, it provides sufficient wet strength so that the sizing step can be eliminated.
• the multimeric PBD fusion proteins of the subject invention have two basic building blocks, a PBD and a second protein, wherein the second protein may or may not be a PBD.
• a PBD can be a protein or a peptide that comprises an amino acid sequence that binds to a polysaccharide such as cellulose or a polymer which contains the basic structural units of the polysaccharide substrate to which the PBD binds, including either backbone sugars and/or terminal sugars and sugars themselves, including monosaccharides and disaccharides. Included within the definition of a PBD are mutants, variants and the like of naturally occurring PBDs, the only requirement being that they bind to a polysaccharide containing substrate.
• PBDs can bind to a substrate polysaccharide either reversibly or i ⁇ eversibly, and the substrate can be natural or synthetic.
• a PBD fusion protein can be a protein molecule having multiple polysaccharide binding domains that may be derived from the same or different polysaccharidases or scaffolding proteins and that may bind to the same or different polysaccharides. When multiple PBDs are present, they preferably occupy separate domains within the PBD fusion protein, and may function independently of each other.
• the term CBD refers to either a domain obtainable from a native protein which is involved in cellulose binding, or to an isolated amino acid sequence or fragment of the native protein which itself binds to cellulose (see, for example, Goldstein et al.
• PBDs and CBDs can be natural, synthetic, or partially synthetic.
• the polysaccharide binding domains including both catalytically competent and incompetent polysaccharidases comprising polysaccharide binding domains, can be obtained by any of a variety of techniques, including biochemical and/or genetic engineering techniques. Thus, they can be obtained by proteolysis (see, for example, Gilkes et al, J.
• One or more predetermined amino acid residues may be substituted, inserted in, or deleted from the amino acid sequence of various PBDs to provide variant or mutated PBDs.
• Amino acid substitutions in a PBD protein or polypeptide sequence can be made in a rational manner based, for example, on similarity or differences in polarity, charge, hydrophobicity, hydrophilicity, and the like of targeted amino acid residue(s). Characteristics such as polarity and hydrophobicity of all amino acids commonly found in proteins are well known in the art, as are techniques for specifically changing (mutating) amino acid sequences.
• the resulting variant or mutated PBDs are deemed to be within the scope of the instant invention.
• Substitutions, insertions and/or deletions can be made to provide a variant PBD having more desirable attributes, for example, for cross-linking or functionalization of particular polysaccharide-containing materials.
• Amino acid sequences co ⁇ esponding only to the polysaccharide binding domain can be used rather than the entire polysaccharidase sequence with specific mutations or modifications.
• PBD is obtained by cleaving the polysaccharidase into functional domains.
• an isolated polysaccharidase is subjected to protease treatment that cleaves the protein into two or more fragments consisting of functional domains.
• the polysaccharidase contains a specific protease site.
• CenA C. fini endoglucanase A
• contains a PT box that is cleaved by conformation-specific C. fini protease.
• the products of that reaction are a polysaccharide binding domain with a PT sequence and a polysaccharidase catalytic domain. If the polysaccharidase is not cleaved by highly sequence specific proteases it will be subject to less specific proteases, and the active fragments can be isolated by chromatography and other peptide purification techniques known to those skilled in the art.
• binding domain Other techniques that can be used to obtain a binding domain include use of amino acid sequence information to generate probes for the cloning of DNA sequences encoding polysaccharidases or polysaccharide binding proteins. These cloned sequences can be used to generate deletion mutants encoding only the polysaccharide binding domains. Conversely, if the cDNA sequence of a polysaccharidase or polysaccharide binding protein is known, then a DNA sequence can be specifically constructed that co ⁇ esponds to the polysaccharide binding domain by using biochemical, amino acid, and DNA sequence data to predict the location of the polysaccharide binding domain based on sequences homologies to other polysaccharidases.
• polysaccharidase genes and polysaccharide binding proteins are known in the art, including synthesis, isolation from genomic DNA, preparation from cDNA or combinations thereof.
• Other techniques that can be used to obtain polysaccharide binding domains include gene fusion, phage display, DNA shuffling and random or site specific mutagenesis.
• Various techniques for manipulation of genes are well known, and include restriction, digestion, resection, ligation, in vitro mutagenesis, primer repair, employing linkers and adapters, and the like (see Molecular Cloning: A Laboratory Manual 2 edition Sambrook et al. (eds.), Cold Spring Harbor Laboratory Press, NY, (1989), which is incorporated herein by reference).
• the nucleic acid encoding a PBD protein of the invention may be obtained from isolated and purified RNA or by genomic cloning. Either cDNA or genomic libraries may be prepared using techniques well known in the art, and may be screened for a particular PBD ennucleotide sequence with probes that are substantially complementary to any portion of the coding sequence. Alternatively, cDNA or genomic DNA may be used as templates for PCR cloning using suitable oligonucleotide primers. Full length clones, i.e., those containing the entire coding sequence of the desired PBD protein may be selected for constructing expression vectors, or overlapping cDNAs can be ligated together to form a complete coding sequence or desired portion thereof, such as the binding domain. Alternatively, DNAs that encode a PBD can be synthesized, in whole or in part, by chemical synthesis using solid-phase techniques well known in the art.
• the PBP can be obtained from a variety of sources, including enzymes which bind to oligosaccharides which find use in the subject invention.
• Table 5 below are listed those binding domains which bind to one or more soluble/insoluble polysaccharides including all binding domains with affinity for soluble glucans ⁇ , ⁇ , and/or mixed linkages.
• the NI cellulose-binding domain from endoglucanase CenC of C. fimi binds to soluble cellosaccharides and one of a small set of proteins which are known to bind any soluble polysaccharides.
• Tables 1 to 4 listed in Tables 1 to 4 are examples of proteins containing putative ⁇ -l,3-glucan-binding domains (Table 1); proteins containing Streptococcal glucan-binding repeats (Cpl superfamily) (Table 2); enzymes with chitin-binding domains (Table 3), and starch-binding domains (Table 4).
• Scaffolding proteins which include a cellulose binding domain protein such as that produced by Clostridium cellulovorans (Shoseyov et al, PCT US94/04132) can also be used for preparing a PBP.
• Several fungi including Trichodenma species and others, also produce polysacchanrdases from which PBP can be isolated.
• New PBPs with interesting binding characteristics and specificities can be identified and screened for in a of ways including spectroscopic (titration) methods such as: NMR spectroscopy (Zhu et al. (1995) Biochemistry 34:, Gehring et al (1991) Biochemistry 30:5524-5531), UV difference spectroscopy (Beishaw et al (1993) Eur. J. Biochem. 211 :717-724), fluorescence (titration) spectroscopy (Miller et al. (1983) J. Biol Chem. 258:13665-13672), UV or fluorescence stopped flow analysis (De Boeck et al. (1985) Eur. J. Biochem.
• affinity methods such as affinity electrophoresis (Mimura et al. (1992) J Chronmatography 597:345-350) or affinity chromatography on immobilized mono or oligosaccharides, precipitation or agglutination analysis including turbidimetric or nephelometric analysis (Knibbs et al. (1993) J. Biol. Chem. 14940-14947), competitive inhibition assays (with or without quantitative IC50 determination) and various physical or physico-chemical methods including differential scanning or isothermal titration calorimetry (Sigurskjold et al. (1992) J Biol. Chem.
• the Ka for binding of the PBP to oligosaccharide is at least in the range of weak antibody-antigen extractions, i.e., 10 -3 , preferably 10 -4 , most preferably 10 " . If the binding of the PBP to the oligosaccharide is exothermic or endothermic, then binding increases or decreases, respectively, at lower temperatures, providing a means for temperature modulation during polysaccharide structure processing.
• Aeromonas sp. Nol0S-24
• Chi D31818 1 Bacillus circulans ( L-12) ChiAl P20533/M57601/A38368 2
• Bacillus circulans (WL-12) ChiD P27050/D 10594 3 Janthinobacterium lividum Chi69 U07025 4 Streptomyces griseus Protease C A53669
• Aeromonas cavia Chi U09139 6 Alteromonas sp (0-7) Chi85 A40633 P32823/D 13762 7 Autographa californica (C6) NPH-128 a P41684/L22858 8 Serratia marcescens ChiA A25090/X03657/L01455/P07254 9 Type III
• Rhizopus oligosporus (IF08631) Chil P29026/A47022/D10157/S27418 10 Rhizopus oligosporus (IF08631) Chi2 P29027/B47022/D10158/S27419 10 Saccharomyces cerevisiae Chi S50371/U17243 11
• Nicotiana tabacum (cv. Havana) Chi P08252/X16939/S08627 27 Nicotiana tabacum (cv. BY4) Chi P24091 X51599/X64519//S13322 26,27,29 Nicotiana tabacum (cv. Havana) Chi P29059/X64518/S20982 26 Oryza sativum (IR36) ChiA L37289 30 Oryza sativum ChiB JC2253/S42829/Z29962 31
• Oryza sativum (cv. Japonicum) Chi X56063 33 Oryza sativum (cv. Japonicum) Chil P24626/X54367/S 14948 34 Oryza sativum Chi2 P25765/S 15997 35 Oryza sativum (cv. Japonicum) Chi3 D16223
• NHP nuclear polyhedrosis virus endochitinase like sequence
• Chi chitinase, "anti-microbial peptide, c pre-hevein like protein, evein, e chitin-binding protein, athogenesis related protein, Swound- induced protein, "wheat germ agglutinin, 'agglutinin (lectin).
• ⁇ -glucanases avicelases, CMCases, Domains* cellodextrinases
• exoglucanses or cellobiohydrolases cellulose binding proteins xylanases mixed xylanases/glucanases esterases chitinases ⁇ -l,3-glucanases ⁇ - 1 ,3-( ⁇ - 1 ,4)-glucanases
• Dextran Binding Domains (Streptococcal) glycosyl transferases 1 ⁇ dextran sucrases (unconfirmed) Clostridial toxins 13,14 glucoamylases" dextran binding proteins ⁇ -Glucan Binding Domains ⁇ - 1 ,3-glucanases 1 - > " ⁇ - 1 ,3-( ⁇ - 1 ,4)-glucanases (unconfirmed) ⁇ -l,3-glucan binding protein
• CBDs are known and are classified into at least 12 Families, any of which can serve as a source of CBDs depending upon the intended use of the CBD.
• Family I contains only CBDs of fungal enzymes. The vast majority of CBDs in the remaining 11 Families are of bacterial origin. The best understood CBDs are those belonging to Families I, II, III, and IV, the CBDs of which are on average 36, 105,
• CBDs of Families I, II, UI, and IV have been characterized as comprising a plurality of anti-parallel ⁇ -sheets folded into jelly rolls CBDs of families I, II, and III bind to both amorphous and crystalline cellulose, whereas CBDs of family IV bind to amorphous cellulose, but not to crystalline cellulose. Only CBDs of family IV bind to solublcellulose derivatives and cellooligosacharides. CBDs that bind to crystalline cellulose and chitin do so with similar affinities, having binding constants in the micromolar range.
• CBDs include those obtainable from strains belonging to the species of Cellulomonas fimi, Trichoderma reesei and M. Bispora (N.R. Gilkes et al, (1988) J. Biol. Chem. 263: 10401-10407; N.R Gilkes et al, (1991), Microbiol. Rev. 55: 303-315); cellulase genes from Cellulomonas fimi (Whittle et al. (1982) Gene 17: 139-145; Gilkes et al (1984) J Gen.
• Microbiol 130: 1377-1384 an exoglucanase (Cex) and an endoglucanase (CenA) from C. fimi and sequences of their genes, cex and cenA (Wong et al. (1986) Gene 44: 315-324; O'Neill et al (1986) Gene 44: 325-330); a 17 KD (peptide) CBD derived from Clostridium cellulovorans described by Shoseyov et al. (1992) (Proc. Natl. Acad. Sci. 89: 3483-3487). Recombinant forms of this CBD exhibit strong affinity for cellulose and chitin (Goldstein et al. (1993) J Bacteriol 175:5762-5768).
• the PBD protein also can be prepared by transforming into a host cell a DNA construct comprising DNA encoding at least a functional portion of the polysaccharide binding region of a polysaccharidase or a polysaccharide binding protein.
• the PBD DNA sequence can be expressed in a host cell, either a eukaryotic or a prokaryotic cell. Expressed and isolated PBD's then can be conjugated to other PBDs and/or one or more functionating protein.
• the isolated polysaccharide binding domain generally is sufficiently pure to exclude catalytic polysaccharidase activity unless this is a desired feature of the intended fusion protein.
• the catalytic activity of such preparation is less than that of crude extracts from cells expressing the polysaccharidase. More preferably, the catalytic activity will reflect a stoichiometry of less than 1 functional catalytic domain per 1000 functional binding domains.
• the binding activity of a PBD can be determined, for example, by binding to microcrystalline cellulose such as Avicel (microcyrstalline cellulose) and showing that the putative binding domain is removed from solution.
• a polypeptide having the desired activity is readily isolated in highly purified form from the cellulose. Binding to Avicel has been used for purification of both native (Gilkes et al, J. Biol. Chem. (1984) 259:10455-10459) and recombinant cellulases (Owolabi et al.,Appl Environ. Microbiol. (1988) 54:518-523).
• the second basic building block of the multimeric PBD fusion protein is a protein which can be a second PBD which can be the same as, or different from, the PBD which is the first building block.
• the multimeric fusion protein can be a dimeric PBD fusion protein encoded by a pair of nucleotide sequences, each encoding a PBD, ligated in frame as is well known in the art (see, for example, U.S. PAT. NO. 5, 856,201 and U.S. PAT. NO. 5,837,814 both to Shoseyov, et al, both of which are incorporated by reference herein in their entirety). Shown in Fig.
• FIG. 5A is an example of a PBD fusion protein in which both the first and the second proteins are CBDs, thus forming a dimeric CBD, wherein the CBDs may be the same (homodimeric-CBDs) or different (heterodimeric-CBDs)).
• Shown in Fig. 5B is the use of the cellulose cross- linking protein of Fig. 5A, wherein one cellulose binding domain is bound to a first polymeric structural unit, and a second cellulose binding domain is bound to a second polymeric structural unit.
• Fig. 6 schematically represents a generic CBD coupler unit including a pair of CBDs linked via a linker unit.
• the second building block can be a PBD that optionally includes one or more functionating group.
• a functionating group is intended a functional group that can modify one or more property of a polysaccharide containing material.
• the functionating group is a protein or a peptide such as a silicon binding peptide, polymer binding peptide or a metal binding peptide (Ljungquist et al, (1989) Eur. J. Biochem. 86: 563-569; Spanner et al, ((1995) Bone 17: 161-165; Slice et al (1990) J Biol Chem. 265: 256-263; Pessi et al (1993) Nature 362: 367-369).
• functionating polypeptides include a starch binding domain which provides a means for crosslinking of polysaccharide fibers and starch molecules; the starch can be an endogenous component of the fibers, or can be applied as a size.
• Starch binding domains can be obtained, for example, from Aspergillus glucoamylase (Chen et al. (1991), Gene 99:121-126).
• polysaccharide and gluten molecules can be crosslinked by using as a second polypeptide a matrix protein such as high molecular weight glutinin (HMWG).
• HMWG high molecular weight glutinin
• the functionating group also can include chemical groups such as one or more thiol group, chromophore, dye, a reactive group such as an aldehyde, a maleimide, a hydrazide, an epoxy, a carbodiimide, or a photo reactive group such as phenyl azide bound to a PBD.
• chemical groups such as one or more thiol group, chromophore, dye, a reactive group such as an aldehyde, a maleimide, a hydrazide, an epoxy, a carbodiimide, or a photo reactive group such as phenyl azide bound to a PBD.
• the first building block of the PBD fusion protein can optionally be linked to the second building block via a linker unit.
• the linker unit of a PBD coupler unit can include various natural or synthetic molecules, including biological polymers such as a protein, a polypeptide, or a polysaccharide, and synthetic polymers such as acrylic polymers and the matrix protein High Molecular Weight Glutinin.
• peptide or protein components of a linker unit examples include JUN protein and FOS protein (see, for example, Gentz et al, (1989) Science 243: 1695-1699); starch binding domain (SBD) (see, for example, Chen et al (1991) Gene 99: 121-126), and S-peptide or S-protein (see, for example, Kim et al. (1993) Protein Science 2: 348-356).
• the first and the second polypeptides (or multiple first and/or second polypeptides) in the fusion protein can be joined directly via a peptide bond, or a larger linker unit, depending in part upon the intended use of the fusion protein.
• FIG. 8 shows schematically a CBD coupler unit (designated CU in the figure) having a first CBD, a second CBD, and a linker unit (LU) linking the first and second CBDs.
• first and second CBDs are depicted in Fig. 8 as being terminal, and as being located at opposite poles of the linker unit, other numbers and a ⁇ angements of CBDs and linker unit(s) are contemplated and are within the scope of the invention.
• the linker unit can be attached to each PBD of a PBD coupler unit by one or a combination of various means, including covalent bonding, ionic bonding, hydrophobic bonding, hydrogen bonding, protein translation, and protein expression.
• a polysaccharide component of a linker unit can be a polysaccharide which is not bound, or bound with low affinity, by a PBD.
• An example of such a polysaccharide is starch.
• a linker unit of a PBD coupler unit can be one or more polysaccharide binding domains other than a PBD.
• Fig. 7A shows a CBD coupler unit having a pair of terminal CBDs linked by a coupler unit which includes a first starch binding domain coupled to a first CBD, a second starch binding domain coupled to a second CBD, and a starch moiety coupled to both the first starch binding domain and the second starch binding domain.
• a linker unit of a PBD coupler unit also can include one or more PBDs.
• Fig. 7B shows a CBD coupler unit having a pair of terminal CBDs linked by a coupler unit which includes a plurality of CBDs, wherein each CBD of the linker unit is coupled to an adjacent CBD via a JUN/FOS bridge (see, for example, Gentz et al, (1989) Science 243: 1695-1699).
• Fig. 7C shows a CBD coupler unit having a pair of terminal CBDs linked by a coupler unit which includes a peptide or protein moiety which does not bind, or binds with only low affinity, to cellulose or related polymers.
• a peptide or protein component of a coupler unit may vary in size from a few hundred Daltons to more than 1 MegaDaltons.
• the linker unit represented in Fig. 7C can be a short peptide of a few amino acids, or a relatively large linker protein, such as HMWG.
• the multimeric PBD fusion protein can be made chemically or recombinantly.
• the polysaccharide binding region or multiples thereof can be produced on its own, purified and then chemically linked to a second protein with or without a functionating group using techniques known to those skilled in the art.
• Methods of protein conjugation include in vitro conjugation chemical reactions to modify the polysaccharide binding domain which can be ca ⁇ ied out while the domain is either bound to a polysaccharide matrix or free from the polysaccharide matrix. Examples include the use of gluteraldehyde conjugation as described by Reichlin in Methods of Enzymology (1980) 70:159-165.
• the polysaccharide binding domain When the polysaccharide binding domain is bound to the matrix, it offers the advantage of protecting the site that actually binds to the matrix while leaving other residues to react with the second moiety, either a second PBD or a functionating protein. If bonding of the chemical moiety to the polysaccharide binding domain results in a diminished capacity to bind the polysaccharide substrate, a reaction procedure requiring the presence of the polysaccharide matrix is prefe ⁇ ed to retain the binding characteristics of the domain. Alternatively the multimeric PBD fusion protein can be made recombinantly.
• nucleotide sequences encoding the components of the PBD fusion protein are used to construct recombinant expression vectors capable of expressing PBD fusion proteins.
• a nucleic acid construct is capable of expressing a protein if it contains nucleotide sequences containing transcriptional and translational regulatory information which are operably linked to nucleotide coding sequences for the protein.
• "Operably linked” refers to a linkage in which the regulatory DNA sequences and the DNA sequence to be expressed are connected in such a way as to permit transcription and translation.
• the polysaccharide binding domain encoding fragment and the DNA encoding the second polysaccharide binding domain or functionating polypeptide are ligated so that the nucleic acid encoding the PBD is joined to the nucleic acid encoding the second protein such that the combined open reading frame of the PBD and the second protein is intact, allowing translation of the entire PBD fusion protein to occur.
• the PBD fusion protein has a protein coupler unit, the nucleotide sequences are operably inserted into the expression construct, between the PBD encoding sequence and the sequence encoding the second protein.
• the resulting ligated DNA can then be manipulated in a variety of ways to provide for expression.
• Vectors for both nucleic acid amplification and for nucleic acid expression are well known in the art. Selection of an appropriate vector depends on various parameters including, the intended function (e.g., amplification or expression), the size of the DNA insert, and the particular host cell to be transformed with the vector.
• Various expression vector/host systems may be utilized by the skilled artisan for the recombinant expression of PBD proteins and PBD fusion proteins. Such systems include microorganisms such as bacteria transformed with recombinant bacteriophage
• DNA, plasmid DNA or cosmid DNA expression vectors containing the desired PBD coding sequence yeast transformed with recombinant yeast expression vectors containing the desired PBD coding sequence; insect cell systems infected with recombinant virus expression vectors (for example, baculovirus) containing the desired PBD coding sequence; plant cell systems infected with recombinant virus expression vectors (for example, cauliflower mosaic virus (CaMV); tobacco mosaic virus, (TMV)) or transformed with recombinant plasmid expression vectors (for example, the Ti plasmid) containing the desired PBD coding sequence; or animal cell systems infected with recombinant virus expression vectors (for example, adenovirus or vaccinia virus) including cell lines engineered to contain multiple copies of the PBD nucleic acid either stably amplified (for example, CHO/dhfr, CHO/glutamine synthetase) or unstably amplified in double-minute chromosomes (
• Plasmid DNA constructs for example, plasmids
• ligation mixtures are used to transform E. coli strains XI- 1 and DH52 and successful transformants are selected by antibiotic (for example ampicillin) resistance, as appropriate.
• Plasmids from the transformants are prepared, and analyzed by restriction and/or sequenced (see, for example, Messing et al, Nucleic Acids Res. 9: 309 (1981); Maxam et al, Methods in Enzymology 65: 499 (1980)).
• expression vectors are capable of replicating efficiently in a host cell, such that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of the protein of interest.
• the expression cassette can be included within a replication system for episomal maintenance in an appropriate cellular host or can be provided without a replication system, where it can become integrated into the host genome.
• the DNA encoding a PBD fusion protein is placed in a vector capable of replication in a host cell, or is propagated in vitro by means of techniques such as PCR or long PCR.
• Replicating vectors can include plasmids, phage, viruses, cosmids, artificial chromosomes and the like.
• Desirable vectors include those useful for mutagenesis of the gene of interest or for expression of the gene of interest in host cells.
• the technique of long PCR has made in vitro propagation of large constructs possible, so that modifications to the gene of interest, such as mutagenesis or addition of expression signals, and propagation of the resulting constructs can occur entirely in vitro without the use of a replicating vector or a host cell.
• a PBD fusion protein For expression of a PBD fusion protein, functional transcriptional and translational initiation and termination regions are operably linked to the DNA encoding the PBD fusion protein. Expression of the fusion protein coding region can take place in vitro or in a host cell. Transcriptional and translational initiation and termination regions are derived from a variety of nonexclusive sources, including the DNA to be expressed, genes known or suspected to be capable of expression in the desired system, expression vectors, chemical synthesis, or from an endogenous locus in a host cell.
• In vitro expression can be accomplished, for example, by placing the coding region for the PBD fusion protein in an expression vector designed for in vitro use and adding rabbit reticulocyte lysate and cofactors; labeled amino acids can be incorporated if desired.
• Such in vitro expression vectors may provide some or all of the expression signals necessary in the system used. These methods are well known in the art and the components of the system are commercially available.
• the reaction mixture can then be assayed directly for the fusion protein, for example by determining its binding activity, or the synthesized fusion protein can be purified and then assayed.
• Transient expression in a host cell can be accomplished in a transient or stable fashion.
• Transient expression can occur from introduced constructs which contain expression signals functional in the host cell, but which constructs do not replicate and rarely integrate in the host cell, or where the host cell is not proliferating.
• Transient expression also can be accomplished by inducing the activity of a regulatable promoter operably linked to the gene of interest, although such inducible systems frequently exhibit a low basal level of expression.
• Stable expression can be achieved by introduction of a construct that can integrate into the host genome or that autonomously replicates in the host cell.
• Stable expression of the gene of interest can be selected for through the use of a selectable marker located on or transfected with the expression construct, followed by selection for cells expressing the marker.
• constructs When stable expression results from integration, integration of constructs can occur randomly within the host genome or can be targeted through the use of constructs containing regions of homology with the host genome sufficient to target recombination with the host locus. Where constructs are targeted to an endogenous locus, all or some of the transcriptional and translational regulatory regions can be provided by the endogenous locus.
• the PBD fusion protein in the source organism When increased expression of the PBD fusion protein in the source organism is desired, several methods can be employed. Additional genes encoding the PBD fusion protein can be introduced into the host organism. Expression also can be increased, for example, by using a stronger promoter by removing destabilizing sequences from either the mRNA or the encoded protein by deleting that information from the host genome, or by adding stabilizing sequences to the mRNA (U.S. Pat. No. 4,910,141).
• Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid encoding the polypeptide or protein of interest. Promoters are untranslated sequences which are located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp of the start codon) and control the transcription and translation of a particular nucleic acid sequence, such as that encoding a PBD fusion protein, to which they are operably linked. Promoters typically fall into two classes: inducible and constitutive.
• Inducible promoters are promoters that initiate increased levels of transcription from nucleic acid under their control in response to some change in culture conditions, for example, the presence or absence of a nutrient or a change in temperature.
• a large number of promoters recognized by a variety of potential host cells are well known in the art.
• the promoter is operably linked to the nucleic acid encoding the fusion protein by removing the promoter from a source nucleic acid by restriction enzyme digestion and inserting the isolated promoter sequence into a vector together with the coding sequence for the fusion protein.
• the promoter can be synthetic, semisynthetic, a native (to the host cell) promoter sequence or a heterologous (to the host cell) promoter can be used to direct amplification and/or expression of the fusion protein.
• Promoters suitable for use with prokaryotic hosts are well known in the art (see, for example, Chang et al (1978) Nature 275:615; Goeddel et ⁇ /.(1979) Nature 281 :544; Goeddel (1980) Nucleic Acids Res. 8:4057; EPO Appln. Publ. No. 36,776; and H. de Boer et al (1983) Proc. Natl Acad. Sci. 80: 21-25).
• nucleotide sequences of such promoters are generally known, thereby enabling the skilled artisan to operably ligate them to a fusion protein-encoding nucleotide sequence (see Siebenlist et al, (1980) Cell 20: 269), using linkers or adapters to supply any required restriction sites.
• Promoters for use in bacterial systems also contain a Shine-Dalgarno (S.D.) sequence operably linked to the PBD-encoding nucleic acid.
• Illustrative transcriptional regulatory regions or promoters include, for bacteria, the lac promoter lambda left and right promoters, tip and lac promoters, tac promoter, and the like.
• the transcriptional regulatory region may additionally include regulatory sequences which allow the time of expression of the fused gene to be modulated, for example the presence or absence of nutrients or expression products in the growth medium, temperature, etc.
• expression of the fusion gene can be regulated by temperature using a regulatory sequence comprising the bacteriophage lambda PL promoter, the bacteriophage lambda OL operator and a temperature sensitive repressor. Regulation of the promoter is achieved through interaction between the repressor and the operator.
• Expression vectors used in prokaryotic host cells also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Expression from certain promoters can be elevated in the presence of certain inducers (for example, zinc and cadmium ions for metallothionein promoters). In this manner, expression of the PBD fusion protein can be controlled. The ability to control expression can be important, for example, if the PBD fusion protein is lethal to a host cell.
• transcriptional and translational regions functional in yeast cells are provided, particularly from the host species.
• the transcriptional initiation regulatory regions can be obtained, for example from genes in the glycolytic pathway, such as alcohol dehydrogenase, glyceraldehyde-3 -phosphate dehydrogenase (GPD), phosphoglucoisomerase, phosphoglycerate kinase, etc. or regulatable genes such as acid phosphatase, lactase, metallothionein, glucoamylase, etc.
• GPD glyceraldehyde-3 -phosphate dehydrogenase
• phosphoglucoisomerase phosphoglycerate kinase
• phosphoglycerate kinase etc.
• regulatable genes such as acid phosphatase, lactase, metallothionein, glucoamylase, etc.
• Any one of a number of regulatory sequences can be used in a particular situation, depending upon whether constitutive or induced transcription is desired, the particular efficiency of the promoter in conjunction with the open-reading frame of interest, the ability to join a strong promoter with a control region from a different promoter which allows for inducible transcription, ease of construction, and the like.
• promoters which are activated in the presence of galactose.
• Galactose- inducible promoters (GALl, GAL7, and GAL 10) have been extensively utilized for high level and regulated expression of protein in yeast (Lue et al, Mol. Cell. Biol. Vol. 7, p. 3446, 1987; Johnston, Microbiol. Rev. Vol. 51, p.
• GAL4 protein Transcription from the GAL promoters is activated by the GAL4 protein, which binds to the promoter region and activates transcription when galactose is present.
• the antagonist GAL80 binds to GAL4 and prevents GAL4 from activating transcription. Addition of galactose prevents GAL80 from inhibiting activation by GAL4.
• nucleotide sequences su ⁇ ounding the translational initiation codon ATG have been found to affect expression in yeast cells. If the desired polypeptide is poorly expressed in yeast, the nucleotide sequences of exogenous genes can be modified to include an efficient yeast translation initiation sequence to obtain optimal gene expression. For expression in Saccharomyces, this can be done by site-directed mutagenesis of an inefficiently expressed gene by fusing it in-frame to an endogenous Saccharomyces gene, preferably a highly expressed gene, such as the lactase gene.
• the termination region can be derived from the 3' region of the gene from which the initiation region was obtained or from a different gene.
• termination regions are known to and have been found to be satisfactory in a variety of hosts from the same and different genera and species.
• the termination region usually is selected more as a matter of convenience rather than because of any particular property.
• the termination region is derived from a yeast gene, particularly Saccharomyces, Schizosaccharomyces, Candida or Kluyveromyces.
• the 3' regions of two mammalian genes, ⁇ interferon and ⁇ 2 interferon are also known to function in yeast.
• Illustrative secretory leaders include the secretory leaders of penicillinase, immunoglobulins, T-cell receptors, outer membrane proteins, and the like. By fusion in proper reading frame the chimeric polypeptide can be secreted into the medium.
• Constructs comprising the coding sequences for the fusion protein can be introduced into a host cell by standard techniques. These techniques include transformation, protoplast fusion, lipofection, transfection, transduction, conjugation, infection, biolistic impact, electroporation, microinjection, scraping, or any other method which introduces the gof interest into the host cell. Methods of transformation which are used include lithium acetate transformation (Methods in Enzymology, Vol. 194, p. 186-187, 1991). Various methods for genetic transformation of prokaryotic and eukaryotic organisms or cells are well known in the art (see, for example, Cohen et al. (1972) Proc. Natl. Acad. Sci.
• Host cells may be transfected, or more preferably transformed, with the above-described expression or cloning vectors of the invention, and the transformed cells may be cultured in conventional nutrient media which may be modified as appropriate for inducing promoters, selecting transformants, or amplifying the gene(s) encoding the desired PBD or PBD fusion protein.
• transformation is meant the introduction of a nucleic acid into an organism such that the nucleic acid is replicable, either as an extra-chromosomal element or by integration into the genome of the host organism.
• the subject host will have at least one copy of the expression construct and may have two or more, depending upon whether the gene is integrated into the genome, amplified, or is present on an extrachromosomal element having multiple copy numbers.
• the subject host is a yeast
• four principal types of yeast plasmid vectors can be used: Yeast Integrating plasmids (Yips), Yeast Replicating plasmids (YRps), Yeast Centromere plasmids (YCps), and Yeast Episomal plasmids (YEps).
• Yips lack a yeast replication origin and must be propagated as integrated elements in the yeast genome.
• YRps have a chromosomally derived autonomously replicating sequence and are propagated as medium copy number (20 to 40), autonomously replicating, unstably segregating plasmids.
• YCps have both a replication origin and a centromere sequence and propagate as low copy number (10-20), autonomously replicating, stably segregating plasmids.
• YEps have an origin of replication from the yeast 2 ⁇ plasmid and are propagated as high copy number, autonomously replicating, i ⁇ egularly segregating plasmids. The presence of the plasmids in yeast can be ensured by maintaining selection for a marker on the plasmid.
• yeast vectors pYES2 (a YEp plasmid available from Invitrogen, confers uracil prototrophy and a GALl galactose-inducible promoter for expression), pRS425-pGl (a YEp plasmid obtained from Dr. T. H. Chang, Ass. Professor of Molecular Genetics, Ohio State University, containing a constitutive GPD promoter and confe ⁇ ing leucine prototrophy), and pYX424 (a YEp plasmid having a constitutive TP1 promoter and confe ⁇ ing leucine prototrophy; Alber and Kawasaki (1982). J. Mol. & Appl. Genetics 1 : 419).
• the transformed host cell can be identified by selection for a marker contained on the introduced construct.
• a separate marker construct may be introduced with the desired construct, as many transformation techniques introduce many DNA molecules into host cells.
• transformed hosts are selected for their ability to grow on selective media. Selective media may incorporate an antibiotic or lack a factor necessary for growth of the untransformed host, such as a nutrient or growth factor.
• An introduced marker gene therefor may confer antibiotic resistance, or encode an essential growth factor or enzyme, and permit growth on selective media when expressed in the transformed host. Selection of a transformed host also can occur when the expressed marker protein can be detected, either directly or indirectly.
• the marker protein may be expressed alone or as a fusion to another protein.
• the marker protein can be detected by its enzymatic activity; for example ⁇ galactosidase can convert the substrate X-gal to a colored product, and luciferase can convert luciferin to a light-emitting product.
• the marker protein can be detected by its light- producing or modifying characteristics; for example, the green fluorescent protein (GFP) of Aequorea victoria fluoresces when illuminated with blue light.
• GFP green fluorescent protein
• Antibodies can be used to detect the marker protein or a molecular tag on, for example, a protein of interest. Cells expressing the marker protein or tag can be selected, for example, visually, or by techniques such as FACS or panning using antibodies. For selection of yeast transformants, any marker that functions in yeast may be used.
• resistance to kanamycin and the amino glycoside G418 are of interest, as well as ability to grow on media lacking uracil, leucine, lysine or tryptophan.
• the host can be grown to express the fused gene in conventional nutrient media (modified as appropriate) for inducing promoters, selecting transformants or amplifying genes.
• Prokaryotic cells used to produce polypeptide or proteins of the instant invention may be cultured in suitable media as described generally in Sambrook et al. (1989) Bacterial Media in Molecular Cloning (Nolan, C. ed.), Cold Spring Harbor Laboratory Press, NY, pp. A.1-4, which is incorporated herein by reference.
• the nutrient medium can be collected and the product isolated by binding to a polysaccharide matrix.
• the cells are harvested, lysed and the product isolated and purified by binding to a polysaccharide substrate.
• a host cell strain is chosen that modulates expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired.
• the term "host cell” may be defined as those cells capable of expressing a PBD protein or PBD fusion protein of interest.
• Host cells can include prokaryotic cells (bacterial) and eukaryotic cells (mammalian, yeast, insect, plant, etc.).
• Modifications (for example, phosphorylation) and processing (for example, cleavage) of protein products may be important for the function of the protein.
• Different host cells often have characteristic or specific mechanisms for the post-translational processing of an expressed protein.
• Appropriate cell lines or host systems may be chosen to ensure the co ⁇ ect modification and processing of the PBD protein or PBD fusion protein expressed.
• the recombinant products can be glycosylated or non- glycosylated, having the wild-type or other glycosylation. The amount of glycosylation depends in part upon the sequence of the particular peptide, as well as the organism in which it is produced.
• expression of the product in E.coli cells results in an unglycosylated product
• expression of the product in insect cells generally results in less glycosylation than expression of the product in mammalian cells.
• Expression in yeast can result in hyperglycosylation.
• the host cell should secrete minimal amounts of proteolytic enzymes.
• expression is to be performed in a eukaryotic host (for example, plants or mammals), it is prefe ⁇ ed that none of the constructs contain potential glycosylation sites.
• Prokaryotic cells of interest include Eschericia, Bacillus, Lactobacillus, cyanobacteria and the like.
• a prokaryotic cell of particular interest for cloning and expression of PBD fusion proteins is E. coli strain BL2(DE3)PLYS.
• Eukaryotic cells include mammalian cells such as those of lactating animals, avian cells such as of chickens, and other cells amenable to genetic manipulation including insect, fungal, plant and algae cells. The cells may be cultured or formed as part or all of a host organism including an animal.
• Viruses and bacteriophage also may be used with the cells in the production of PBD fusion proteins, particularly for gene transfer, cellular targeting and selection.
• host animals include mice, rats, rabbits, chickens, quail, turkeys, bovines, sheep, pigs, goats, yaks, etc., which are amenable to genetic manipulation and cloning for rapid expansion of the transgene expressing population.
• the PBD fusion protein coding sequence can be adapted for expression in target organelles, tissues and body fluids, such as the breast milk of the host animal, through modification of the gene regulatory regions.
• host microorganisms include Saccharomyces cerevisiae, Saccharomyces carlsbergensis, or other yeast such as Candida, Kluyveromyces or other fungi, for example, filamentous fungi such as Aspergillus, Neurospora, Penicillium, etc. Desirable characteristics of a host microorganism are, for example, that it is genetically well characterized, and can be used for high level expression of the product using ultra-high density fermentation.
• gene transfer can be performed by introducing a nucleic acid sequence encoding a PBD fusion protein into the cells following procedures known in the art. If a- transgenic animal is desired, pluripotent stem cells of embryos can be provided with a vector carrying a PBD fusion protein encoding transgene and developed into an adult animal (U.S. Pat. No. 5,162,215; Ono et al (1996) Comparative Biochemistry and Physiology A 113(3):287-292; WO 9612793; WO 9606160). In most cases, the transgene is modified to express high levels of the PBD fusion protein.
• the transgene can be modified, for example, by providing transcriptional and/or translational regulatory regions that function in avian cells, such as promoters which direct expression in particular tissues and egg parts such as yolk.
• the gene regulatory regions can be obtained from a variety of sources, including chicken anemia or avian leukosis viruses or avian genes such as a chicken ovalbumin gene.
• Production of PBD fusion proteins in insect cells can be conducted using baculovirus expression vectors harboring a PBD fusion protein transgene.
• Baculovirus expression vectors are available from several commercial sources such as Clonetech.
• the timing, extent of expression and activity of the PBD fusion protein transgene can be regulated by fitting the polypeptide coding sequence with the appropriate transcriptional and translational regulatory regions selected for a particular use.
• promoter regions which can be induced under preselected growth conditions. For example, introduction of temperature sensitive and/or metabolite responsive mutations into the transgene coding sequences, its regulatory regions, and/or the genome of cells into which the transgene is introduced can be used for this purpose.
• the transformed host cell is grown under appropriate conditions adapted for a desired end result.
• the conditions are typically optimized to produce the greatest or most economical yield of PBD fusion proteins.
• Media conditions which may be optimized include: carbon source, nitrogen source, addition of substrate, final concentration of added substrate, form of substrate added, aerobic or anaerobic growth, growth temperature, inducing agent, induction temperature, growth phase at induction, growth phase at harvest, pH, density, and maintenance of selection.
• Microorganisms such as yeast are preferably grown using selected media of interest, which include yeast peptone broth (YPD) and minimal media (contains amino acids, yeast nitrogen base, and ammonium sulfate, and lacks a component for selection, for example uracil).
• substrates to be added are first dissolved in ethanol.
• expression of the polypeptide of interest may be induced, for example by including or adding galactose to induce expression from a GAL promoter.
• Transient expression can be accomplished via known methods, for example infection or lipofection, and can be repeated in order to maintain desired expression levels of the introduced construct (see Ebert, PCT publication WO 94/05782).
• Stable expression can be accomplished via integration of a construct into the host genome, resulting in a transgenic animal.
• the construct can be introduced, for example, by microinjection of the construct into the pronuclei of a fertilized egg, or by transfection, retroviral infection or other techniques whereby the construct is introduced into a cell line which may form or be incorporated into an adult animal (U.S. Pat. No. 4,873,191; U.S. Pat. No.
• transgenic animals are identified, for example, by the presence of an introduced marker gene, such as for coat color, or by PCR or Southern blotting from a blood, milk or tissue sample to detect the introduced construct, or by an immunological or enzymological assay to detect the expressed protein or the products produced therefrom (U.S. Pat. No. 4,873,191; U.S. Pat. No. 5,530,177; U.S. Pat. No. 5,565,362; U.S. Pat. No. 5,366,894; Wilmut et al (supra)).
• the resulting transgenic animals may be entirely transgenic or may be mosaics, having the transgenes in only a subset of their cells.
• the transgene can be adapted for expression so that it is retained in the mammary cells, or secreted into milk, to form the PBD fusion proteins localized to the milk (PCT publication WO 95/24488).
• Expression can be targeted for expression in mammary tissue using specific regulatory sequences, such as those of bovine ⁇ -lactalbumin, ⁇ -casein, ⁇ -casein, ⁇ -casein, ⁇ -casein, ⁇ - lactoglobulin, or whey acidic protein, and may optionally include one or more introns and/or secretory signal sequences (U.S. Pat. No. 5,530,177; Rosen, U.S. Pat. No. 5,565,362; Clark et al, U.S. Pat. No.
• PBD fusion proteins are readily purified by affinity chromatography using a substrate polysaccharide.
• polysaccharide structures are modified using
• PBD fusion proteins by contacting a polysaccharide structure with a sufficient amount of the PBD fusion protein for a time sufficient to achieve a desired modification under appropriate conditions of reagents, temperature and the like.
• Conditions of modification generally are optimized to provide for Km, Vmax, and kcat and other biochemical parameters such as pH optima of the PBD.
• the interaction of the PBD with substrate generally is extremely rapid. To achieve a desired effect, it therefore is necessary to evaluate various concentrations of PBD fusion protein, and/or time and/or temperature of treatment to achieve a desired effect.
• the conditions used are determined empirically and are based upon the requirements of the PBD fusion protein used and the desired end result.
• typical conditions for PBD fusion proteins comprising a PBD derived from an endoglucanase include mM phosphate, pH 7.0, a concentration of PBD generally of about 0.1-10 mg/ml per 25 mg of cellulose fiber such as cotton.
• the temperature is generally about 20-37 °C, preferably about 25 °C.
• the time of treatment varies from 5 minutes to up to 12 hours, although longer treatments may be used so long as the polysaccharide structures are not damaged. Generally as appropriate, the mixture is gently agitated to facilitate uniform treatment of the structures. Following treatment of the structures, the structures are dried and then used for preparation of an end product such as paper or textile. Alternatively or additionally, an end product such as paper or textile is treated by PBD, taking into account considerations similar to those listed above.
• An assay of the progress of modification or the rate of reaction can be used for the detection of inhibitory end products that might be formed during the modification treatment and for the detection of intermediate or final desirable properties that are produced during treatment. For example, it may be desirable not to fully crosslink the fibers. Rather, it may be preferable to stop the reaction at an intermediate point to obtain polysaccharide structures having desirable properties that are present due to incomplete crosslinking of the structure, for example to obtain a less rigid yarn for weaving.
• a number of objective tests are known to those of skill in the art for evaluating the PBD treatment, including Young's modulus, strain at maximum load, energy to break point, and toughness.
• the type of modification of the polysaccharide structure that is achieved depends at least in part upon the nature of the protein that is fused to the binding domain. Modification by a PBD is defined as an observable (detectable) change in the structure of the polysaccharide. This includes aggregation of the polysaccharide structure leading to observable modifications such as increased wet strength, change in surface properties such as hydrophobicity, hydrophilicity, wetability, surface texture and the like. Electrical properties of a polysaccharide containing material that can be changed include surface charge (positive or negative) and electrical conductivity.
• Chemical properties of a polysaccharide containing material that can be changed include the introduction of various chemically and photochemically reactive chemical groups to at least the surface of the polysaccharide containing material.
• Mechanical properties of a polysaccharide containing material that can be changed include tensile strength, resistance to shear, abrasion resistance, frictional coefficient, and elasticity.
• a reagent or composition having two or more CBDs per molecule can be used to cross-link cellulose fibers.
• Figs. 9A-C schematically represent some of the ways in which a CBD coupler unit of the invention can interact with and bind to a polymeric structural unit of a polysaccharide.
• FIG. 9A schematically represents a CBD coupler unit having a first CBD bound to a first polymeric structural unit, and a second CBD bound to a second polymeric structural unit. It is to be expected that, at least in the case of a linker unit having a high degree of flexibility is used, both a first and a second CBD of a CBD coupler unit can bind to the same polymeric structural unit.
• Fig. 9B shows a CBD coupler unit having a flexible linker unit, wherein both the first and second CBDs are bound to a single polymeric structural unit.
• Fig. 9C schematically represents how a plurality of polymeric structural units can be cross-linked by a plurality of CBD coupler units to form a three dimensional network of polymeric material.
• aggregates of filamentous polysaccharide for example, cellulose filaments
• Materials constructed from cellulosic materials such as paper, cotton yarn and cotton fabric (both woven and non-woven), which are cross-linked via CBD coupler units have altered mechanical properties, such as Young's modulus.
• Cross- linking of cellulosic materials can be performed at various stages in manufacture of a cellulose-containing material.
• cross- linking can be performed by treating cellulose fibers with a CBD coupler unit composition at various stages in the paper making process, or a formed paper product can be treated with a CBD coupler unit.
• cotton yarn or cotton fabric can be cross-linked with a CBD coupler unit composition to provide yarn or cotton fabric having improved surface and/or mechanical properties.
• Fig. 8 schematically represents a CBD functionating moiety that includes at least one CBD and a functional moiety (FM) attached thereto.
• the functional moiety can be any of numerous chemical species, including: a hydrophobic moiety, such as a hydrophobic amino acid sequence or peptide or a fatty acid derivative; to decrease wet-ability and to provide increased tolerance of the material to moisture and water, a hydrophilic moiety; an electrically charged or ionic moiety; a silicon binding moiety; a polymer binding moiety; a metal or metal binding moiety to provide for binding to a metal substrate (examples of metal binding proteins include bacterial siderophores, metallothioneins and metallothionein-like proteins (Slice et al. (1990) J Biol. Chem. 265: 256-263), fe ⁇ itin (Spanner et al.
• a chemically reactive group of the invention can include, for example, an aldehyde, a maleimide, a hydrazide, an epoxide, or a carbodiimide.
• a photo-chemically reactive group of the invention can include a phenylazide.
• a composition having a hydrophobic moiety for example, a hydrophobic polypeptide, a long chain hydrocarbon or hydrocarbon derivative, can be used to confer hydrophobicity to cellulose fibers or products made from cellulose fibers.
• Hydrophobicity leads to decreased wet-ability of a material constructed from the modified cellulose fibers, and indirectly results in increased durability of the material in the presence of water.
• cross-linking of cellulose fibers directly leads to increased wet strength of the material.
• herein reference to strength of paper or other cellulose containing materials is used nonspecifically to include wet strength of such materials.
• the types of polysaccharide materials that can be modified using the subject process are varied.
• Examples include wood products, paper products derived from cellulose fibers, and products derived from cotton or ramie, such as yarn and fabric.
• the term “paper” includes sheet-like masses and molded products made from fibrous cellulosic materials and combinations of cellulosic materials and synthetic materials. Examples of paper include tissue paper, office paper, newsprint, fluting paper, paper towel, laminated paper, and paperboard.
• the term "polysaccharide material” or “polysaccharide-containing material” refers to a material that comprises at least one polysaccharide, generally a substantial amount of least one polysaccharide, such as cellulose, or chitin.
• CBD-containing compositions can also find applications in paper making processes.
• a CBD-containing composition can be used to treat cellulosic material at different stages of a paper making process. For example, treatment may be performed at the forming stage, or at the sizing stage. Treatment at the forming stage of paper making may be performed by adding a CBD cross-linking composition (for example, a CBD coupler unit composition or a cellulose cross- linking (fusion) protein (CCP)) to a suspension of cellulose fibers.
• a CBD cross-linking composition for example, a CBD coupler unit composition or a cellulose cross- linking (fusion) protein (CCP)
• CCP cellulose cross- linking protein
• treatment of cellulosic material with a CBD-containing composition occurs at, or before, the forming stage.
• a functional moiety can be attached, conjugated, or coupled to a CBD according to methods described hereinabove for attachment of a linker unit to a CBD to form a CBD coupler unit (Figs. 4A-G, 5A-B) (see also, for example, U.S. PAT. NO. 5,962,289).
• E. coli pET-CBD was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, M, VA 20110-2209 on Apr. 12 1993, and has been assigned the accession number 75444.
• CBDclos Construction and over-expression of the cellulose binding domain of Cellulose
• pET-CCP-180 (Figs. 2A-E) was constructed from pET-CBD (Figs. 1A-C, M.A. Goldstein et al (1993; J Bacteriol 175: 5762-5768) and pET-CBD-180 (Figs. 1D-G, E. Shpigel et al. (1999) Biotech. Bioeng. 65: 17-23 [pET-CBD and pET-CBD- 180 were digested with Ncol and BamHl and the resulting D ⁇ A fragments separated on 1.2 % and 0.6 % agarose gels, respectively.
• the 500 bp fragment of pET-CBD and the 5 Kb fragment of pET-CBD-180 were extracted from the gel using a Qiaex D ⁇ A gel extraction kit (Qiagen, Ine, California), and the two fragments were ligated.
• the ligation mixture was transformed into E.coli XL 1 -blue competent cells, followed by transformation into the expression host E.coli BL21 (DE3).
• the positive clone containing two CBDs fused in frame was designated pET-CCP-180 and confirmed by sequencing. Expression of CCP- 180 was conducted as described by M.A. Goldstein et al. (1993) J Bacteriol. 175: 5762-5768 for CBDclos
• CBD was PCR amplified using the cbpA gene (Shoseyov et al, (1992) Proc.
• primer A ( ⁇ -terminal primer): 5'- GGGGGAATTCCATGGCAGCGACAT-3' (SEQ ID NO: 11) containing an EcoRI site
• primer B (C-terminal primer): 5'-GGGGGATCCTATGGTGCT-3' (S ⁇ Q ID NO: 12) containing a stop codon followed by a BamHl site.
• the primers were designed to enable EcoRI/if ⁇ mHI force cloning of the 500 bp DNA fragment of into the plasmid pRIT2, fused in frame to the C-terminal of the Protein A gene.
• PCR conditions were as described in Innis et al, PCR Protocols: A Guide to Methods & Applications. Innis et al. ⁇ d., Academic Press, San Diego, 1990) with the following modifications: 2 ng of template DNA and ImM MgCl 2 were used in the reaction mixture.
• the reaction was conducted using a programmable thermal controller (M&J Research, Inc.,). Standard DNA manipulations were conducted according to Sambrook et al, Eds. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press.
• the PCR amplified product was digested with EcoRI and BamHl, and the expected 500 bp DNA fragment was isolated from 1.5 % agarose gel using a Qiaex gel extraction kit (Qiagen, Inc.).
• the EcoRI/_ ⁇ mHI fragment was ligated into EcoRI/5amHI-predigested pRIT2 using T4 ligase.
• the ligation mixture was used to transform E. coli strain 2097 competent cells, and transformed colonies were selected on LB agar plates containing 100 mg/L ampicillin.
• the successful construct containing the DNA insert of interest was designated pRIT2-CBD.
• Prot A-CBD was cloned into the T7 mediated over-expression vector pET3d (F. Studier et al, (1986) J Mol Biol. 189: 113-130).
• the Prot A-CBD was PCR amplified using pRIT2-CBD as a template using the following primers: C-terminal: as described above (i.e., 5'-GGGGGGATCCTATGGTGCT-3' SEQ ID NO: 12); and N- terminal: 5'-GGGGGGTACCATGGAACAACGC-3' (SEQ ID NO: 13), containing an initiation site within the Ncol site.
• the PCR product was partially digested with Ncol.
• the recovered D ⁇ A was digested with _ ⁇ mHI, and the 1.3 Kb D ⁇ A fragment was cloned into pET3d.
• the ligation mixture was used to transform E. coli XL 1 -Blue competent cells, and transformed colonies were selected on LB agar plates containing 100 mg/L ampicillin.
• the successful construct containing the D ⁇ A insert was designated pET-ProtA-CBD (Figs. 3A-G).
• pET-ProtA-CBD was transformed into E. coli BL21 (DE3) competent cells. Expression of the fusion protein was conducted as described by ⁇ ilsson et al, (1985), EMBO J. 4: 1075-1080.
• All the cells were grown in shake flasks at 250 rpm in a volume of 40 ml of LB, supplemented with 50 mg/L ampicillin, inoculated with 400 ⁇ l of an overnight culture of E. coli 2097 containing pRIT2-CBD.
• the culture was grown at a temperature of 30 °C until it attained an O.D. 6 oo nm of 0.4. The temperature was then raised to 42 °C for 45 minutes, and then decreased to 37 °C for an additional 2 hours.
• Cells were suspended at a concentration of 0.1 g/ml in 50 mM Tris/HCl, 10 mM EDTA, pH 8, and were disrupted by a RANNIE high pressure laboratory homogenizer (MINI/LAB Type 8.30 H). The suspension was centrifuged, and 1 liter of supernatant at a protein concentration of 5 mg/ml was applied to a cellulose (Avicel 200 Sigma) column (2.6 x 32 cm). The column was equilibrated with PBS (15 mM phosphate buffer, 150 mM NaCl, 3 mM KC1, pH 7.4). The column was washed at a flow rate of 5 ml/min until the absorbancy at 280 nm was less than 0.05.
• PBS 15 mM phosphate buffer, 150 mM NaCl, 3 mM KC1, pH 7.4
• ProtA-CBD was eluted with 50 mM Tris/NaOH solution, pH 12.5. The eluted ProtA-CBD was immediately titrated to pH 8 with HCl and lyophilized.
• the ProtA-CBD peak showed a single band at about 45 kD.
• Young's modulus is an important property of materials. Young's modulus may be loosely defined as the force required to elongate a material in the elastic regime using relatively small forces that do not i ⁇ eversibly stretch the material.
• Rectangular strips of tissue paper were treated by immersion for 10 minutes in solutions of CBDclos ,CCP, Prot-A- CBD, Ab-ProtA-CBD- at a concentration of 2.5 mg/ml and 2.0 mg/ml, respectively, in 20 mM Tris base, pH 7.
• Control treatment consisted of immersion in a solution of 20 mM Tris base, pH 7, also for 10 minutes. After immersion the treated and control strips were removed from the liquids and dried for 2 days under vacuum.
• Fig. 10A Young's modulus values for control, CBDclos-treated and CCP-180-treated samples of paper are given in Fig. 10A.
• the paper treated with CBD had a Young's modulus significantly greater than that of the control (untreated) paper.
• the paper treated with CCP had a Young's modulus even greater than that of the CBD treated paper.
• CCP- 180 are shown in Fig. IOC. Energy to breake point of the CBD treated paper was substantially the same as that of the control. However, the paper treated with the crosslinking protein, CCP- 180 showed significantly increased energy to brake point as compared with the control.
• the cotton yarn used in this study was 100 % gray cotton double yarn fiber (34/2) with low T.P.U. (turns per inch). Yarn diameter was 0.5 mm and the weight per length was 0.8 mg/cm.
• the yarn treatment apparatus is schematically represented in Fig. 11.
• the apparatus includes a feeder wheel, a collecting wheel, a first bath A, a second bath B, and an engine engaged with the collecting wheel.
• the feeder wheel and collecting wheel may be interchanged, thereby allowing yarn to be re-passed through baths A and B.
• the engine can be operated at various selected speeds, thereby allowing the immersion time of a yarn sample to be determined.
• Lengths of yarn may be connected between the feeder wheel and the collecting wheel, and the yarn may be moved from the feeder wheel to the collecting wheel by passing the yarn through liquid(s) contained within bath A and bath B. In this way, yarn may be immersed in a single liquid (present in both baths) or in two different liquids for a particular time period.
• Lengths of cotton yarn (3-4 meters) were wound onto the feeder wheel of the YTA, one end of the yarn was connected to the collecting wheel, and the yarn was advanced through bath A and bath B by means of the engine. Dipping duration of the yarn was approximately 45 seconds.
• Treatments of the cotton yarn described above were as follows: (i) Treatment with CCP- 180: yam fibers were immersed (bath A) in a solution of CCP-180 (lmg/ml in 20mM Tris base, pH 8).
• CBD-PA Protein A-CBD
• Control yam fibers were immersed (bath A) in 20 mM Tris base, pH 8 for 45 seconds.
• Young's modulus values for control, CCP-180-treated, ProteinA-CBD-treated and Ab-ProteinA-CBD-treated samples of yam are given in Fig. 12A.
• the yam treated with CCP-180 and Ab-ProteinA-CBD had Young's modulus values significantly greater than that of the control (untreated) yam.
• These data indicate that treatment of yam with Ab-ProteinA-CBD and CCP resulted in increased tensile strength (as determined by Young's modulus values) of the treated yarn, as compared with the control.
• the yam treated with CBD-PA had a Young's modulus value much lower than that of the control yarn.
• Results showing strain at maximum load for yarn samples treated with CCP- 180, CBD-PA, and CBD-PA-Ab are shown in Fig. 12B.
• Yam treated with either CCP- 180 or CBD-PA-Ab had lower values of strain at maximum load as compared with the control, thus indicating that these treatments rendered the yarn less elastic as compared with the control.
• the yam treated with CBD-PA had a strain at maximum load similar to that of the control.
• a CBD functional moiety is prepared by coupling a CBD to a functional moiety having affinity for a heavy metal, such as a metal-binding protein.
• a stream of liquid containing an excessive level of a heavy metal is passed over the metal binding filter medium, whereby the concentration of the heavy metal in the liquid stream is greatly decreased to a non-toxic level.
• a CBD functional moiety is prepared by coupling a CBD to a hydrophobic functional moiety.
• Cellulose fibers suitable for paper making are treated with the CBD hydrophobic functional moiety under conditions (pH, temperature, ionic concentration) such that the CBD component of the CBD functional moiety binds to the cellulose fibers to provide cellulose fibers having a hydrophobic moiety attached thereto. Paper produced from the treated cellulose fibers is hydrophobic and resistant to water.
• paper produced from untreated (non-functionalized) cellulose fibers is functionalized with a CBD-linked hydrophobic moiety, either before or after drying the paper.
• Paper treated with the CBD hydrophobic functional moiety is hydrophobic and resistant to water.
• a CBD functional moiety is prepared by coupling a CBD to a hydrophilic functional moiety.
• Tissue paper is treated (functionalized) with the CBD hydrophilic functional moiety, either before or after the first or second drying stages of a paper making process.
• Tissue paper treated with the CBD hydrophilic functional moiety is hydrophilic and shows increased absorption of water and aqueous liquids.
• SSC S-protein-CBD-S-peptide
• Fig. 13 shows the results of expression of SCS in E.coli.
• E.coli proteins before induction with IPTG are shown in lane 2
• total E.coli proteins after induction with IPTG are shown in lane 3
• inclusion bodies containing the SCS protein are shown in lane 4.
• Fig. 14 shows a Young's modulus map of the results of treating Whatman papers with CBD, CCP, or SCS. Note that treatment of Whatman papers with CBD or CCP in all concentrations tested resulted in increased Young's modulus.
• Fig. 15 shows the energy to break points of CBD, CCP, and SCS treated Whatman papers. Note that use of CCP in the concentration of 2.5 mg/ml resulted in about 30 % increase in the energy to break point. In addition, treatment with SCS at all concentrations tested resulted in increased energy to break point.
• Fig. 16 shows the results of a toughness test of CBD, CCP, and SCS treated Whatman papers. Note that use of CCP in the concentration of 2.5 mg/ml resulted in about 40 % increase in toughness. In addition, treatment with SCS at all concentrations tested resulted in increased toughness.
• Fig. 17 shows the stress at maximum load of CBD, CCP, and SCS treated
• Model 1011 (High Wycombe, UK) in tensile mode.
• the rectangular papers were inserted into the upper and lower tensile grips (screw-action grips, Instron Corp., Canton, MA) to enable a proper grip during the tension experiments. All measurements were taken at 23 °C, 65 % relative humidity and a constant deformation rate of 20 mm/min.
• the tensile properties measured included stress at failure, strain at failure, stretch at break point and energy absorption. All calculations were performed according to Hayden, W., Moffatt, W.G. & Wulff, J. Mechanical Behavior. 1-22 (Johan Wiley & Sons, Inc., NY; 1956) and Dufresne, A., Cavaille, J.Y. & Vignon, M.R. Mechanical behavior of sheets prepared from sugar beet cellulose microfibrils. J. Appl. Polym. Sci. 64, 1185-1194 (1996).
• Fig. 18 shows typical stress versus strain curves of pre-formed Whatman papers treated with CBD or CCP.
• the deformation behavior of the treated paper under an applied load could be deduced from the stress-strain curve. Up to 0.02 strain, a linear relationship between stress and strain was observed. However, at strains higher than 0.02, a nonlinear relationship was found. It is evident from Fig. 18 that the tensile stress increases from control to CBD and to CCP, respectively.
• the tensile strength value of the CCP-treated paper was about 40 % higher than the non- treated paper and 14 % higher than the CBD-treated paper.
• the CBD-treated paper strength was about 25 % higher than that of the non- treated paper. In both treatments, the differences were statistically significant (Table 6). Table 6
• Fig. 19 shows water-absorption time of pre-formed Whatman papers treated with CBD or CCP at different concentrations.
• Fig. 20 shows time-lapse photographs of water abso ⁇ tion on pre-formed Whatman paper treated with CCP. Distilled water
• CBD- and CCP-treated papers increased with increasing protein amount.
• water abso ⁇ tion time was two orders of magnitude higher than with paper treated with CBD at the same concentration (580 seconds for CCP versus 5 seconds for CBD) and at least four orders of magnitude higher then the non-treated paper (580 seconds for CCP versus less than a second for the control).
• Optical contact angle meter was used to visualize the dynamics of water dropped onto CCP-treated paper.
• Fig. 20 (photograph F) illustrates the contact of the water droplet with non-treated paper after 25 ms. It is clear that water absorbs into the paper immediately after contact.
• Fig. 20 (photographs A to E) further illustrates the abso ⁇ tion of a water droplet by CCP-treated paper versus lapsed time. In the first 2 minutes, no abso ⁇ tion could be detected, and the contact angle remained at > 90 °. Only after 4 min was abso ⁇ tion into the paper observed. Even after 8 min, the water was not completely absorbed by the paper. Full abso ⁇ tion into the paper was observed only after 10 min (Fig. 19).
• a CBD coupler unit composition or reagent is prepared by linking at least two CBDs with a linker unit.
• a suspension of cellulose fibers which includes a substantial amount of fine cellulose fibers capable of passing through the forming fabric (filter), are treated with the CBD coupler unit composition prior to passing the suspension through the forming fabric.
• CBD coupler units of the CBD coupler unit composition crosslink with the fine cellulose fibers to form a plurality of three-dimensional aggregates of cellulose fibers. After passage of the treated suspension through the forming fabric, the three-daggregates of cellulose fibers are retained by the filter, thereby allowing for greatly enhanced recovery of raw material (cellulose fibers).
• PBD fusion proteins that include a dual or dimeric PBD (e.g., a fusion product of two CBDs, for example, cellulose cross-linking protein (CCP)), a fusion product of a CBD with Protein A, and a Speptide-CBD- Sprotein fusion can be prepared and used to modify polysaccharide structures.
• a dual or dimeric PBD e.g., a fusion product of two CBDs, for example, cellulose cross-linking protein (CCP)
• CCP cellulose cross-linking protein
• Speptide-CBD- Sprotein fusion e.g., Speptide-CBD- Sprotein fusion
• certain features of the invention which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment.
• various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

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