WO1992001797A1 - PRODUCTION D'IMMUNOGLOBULINES PAR LES $i(TRICHODERMAS) - Google Patents

PRODUCTION D'IMMUNOGLOBULINES PAR LES $i(TRICHODERMAS) Download PDF

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Publication number
WO1992001797A1
WO1992001797A1 PCT/FI1991/000218 FI9100218W WO9201797A1 WO 1992001797 A1 WO1992001797 A1 WO 1992001797A1 FI 9100218 W FI9100218 W FI 9100218W WO 9201797 A1 WO9201797 A1 WO 9201797A1
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Prior art keywords
gene
trichoderma
immunoglobulin
immunologically active
dna
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PCT/FI1991/000218
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English (en)
Inventor
Eini NYYSSÖNEN
Sirkka KERÄNEN
Merja Penttilä
Kristiina Takkinen
Jonathan K. C. Knowles
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Oy Alko Ab
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Priority to JP91511365A priority Critical patent/JPH05508768A/ja
Priority to AU80938/91A priority patent/AU657911B2/en
Publication of WO1992001797A1 publication Critical patent/WO1992001797A1/fr
Priority to FI925724A priority patent/FI925724A/fi

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6478—Aspartic endopeptidases (3.4.23)
    • C12N9/6481—Pepsins (3.4.23.1; 3.4.23.2; 3.4.23.3)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi

Definitions

  • the present invention is related to heterologous protein production by Trichoderma, and specifically to immunoglobulin production by Trichoderma . Methods for the production of such 15 immunoglobulins by genetically engineering members of the species Trichoderma are disclosed. These preparations are especially useful for the treatment of medical conditions.
  • Filamentous fungi are lower eukaryotes widely used in * biotechnology to make various fermentation products. Fungi secrete many industrially important enzymes such as gluco- amylase, proteases, lactase, pectinases and glucose oxidase.
  • Filamentous fungi have a number of advantages for biotechnology. They generally produce high amounts of proteins, cultivation in large scale is not complicated and separation of the mycelium from the culture liquid after the fermentation is easy. For example, certain hypercellulolytic strains of the filamentous fungi Trichoderma are capable of secreting over 40 grams of cellulases per liter of culture medium (Durand, H. et al. , in Biochemistry and Genetics of Cel lulase Degradation, Academic Press, San Diego, 135-151
  • Trichoderma does not hyperglycosylate proteins as Saccharomyces yeast does (EP 215,594; Penttila, M., Construction and Characterization of Cel lulolytic Yeasts, VTT
  • Trichoderma reesei (formerly T. viride) (Simmons, E.G., In: Abstracts of Second International Mycological Congress, Tampa, Florida, Aigelow,
  • the amount of extracellular protein produced by the best T. reesei mutants is more than 50% of the total cell protein.
  • a major part of the secreted protein comprises cellulases among which the CBH I component is most abundant, representing up to 60% of the secreted cellulase proteins.
  • the gene for CBH I has been cloned by Shoemaker et al . (Shoemaker, S., et al . , Bio/Technology J:691-696 (1983)) and Teeri et al. (Teeri, T., et al . , Bio/Technology 1:696-699 (1983)) and the entire nucleotide sequence of the gene has been published (Shoemaker, S., et al . , Bio/Technology 1:691- 696 (1983)). From T.
  • Transformation systems have been developed for a number of filamentous fungi, for example, Neurospora crassa (Case, M., et a7., Proc. Natl . Acad. Sci . USA 75:5259-5263 (1979)), A. nidulans (Ballance, J., et al . , Biochem. Biophys. Res. Commun. 112:284-289 (1983); Tilburn, J., et al.
  • the genus Aspergillus is included in the class Ascomycetes, sub-class Euascomycetes.
  • Euasco ycetes are divided into three groups, Ple omycetes, Pyreno ycetes and Disco ycetes on the basis of the fruiting bodies. The most important genera are Aspergillus and Penicillium (Doi, Y., Bull . Nat. Sci . Mus. Tokyo 11:185-189 (1968)).
  • Trichoderma instead, is classified as a member of Fungi imperfecti. Fungi imperfect!
  • Trichoderma is a catch-all category of fungi which have no sexual reproduction or obvious affinities with sexually reproducing genera, such as the highly charac ⁇ teristic Aspergillus.
  • Trichoderma has been reported to possess a poorly defined sexual stage being an imperfect state of the perfect ascomycete species Hypocrea (Beja da Costa, M., et a7., Biotechnol. Biogen. 22:2429-2432 (1980)), the genera Aspergillus and Trichoderma are clearly to be considered taxono ically very different.
  • Trichoderma reesei Due to its exceptional ability to secrete proteins into the growth medium Trichoderma reesei is a good candidate as a possible host for the production of proteins.
  • genetic studies of T. reesei have so far been directed almost exclusively to improving the cellulase-producing properties of the fungus and the only technique used for the development of hypercellulotic Trichoderma strains has been traditional mutagenesis and screening.
  • transformation is carried out using protoplasts.
  • the transforming DNA is usually integrated into the host genome.
  • the vector system must carry a functional gene (a selection marker) which either complements a corresponding mutation of the host genome or supplies an activity, usually an enzyme, required for the growth of the prototrophic strain on a particular growth medium.
  • Immunoglobulins consist of two identical light chains and two identical heavy chains linked by sulphur bridges.
  • the polypeptide chains of immunoglobulins are formed from successive domains, which can fold up independently.
  • Light chains consist of two domains, heavy chains usually four- five domains.
  • the ability of immunoglobulins to recognize and specifically bind their antigens is due to differences in the variable domai. which reside at the N-terminal ends of both the light and heavy chains.
  • An antibody molecule can be proteolysed into two parts, the so-called Fc and Fab fragments.
  • the Fab fragment includes the sites from the variable domains of both the light and heavy chains which are responsible for the identification and binding of antigens.
  • the domains of the Fc fragment play roles in the activation of the immune response in a cell. Biochemical tests have shown that the Fc fragment is not necessary for antigen binding.
  • Monoclonal antibodies are produced in hybridoma and myeloma tissue cultures, often with human or mouse hybridoma cell lines.
  • the methods are fairly expensive and the yields of monoclonal antibodies from human hybridoma cell lines are relatively low (1 ⁇ g/ml for human hybridomas compared to 100 g/ml for mouse hybridomas).
  • the present invention describes a recombinant DNA cloning vector and • ⁇ ctor system for the transformation of Trichoderma for the expression of such heterologous proteins, and especially for the expression of recombinantly produced immunoglobulins, immunoglobulin peptides, and biologically active portions of such immunoglobulins and immunoglobulin peptides. Transformation of Trichoderma with the present vector or vector system will provide a high level expression and secretion of the desired heterologous protein when the transformed microorganism is grown in a suitable culture medium.
  • the present invention provides a vector, such vector comprising: a) at least one gene encoding a desired expressible protein, or biologically active fragment thereof, and b) a selection marker capable of providing selection of Trichoderma hosts which have been transformed with such vector.
  • the present invention provides a vector system of one or more plasmids, such vector system comprising: a) at least one gene encoding a desired protein product, or biologically active fragment thereof, and b) at least one selection marker capable of providing selection of Trichoderma hosts which have been transformed with such vector, such selection markers being provided to such
  • the gene encoding a desired protein product or biologically active fragment thereof is a gene encoding an immunoglobulin or immunologically active fragment thereof.
  • the gene encoding a desired protein product or biologically active fragment thereof is flanked on its 5' end, and operably linked in reading phase to, a signal or leader sequence, such signal or leader sequence being is capable of directing secretion of such desired protein out of such Trichoderma host cell, such amino acid sequence being capable of cleavage from said desired protein or biologically active fragment thereof.
  • the gene encoding a desired protein product or biologically active fragment thereof is flanked, and operably linked to, (and, if necessary, in reading phase with,) one or more DNA regulatory elements selected from the group consisting of promoters, enhancers, signal/leader sequences, terminators, and combinations thereof, such DNA sequences being operably linked to such gene to control expression and/or secretion of the desired protein product.
  • the DNA regulatory sequences which are operably linked to the gene encoding a desired protein product or biologically active fragment thereof are DNA sequences homologous to Trichoderma genomic sequences.
  • the homologous DNA regulatory sequences which are operably linked to the gene encoding a desired protein product or biologically active fragment thereof are selected from the DNA regulatory regions of a gene expressed on glucose, the cbhl gene, the cbh2 gene, the eg71 gene and the eg72 gene.
  • the selection marker capable of providing selection of Trichoderma hosts which have been transformed with such vector is selected from the group consisting of phleomucin resistance, amdS, trpC and argB selection.
  • the present invention provides a method for transformation of Trichoderma, wherein a suitable Trichoderma strain is transformed with the present vector or vector system.
  • the present invention provides a method for the production of a heterologous protein, in Trichoderma, such method comprising transforming
  • Trichoderma with the present vector or vector system, culturing the transformed strain in a suitable medium and recovering the expressed and secreted product from the medium.
  • such heterologous protein is selected from the group consisting of an i munoglobulin, an immunoglobulin light chain, an immunoglobulin heavy chain, and i munologically active fragments of such immunoglobulin, immunoglobulin light chain, and immunoglobulin heavy chain.
  • the present invention provides a method for the production of an immunoglobulin, an immunoglobulin heavy chain, an immunoglobulin light chain, or biologically active fragments of such immunoglobulin or immunoglobulin heavy chain or immunoglobulin light chain, in
  • Trichoderma such method comprising transforming Trichoderma with the present vector or vector-system, culturing the transformed strain in a suitable medium and recovering the expressed and secreted immunoglobulin product from the medium.
  • such immunoglobulin such immunoglobulin, such immunoglobulin heavy chain, immunoglobulin light chain, or biologically active fragments of such immunoglobulin or immunoglobulin heavy chain or immunoglobulin light chain are reassociated in vitro with another immunoglobulin, an immunoglobulin heavy chain, an immunoglobulin light chain, or biologically active fragments of such immunoglobulin or immunoglobulin heavy chain or immunoglobulin light chain.
  • the present invention provides Trichoderma strains which have been stably transformed with a desired gene using the vector and vector systems of the invention.
  • the present invention provides fusion proteins
  • the present invention provides compositions which are enriched in such heterologous proteins, and especially in immunoglobulins, immunoglobulin heavy chains, immunoglobulin light chains, or biologically active fragments of such immunoglobulins, immunoglobulin heavy chains and immunoglobulin light chains.
  • Fig. 1 shows the principle of the inactivation of a chromosomal cellulase gene.
  • Fig. 2 shows the construction of plasmids pMS4 used for insertion mutagenesis of the T. reesei chromosomal CBHI locus. Position of a frameshift mutation generated by inactivation of EcoRI site is marked with *.
  • Fig. 3 shows the construction of plasmid p285' proC
  • Fig. 4 shows the construction of plasmids pMT648 and pMT813.
  • Fig. 5 shows the construction of plasmid pMT837.
  • Fig. 6 shows the restriction map of plasmid pR27.
  • Fig. 7 shows the restriction map of plasmid pAMHIOO.
  • Fig. 8 shows the construction of plasmid pAMH105.
  • Fig. 9 shows the construction of plasmids pAMH1103, pAHM1106 and pAMHllOl by loop-mutagenesis using synthesized oligonucleotides 0AMH1, 0AMH2 and 0AMH3.
  • Fig. 10 shows the linkers NOR 202, NOR 203, 0AMH1, 0AMH2 and 0AMH3.
  • Fig. 11 shows the restriction maps of plasmids pAMH102 and pAMH104.
  • Fig. 12 shows the restriction map of plasmid pAMH1103 and the construction of pAMH103.
  • Fig. 13 shows the restriction map of plasmid pAMH1106 and the construction of pAMHIO ⁇ .
  • Fig. 14 shows the restriction map of plasmid pAMHllOl and the construction of pAMHIOl.
  • Fig. 15 shows the construction of plasmid pAHMHO.
  • Fig. 16 shows the construction of plasmid pAMHlll used for expression of EGI under CBHI promoter function.
  • Fig. 17 shows the expression of chymosin in T. reesei , Western blot.
  • Lane 1 purified prochymosin control, includes traces of pseudochy osin and chymosin.
  • Lane 2 culture supernatant from growth of strain including pAMH102.
  • Lane 3 control supernatant from strain without plasmids.
  • Lane 4 ycelia from strain including pAMH102.
  • Figure 18 is a diagram of plasmid pLTl which carries a subclone of the light chain cDNA.
  • Figure 19 is a diagram of plasmid pLT2, which carries a subclone of the IgG3 heavy chain cDNA (V/, C#, partly C ⁇ ) •
  • Figure 20 is a diagram of plasmid pT18, which carries a subclone of the IgGl heavy chain cDNA (V# and C// j ).
  • Figure 21 is a diagram of plasmid pENlOl, which carries a subclone of the chromosomal cbhl -gene.
  • Figure 22 diagrams the construction of plasmids pEN304 and pEN305, fungal expression vectors for the light chain cDNA.
  • Figure 23 diagrams the construction of plasmid pEN207, an intermediate vector for construction of plasmid pEN209.
  • Figure 24 diagrams the construction of plasmid pEN209, an expression vector for the cbhl - heavy chain Fab fusion.
  • Figure 25 diagrams the construction of plasmid pAJ202, an expression vector for the heavy chain Fab cDNA.
  • Figure 26 diagrams the construction of plasmid pUJIO.
  • Figure 27 shows the construction of plasmids pEN401 and pEN402.
  • Figure 28 shows the construction of plasmid pMPH31.
  • Figure 29 diagrams the elements of plasmid pML5, which carries a structural gene of single chain antibody, having cbhl hinge as a linker.
  • rDNA recombinant DNA
  • RNA sequence containing a template for a RNA polymerase RNA that codes for a protein is termed messenger RNA (mRNA) and, in eukaryotes, is transcribed by RNA polymerase II.
  • mRNA messenger RNA
  • cDNA complementary DNA gene includes recombinant genes synthesized by reverse transcription of mRNA and from which intervening sequences (introns) have been removed.
  • a protein homologous to a Trichoderma host of the invention is meant that an untransfor ed Trichoderma of the same species as the host species naturally produces some amount of the native protein; by a gene homologous to a Trichoderma host of the invention is meant a gene found in the genome of an untransformed Trichoderma of the same species as the host species.
  • a protein heterologous to a Trichoderma host of the invention is meant that an untransformed Trichoderma of the same species as the host species does not naturally produce some amount of the native protein; by a gene heterologous to a Trichoderma host of the invention is meant a gene not found in the genome of an untransformed Trichoderma of the same species as the host species.
  • Cloning vehicle A plasmid or phage DNA or other DNA sequence (such as a linear DNA) which provides an appropriate nucleic acid environment for the transfer of a gene of interest into a host cell.
  • the cloning vehicles of the invention may be designed to replicate autonomously in prokaryotic and eukaryotic hosts. In Trichoderma, the cloning vehicles generally do not autonomously replicate and instead, merely provide a vehicle for the transport of the gene of interest into the Trichoderma host for subsequent insertion into the Trichoderma genome.
  • the cloning vehicle may be further characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vehicle, and into which DNA may be spliced in order to bring about replication and cloning of such DNA.
  • the cloning vehicle may further contain a marker suitable for use in the identification of cells transformed with the cloning vehicle.
  • the word "vector” is sometimes used for "cloning vehicle.”
  • such markers may be provided on a cloning vehicle which is separate from that supplying the gene of interest.
  • Expression vehicle A vehicle or vector similar to a cloning vehicle but which is capable of expressing a gene of interest which has been cloned into it, after transformation into a desired host.
  • such expression vehicle provides for an enhanced expression of a gene of interest which has been cloned into it, after transformation into a desired host.
  • the gene of interest which it is desired to express is provided to a fungal host as part of a cloning or expression vehicle and integrates into the fungal chromosome. Sequences which derive from the cloning vehicle or expression vehicle may also be integrated with the gene of interest during the integration process.
  • Expression vehicles also include plasmids or other vectors which carry a gene of interest, for example, a functionally complete constant heavy or light chain sequence, or a desired biologically active portion thereof, or a portion thereof, which, when reassociated with another peptide results in a biologically active protein product.
  • genes of interest usually have appropriate restriction sites engineered so that any sequence with the appropriate cohesive ends can be easily inserted thereinto.
  • Such vehicles can be used as intermediates for the expression of any desired protein in any appropriate Trichoderma host.
  • Immunoglobulin is meant to include proteins which possess immunological properties (such as antigen binding),for example, a multisubunit antibody containing two heavy chains and two lights chains. As used herein, the term also refers to a protein which contains the amino acid sequence of the individual light or heavy chain of such an antibody, such light or heavy chains being capable of being modified (for example, by reassociation with another immunoglobulin) to reveal their inherent immunological activity.
  • immunoglobulin gene refers to a DNA sequence which encodes such an immunoglobulin heavy or light chain.
  • Constant or Variable are used functionally to denote those regions of the immunoglobulin chain, either heavy or light chain, which code for properties and features possessed by the variable and constant regions on natural antibodies. It is not necessary for the complete coding regions for variable or constant regions to be present, as long as a functionally operating regions is present and available.
  • domain is used to describe an independently folding part of a protein which may or may not function independently. General definitions for domain borders in natural proteins are found in Argos, P., J. Mol . Biol . 211:943-958 (1990).
  • a "functional derivative" of a protein is a smaller peptide of that protein, such peptide possessing a desired biological activity (either functional or structural), or modifiable in a known way to reveal such activity.
  • a functional derivative of an immunoglobulin heavy chain may be that fragment of the heavy chain which is found in the Fab fragment and which does not reveal its inherent activity until, with an appropriate functional derivative of a light chain, it is reassociated into an immunologically active Fab fragment.
  • a protein is said to be a "chemical derivative" of another protein when it contains additional chemical moieties not normally a part of the protein. Such moieties may improve the protein's solubility, absorption, biological half life, etc.
  • the moieties may alternatively decrease the toxicity of the protein, eliminate or attenuate any undesirable side effect of the protein, etc. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980). Procedures for coupling such moieties to a protein are well known in the art.
  • a functional derivative of a protein may or may not contain post-translational modifications such as covalently linked carbohydrate, depending on the necessity of such modifications for the performance of a specific function.
  • the term “functional derivative” is intended to include the "frag ⁇ ments,” “variants,” “analogues,” or “chemical derivatives” of a molecule. Fragment.
  • a “fragment” of a protein is meant to refer to a peptide which possesses some but not all of the amino acid sequence of a longer protein.
  • a protein is said to be an immunologically active fragment of a protein if such protein possesses a function found in a native immunoglobulin, or can be modified (for example, by reassociation with another immunoglobulin fragment) to reveal such function.
  • variants of a protein are meant to refer to a protein substantially similar in structure and biological activity to a protein which is found in nature, or a fragment thereof, but which has been engineered to contain a different amino acid sequence. Thus, provided that two proteins possess a similar activity, they are considered variants as that term is used herein even if the primary structure of one of the protein does not possess the identical amino acid sequence to that found in the other.
  • An antibody is said to be “capable of binding” a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody.
  • epitope is meant to refer to that portion of an antigen which can be recognized and bound by an antibody.
  • An antigen may have one, or more than one epitope.
  • An "antigen” is capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen.
  • the specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.
  • immunoglobulin immunoglobulin
  • antibody antibody
  • Mob monoclonal antibody
  • immunoglobulin immunoglobulin
  • Fab and F(ab') 2 fragments which are capable of binding an antigen.
  • Fab and F(ab') fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et a7., J. Nucl . Med. 24:316-325 (1983)).
  • immunoglobulins usually contain two pairs of chains bound together by disulphide bridges
  • immunoglobulin is meant herein to encompass the individual immunoglobulin chains ("immunoglobulin peptides") even though such chains may require reassociation with a second immunoglobulin chain to reveal a biological activity inherent in their structure.
  • the immunoglobulins of the invention may be of any type (class) such as, for example, IgM, IgG, IgA, IgD and IgE as well as any of the various subtypes of the individual classes.
  • the light chain may be either K (kappa) or ⁇ (lambda).
  • the heavy chains may be ⁇ (mu), ⁇ (gamma), S (delta), ⁇ (alpha) or £ (epsilon).
  • different gene families encode the light and heavy chains.
  • Biologically active portions of such chains, or domains from the various chains may be genetically recombined as desired. Light and heavy chains are divided into domains of structural and functional homology.
  • variable regions of both light (VL) and heavy (V ⁇ ) chains determine immunoglobulin's recognition and specificity.
  • and heavy (C ⁇ ) chains confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and the like.
  • V (variable) region gene sequences conferring antigen specificity and binding are located in separate germ line gene segments called V ( -
  • V ⁇ -D-J ⁇ V segments then encode the complete variable regions or antigen binding domains of light and heavy chains, respectively.
  • a hybridoma source of a monoclonal antibodies which it is desired to clone and express according to the present invention is prepared by any of a variety of methods.
  • Monoclonal antibodies can be prepared using hybridoma tech ⁇ nology (Kohler et al. , Nature 255:495 (1975); Kohler et a7., Eur. J. Immunol. 5:511 (1976); Kohler et a7., far. J. Immunol. 5:292 (1976); Hammerling et a7., In: Monoclonal
  • Such procedures involve immunizing an animal with the desired protein.
  • the splenocytes of such animals are extracted and fused with a suitable myeloma cell line.
  • Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP2O), available from the American Type Culture Collection, Rock- ville, Maryland.
  • SP2O parent myeloma cell line
  • the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution, for example, as described by Wands, J.R., et a7., Gastroenterol ogy 50:225-232 (1981), which reference is herein incorporated by reference.
  • the hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the desired protein.
  • hybridomas A wide range of source hybridomas are immediately available for the preparation of the recombinant immunoglobulin constructs of the invention. For example, see the catalogue ATCC Cel l Lines and Hybridomas, American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A., and the ECACC Catalogue, 2nd edition, PHLS CAMR Porton Down, Salisbury, Wills, U.K. Hybridomas secreting monoclonal antibodies reactive to a wide variety of antigens are listed therein, are available from the collection, and usable in the invention. Of particular interest are hybridomas secreting antibodies which are reactive with viral antigens, bacterial antigens, and tumor antigens.
  • polyclonal antibodies are desired, cells expressing a desired protein, or an antigenic fragment thereof, can be administered to an animal in order to induce the production of sera containing polyclonal antibodies that are capable of specifically binding the protein.
  • a protein is isolated or a peptide fragment is chemically synthesized and purified by HPLC to render it substantially free of contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of high specific activity.
  • polyclonal antibodies may be especially useful in identifying clones which secrete a desired monoclonal antibody which is produced according to the methods of the invention.
  • the host strain may either be a prototrophic or an auxotrophic Trichoderma strain depending on the selection marker used in the transformation procedure. If a selection marker is used which complements a corresponding mutation of the host genome auxotrophic Trichoderma mutants must be constructed.
  • Auxotrophic Trichoderma mutants may be produced by known methods for producing mutant strains. Auxotrophic Trichoderma mutants requiring uracil, tryptophan or arginine for growth may be isolated by a filtra ⁇ tion enrichment technique as described by Nevalainen (Neva- lainen, H., Technical Research Center of Finland, Publication 26 (1985)). From arginine requiring auxotrophs, mutants deficient in the argB gene were identified by using a series of minimal plates supplied by different intermediates in the arginine biosynthesis.
  • Mutants having a trpC gene defect can be characterized by the lack of the PRA iso erase-InGP synthetase activity in their mycelia (Creighton, T.E., Biochem. J. 120:699-707 (1970)).
  • the trpC-character of the mutants showing no enzyme activity may be confirmed by e.g. transformation and complementation with the trpC-plasmid of A. nidulans (Yelton, M., et al. , Proc. Natl. Acad. Sci. USA 51:1470-1474 (1984)).
  • genetic sequences are capable of encoding a desired protein and through the expression of such genetic sequences.
  • genetic sequences is intended to refer to a nucleic acid molecule (preferably DNA). Genetic sequences which are capable of encoding a desired protein are derived from a variety of sources. These sources include genomic DNA, cDNA, synthetic DNA, and combinations thereof, preferably from sources which naturally the desired protein.
  • Hybridoma cell lines which produce immunoglobulins and especially monoclonal antibodies of defined specificity are especially useful for the isolation of mRNA or genomic DNA for subsequence cloning of genes capable of expressing immunoglobulins according to the invention.
  • mRNA or genomic DNA can be isolated by techniques known in the art, for example, as described in Maniatis, T. et a7., Molecular Cloning (A Laboratory Manua l) , Cold Spring Harbor Laboratory, 2nd edition, 1989. A wide range of source hybridomas are available for the preparation of mRNA or genomic DNA.
  • Hybridomas secreting immunoglobulins and especially monoclonal antibodies reactive with a wide variety of antigens are listed therein, are available from the collection, and usable in the invention.
  • hybridomas secreting antibodies which are reactive with viral antigens, bacterial antigens, and tumor antigens.
  • the recombinant protein preparations according to this invention are produced by the fungus Trichoderma which has been modified by recombinant DNA techniques.
  • the Trichoderma hosts of the invention are modified so as to express a natural or chimeric immunoglobulin.
  • natural immunoglobulin is meant an immunoglobulin whose coding sequence was originally formed as a result of genomic rearrangements in an animal cell.
  • chimeric immunoglobulin is meant an immunoglobulin whose coding sequence was originally formed as a result of in vitro recombinant DNA techniques.
  • the Trichoderma hosts of the invention may be modified to produce an increased amount of one or more immunoglobulins and especially immunoglobulin chains. According to this invention, it is possible to enrich Trichoderma hosts for a desired immunoglobulin by inserting a gene encoding such immunoglobulin such that expression of the desired gene is operably linked to a promoter.
  • the promoter is a strong homologous promoter.
  • the desired gene is inserted into the cbhl locus or a cassette comprising a desired gene already operably linked to the homologous cbhl promoter is inserted into the cbhl locus (U.S. Application No. 07/524,308, incorporated herein by reference).
  • a promoter derived from a gene for a protein heterologous to Trichoderma is the Aspergillus glucoamylase promoter, and the
  • Aspergillus amdS, gpd, argB promoters (Penttila, M. et a7., Gene 51:155-164 (1987) and trpC promoters.
  • Synthetic promoters may be constructed containing regulatory regions derived from two or more different natural promoters. Eliminating the activity of any of the cellulase genes will result in a host which is partially or completely deficient in its ability to degrade cellulose to glucose.
  • cellulase deficient strains can be obtained, for example, by transformation of Trichoderma with a plasmid containing only part of the homologous coding region (insertional activation). This is described in EP 244,234.
  • cellulase deficient strains can also be prepared by gene replacement strategy in which the 5' and 3' flanking DNA is utilized to obtain homologous recombination. In between the flanking regions, a marker gene or gene coding for the desired protein product can be placed. 7.
  • reesei strains deficient in different cellulase enzymes can be identified by using, for example, an ELISA test which uses antibodies, and especially monoclonal antibodies, against the different cellulases to characterize the presence or absence of a specific cellulase (U.S. Application No. 07/524,308).
  • Integration of a desired a gene with homologous recombination may be done with a circular DNA, which integrates in a colinear manner into the Trichoderma chromosomal DNA.
  • the use of a linear DNA especially helps in directing integration into a homologous locus.
  • the integration of a desired gene is directed into the Trichoderma cbhl locus.
  • Clones of the cellulase enzymes which may be used to provide a source of homologous transcriptional and translational regulatory sequences for the hosts of the invention are known in the art.
  • the gene for the native cellobiohydrolase CBH I sequence has been cloned by Shoemaker et a7.
  • the genetic constructs which encode a desired protein may be introduced into the genome of Trichoderma.
  • Enhanced expression can also be achieved by using strong promoters such as cbhl and, if desired, additional or modified regulatory regions, such as, for example, enhancer sequences may be used.
  • promoters such as cbhl
  • additional or modified regulatory regions such as, for example, enhancer sequences may be used.
  • such regulatory sequences are homologous to Trichoderma.
  • a regulatory region, and especially a promoter may be modified to contain only those sequence elements needed for expression and/or to retain a region which is responsible for high expression levels.
  • Enhancer sequences may be introduced concurrently with the gene of interest as a separate DNA element but operably-1inked - to such gene of interest, for example, as a DNA sequence which is colinear with that providing the gene of interest (for example, in a 5' or 3' non-translating sequence, or in an intron).
  • increased amounts of a desired protein are achieved by introducing the gene producing such desired protein into a specific locus and/or introducing the gene in multicopies into the genome of Trichoderma as described above.
  • a gene encoding a desired protein can also be integrated into the genome of Trichoderma by using a vector which provides cloning sites allowing insertion of a gene of interest into an expression cassette in a manner which operably links the gene of interest to a promoter and terminator sequence.
  • a promoter and terminator sequence Preferably, the cbhl gene promoter and terminator are used.
  • the cassette may further provide a stuffer fragment between the promoter and terminator sequences which can be removed.
  • pAMHUO which is described in the patent application EP 244,234 may be used.
  • pAMHUO is derived from pUC19 (Yanish-Perron et al . , Gene 33:103-119 (1985)).
  • the stuffer fragment in pAMHUO may be removed by digestion with SadI and M-tel. After the ends of a double-stranded DNA sequence which encodes a desired protein are made blunt, any DNA, cDNA or chromosomal DNA can be inserted between the promoter and terminator.
  • the desired gene can be inserted to the cbhl expression cassette in the plasmid pAMHUO between the cbhl promoter and terminator.
  • Transcriptional regulatory elements of other genes may be used where it is desired not to use the cbhl elements.
  • a vector construction comprising the 3- phosphoglycerate kinase gene [pgk) transcriptional regulatory regions may be used as 3-phosphoglycerate kinase, a key enzyme for ATP generation by glycolysis, is expressed in the presence of glucose under which conditions the synthesis of cellulases is repressed.
  • the DNA constructions prepared according to this invention can be used to transform any Trichoderma strain.
  • Such strains include, for example, 7. reesei strains QM9414 (ATCC 26921), RUT-C-30 (ATCC 56765), and highly productive mutants like VTT-D-79125, which is a descendant of QM9414 (Nevalainen 1985, Technical Research Centre of Finland Publications 26, (1985), Espoo, Finland).
  • Trichoderma may be performed by any technique known in the art and especially by the technique taught in U.S. Application No. 07/044,077, filed April 29, 1987.
  • the Trichoderma host cells may be cultivated and the desired enzymes produced by cultivating the host strain having the desired properties under any conditions which allow expressing of the desired enzymes.
  • a Trichoderma host strain having the desired properties may be cultivated in a liquid cultivation medium, which may comprise, for example, 6% Solka Floe cellulose, 3% distiller's spent grain, 0.5% KH 2 P0 4 , 0.5% (NH 4 ) 2 S0 , 0.1% struktol .
  • Trichoderma cultivation should be kept at approximately pH 5 by the addition of phosphoric acid or ammonia and the temperature may be kept at 30'C during the cultivation. However, the temperature and pH should be adjusted according to the strain and according to the protein to be produced (Merivuori et a7., Biotechnology Lett . 12:117-120 (1990)).
  • Vector systems may be used in the method of producing the recombinant proteins, and especially the immunoglobulins of the invention.
  • Such vector systems may be constructed so as to provide (a) on a first vector, a gene which encodes at least one desired gene, for example, a gene encoding an immunoglobulin chain or i munologically active fragment thereof, to be integrated to the genome of Trichoderma and (b) on a second vector, another gene which encodes at least one desired gene, for example, a gene encoding another immunoglobulin chain or immunologically active fragment thereof, to be integrated to the genome of Trichoderma; and (c) a selectable marker which provides selection of Trichoderma which contain (a) and/or (b).
  • a selectable marker may be provided on a separate vector.
  • two cassettes encoding two different genes of interest are-provided at different sites on the same vector.
  • the cloned DNA which is used in the hosts of the invention may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained in association with the native 5' promoter region of the DNA genetic sequences and/or with the 3' transcriptional termination region if such sequences are capable of functioning in Trichoderma. Further, such genomic DNA may be obtained in association with the genetic sequences which encode the 5' non-translated region of the mRNA and/or with the genetic sequences which encode the 3' non-translated region.
  • Genomic DNA can be extracted by means well known in the art (for example, see Guide to Molecular Cloning Techniques, S.L. Berger et a7., eds., Academic Press (1987)).
  • mRNA can be isolated from any cell which produces or expresses the desired protein, and used to produce cDNA by means well known in the art (for example, see Guide to Molecular Cloning Techniques, S.L. Berger et a7., eds., Academic Press (1987)).
  • the mRNA preparation used will be enriched in mRNA coding for a desired protein, either naturally, by isolation from a cells which are producing large amounts of the protein, or in vitro, by techniques commonly used to enrich mRNA preparations for specific sequences, such as sucrose gradient centrifugation, or both.
  • Such suitable DNA preparations are randomly sheared or enzyma- tically cleaved, respectively, and ligated into appropriate vectors to form a recombinant gene (either genomic or cDNA) 1ibrar .
  • a DNA sequence encoding a desired protein may be inserted into a DNA vector in accordance with conventional techniques, including blunt-ending or staggered-ending termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Maniatis et a7., Molecular Cloning (A Laboratory Manual) , Cold Spring Harbor Laboratory, 1982), and are well known in the art.
  • Libraries containing clones encoding a desired protein, such as an immunoglobulin chain and especially a chain of a monoclonal antibody, may be screened and a clone to the desired protein identified by any means which specifically selects for that protein's DNA such as, for example, a) by hybridization with an appropriate nucleic acid probe(s) containing a sequence specific for the DNA of this protein, or b) by hybridization-selected translational analysis in which native mRNA which hybridizes to the clone in question is translated in vitro and the translation products are further characterized, or, c) if the cloned genetic sequences are themselves capable of expressing mRNA, by immunoprecipitation of a translated protein product produced by the host containing the clone.
  • a desired protein such as an immunoglobulin chain and especially a chain of a monoclonal antibody
  • Oligonucleotide probes specific for the desired protein which can be used to identify clones to such protein can be designed from knowledge of the amino acid sequence of the protein and if desired, obtained commercially.
  • the genetic code is degenerate, more than one codon may be used to encode a particular amino acid (Watson, J.D., In: Molecular Biology of the Gene, 3rd Ed., W.A. Benjamin, Inc., Menlo Park, CA (1977), pp. 356-357).
  • the peptide fragments are analyzed to identify sequences of amino acids which may be encoded by oligonucleotides having the lowest degree of degeneracy. This is preferably accomplished by identifying sequences that contain amino acids which are encoded by only a single codon.
  • amino acid sequence may be encoded by only a single oligonucleotide sequence
  • amino acid sequence may be encoded by any of a set of similar oligonucleotides.
  • all of the members of this set contain oligonucleotide sequences which are capable of encoding the same peptide fragment and, thus, potentially contain the same oligonucleotide sequence as the gene which encodes the peptide fragment
  • only one member of the set contains the nucleotide sequence that is identical to the exon coding sequence of the gene.
  • this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it is possible to employ the unfractionated set of oligonucleotides in the same manner in which one would employ a single oligo- nucleotide to clone the gene that encodes the peptide.
  • oligonucleotides can be identified from the amino acid sequence, each of which would be capable of encoding the protein.
  • the probability that a particular oligonucleotide will, in fact, constitute the actual protein's sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic cells.
  • Such "codon usage rules" are disclosed by Lathe, R., et a7., J. Mo lec. Biol . 153:1-12 (1985).
  • the suitable oligonucleotide, or set of oligonucleotides, which is capable of encoding a fragment of the protein's gene (or which is complementary to such an oligonucleotide, or set of oligonucleotides) may be synthesized by means well known in the art (see, for example, Synthesis and Application of DNA and RNA, S.A. Narang, ed., 1987, Academic Press, San Diego, CA) and employed as a probe to identify and isolate the desired cloned gene by techniques known in the art. Techniques of nucleic acid hybridization and clone identification are disclosed by Maniatis, T., et a7.
  • the above-described DNA probe is labeled with a detectable group.
  • detectable group can be any material having a detectable physical or chemical property. Such materials have been well-developed in the field of nucleic acid hybridization and in general most any label useful in such methods can be applied to the present invention. Particularly useful are radioactive labels, such as -'- ' ?, ⁇ , ⁇ , 35s ? 125 ⁇ or t (- e ii e. Any radioactive label may be employed which provides for an adequate signal and has a sufficient half-life.
  • the oligonucleotide may be radioactively labeled, for example, by "nick-translation" by well-known means, as described in, for example, Rigby, P.J.W., et al . , J. Mol . Biol. 113:237 (1977) and by T4 DNA polymerase replacement synthesis as described in, for example, Deen, K.C, et al . , Anal . Biochem. 135:456 (1983).
  • polynucleotides are also useful as nucleic acid hybridization probes when labeled with a non-radioactive marker such as biotin, an enzyme or a fluorescent group.
  • a non-radioactive marker such as biotin, an enzyme or a fluorescent group.
  • the actual identification of protein's sequence permits the identification of a theoretical "most probable" DNA sequence, or a set of such sequences, capable of encoding such a peptide.
  • a DNA molecule or set of DNA molecules, capable of functioning as a probe(s) for the identification and isolation of clones containing the protein's gene.
  • a library is prepared using an expression vector, by cloning DNA or, more preferably cDNA prepared from a cell capable of expressing a desired protein, into an expression vector.
  • the library is then screened for members which express the protein, for example, by screening the library with antibodies to the protein, and especially, in this case, antibodies which recognize a desired immunoglobulin or fragment thereof.
  • the above discussed methods are, therefore, capable of identifying genetic sequences which are capable of encoding a desired immunoglobulin or fragments of this protein.
  • Such expression identifies those clones which express proteins possessing characteristics of the desired protein, and especially, the desired immunoglobulin.
  • Such characteris ⁇ tics may include the ability to specifically bind antibodies directed against the protein, the ability to elicit the production of antibody which are capable of binding the protein, and the ability to provide a function specific to the protein, among others.
  • the cloned protein encoding sequences obtained through the methods described above, and preferably in a double- stranded form, may be operably linked to sequences controlling transcriptional expression in an expression vector, and introduced into a Trichoderma host cell to produce recombinant protein or a functional derivative thereof.
  • a nucleic acid molecule such as DNA, is said to be "capable of expressing" a polypeptide if it contains expression control sequences which contain transcriptional regulatory information and such sequences are “operably linked” to the nucleotide sequence which encodes the polypeptide.
  • An operable linkage is a linkage in which a sequence is connected to a regulatory sequence (or sequences) in such a way as to place expression of the sequence under the influence or control of the regulatory sequence.
  • Two DNA sequences are said to be operably linked if induction of promoter function results in the transcription of the protein encoding sequence mRNA and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the expression regulatory sequences to direct the expression of the mRNA or protein, or (3) interfere with the ability of the template to be transcribed by the promoter region sequence.
  • a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
  • the precise nature of the regulatory regions needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribing and 5' non-translating (non-coding) sequences involved with initiation of transcription and translation respectively.
  • Expression of the protein in the Trichoderma hosts requires the use of regulatory regions functional in such hosts.
  • a wide variety of transcriptional and translational regulatory sequences can be employed, since Trichoderma generally recognize transcriptional controls, such as, for example, those of other filamentous fungi.
  • control regions may or may not provide an initiator methionine (AUG) codon, depending on whether the cloned sequence contains such a methionine.
  • AUG initiator methionine
  • Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis in the host cell.
  • a promoter region sufficient to direct the initiation of RNA synthesis in the host cell.
  • translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes the protein, or a functional derivative thereof, does not contain any intervening codons which are capable of encoding a methionine.
  • the secretion of the expressed product the gene for the desired product is preferably provided with a preregion ensuring effective direction of the expressed product into the secretory pathway of the cell.
  • a desired protein is secreted into.the surrounding medium due to the presence of a homologous Trichoderma secretion signal sequence.
  • Such preregion may provide the signal sequence of an immunoglobulin molecule or may provide a signal sequence of a different protein.
  • the preregion might be a naturally occurring signal or leader peptide or parts thereof or a synthetic signal/leader sequence, especially a synthetic sequence containing codons preferred in the desired host.
  • the preregion is generally cleaved off from the desired product during secretion whereupon the mature product can be isolated from the culture medium.
  • the signal/leader sequence may be derived from the signal/leader sequence of the gene encoding the desired protein or may be derived from genes for other secreted proteins either from Trichoderma or from any other source of organism.
  • Examples of signal/leader sequences derived from genes encoding a protein secreted by Trichoderma are cbhl signal sequence or the eg71 signal sequence or parts thereof.
  • Signal/leader sequences derived from genes encoding secreted proteins heterologous to Trichoderma may be derived from genes for Aspergil lus amylases or glucoamylase.
  • the desired coding sequence of a protein may be linked to any signal sequence which will allow secretion of the protein from a Trichoderma host, for example, a yeast sequence (Wood, C.R., et a7., Nature 314:446-449 (1985); Kaiser, CA. et a7., Science 235:312-317 (1987)).
  • Transcriptional initiation regulatory signals can be selected which allow for repression or activation, so that expression of the operably linked genes can be modulated.
  • regulatory signals which are temperature- sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical regulation, e.g., substrate or metabolite regulation.
  • the non-transcribed and/or non-translated regions 3' to the sequence coding for a protein can be obtained by the above-described cloning methods.
  • the 3'-non- transcribed region may be retained for its transcriptional termination regulatory sequence elements; the 3-non-translated region may be retained for its translational termination regulatory sequence elements, or for those elements which direct polyadenylation in eukaryotic cells.
  • genetically stable transformants of Trichoderma are constructed whereby a desired monoclonal antibody's DNA is integrated into the host chromosome.
  • the coding sequence for the desired monoclonal antibody may be from any source. Such integration may occur de novo within the cell or, in a most preferred embodiment, be assisted by transformation with a vector which functionally inserts itself into the host chromosome, for example, DNA elements which promote integration of DNA sequences in chromosomes.
  • Cells which have stably integrated the introduced DNA into their chromosomes are selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector in the chromosome.
  • the selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Different selection markers may be used, e.g., argB [A. nidulans or 7. reesei ) , and amdS [A. nidulans) .
  • a dominant selection marker is used such as amdS (Kelly, J.M. et a7., EMBO J. 4:475-479 (1985); Tiburn, J. et al .
  • Auxotrophic selection markers are also available, for example, trpC or argB (Penttila, M. et a7., Gene 51:155-164 (1987)).
  • a desired monoclonal antibody is expressed from two different expression cassettes on two different plasmids (one cassette expressing the light chain and one cassette expressing the heavy chain), it may be desired to utilize two different selection markers to ensure that the host has been transformed with both plasmids.
  • reesei grows poorly on acetamide as the sole nitrogen source and the growth can be further inhibited by adding CsCl to the medium.
  • the growth on acetamide as the sole nitrogen source in the presence of CsCl can accordingly be used as a selection medium to identify AmdS + transformants.
  • Factors of importance in selecting a particular plasmid include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • the DNA construct(s) is introduced into an appropriate host cell by any of a variety of suitable means, including transformation as described above.
  • recipient cells are grown in a selective medium, which selects for the growth of transformed cells. Expression of the cloned gene sequence(s) results in the production of the desired protein, or in the production of a fragment of this protein. This expression can take place in a continuous manner in the transformed cells, or in a controlled manner.
  • immunoglobulin molecules may be provided including divalent, dispecific (i.e., with different variable regions), molecules with chimeric heavy chains and non-chi eric light chains, or molecules with variable binding domains attached to peptide moieties carrying desired functions.
  • Antibodies having chimeric heavy chains of the same or different variable regions binding specificity, and non- chimeric (i.e. all human or all non-human) light chains can be prepared by appropriate association of the needed polypeptide chains. These chains can be prepared by using two Trichoderma immunoglobulin-expressing clones of the invention (one expressing the heavy chain and one expressing the light chain) or by using one clone which expresses both of the desired chains. The light and heavy chains of such monoclonal antibodies may be allowed to associate in the Trichoderma host cell or may be combined in vitro, by allowing the disulfide bridges which characterize the chain association of such antibodies to form.
  • Class switching is useful when it is desired to change the association, aggregation or other properties of antibodies obtained from cell fusion or hybridoma technology.
  • a balanced synthesis of both light and heavy chain protein within the host cell is preferred.
  • One approach is to place the light and heavy chain genes on separate cassettes on separate vectors, preferably with the same promoter being used to drive the expression of both chains.
  • different promoters are used, they should be balanced such that one promoter is not so much stronger or weaker than the other promoter that a disproportionate synthesis of one chain over the other chain occurs.
  • Efficient secretion of the immunoglobulin chains can be achieved by constructing fusion proteins, where either one or both immunoglobulin chains are preceded by an amino acid sequence which functions as secretion signal in Trichoderma.
  • such amino acid sequence is derived from an efficiently secreted Trichoderma protein or parts of such protein.
  • Efficiently secreted cellulases of Trichoderma are well suited for fusion constructions. After secretion, the Trichoderma protein part of the fusion could be removed, for example, by adding or otherwise engineering a proteolytic cleavage site between the two protein sequences.
  • DNA-sequences may be modified by amendment or deletion of a couple of bases non-essential to the encoded product or encoded function.
  • DNA sequences substantially similar to cbhl or egll signal or promoter sequences may be used as long as they exhibit the intended function in Trichoderma.
  • genes for the desired proteins may be altered as long as this has no deleterious effect on the activity of the protein.
  • the transformation technique is adapted from the methods for transformation of A. nidulans (Ballance, J., et a7., Biochem. Biophys. Res. Commun. 112:284-289 (1983); Tilburn, J., et a7., Gene 25:205-221 (1983)). This is described in Penttila, M. et a7., Gene 51:155-161 (1987).
  • Protoplasts are prepared by known methods by growing mycelium on agar plates and suspending mycelium in a buffered solution of Novozy ⁇ 234.
  • a sorbitol solution may advantageously be used.
  • Transforming DNA is then added to the protoplast solution as described in further detail in the experimental part of the specification.
  • the transformation is usually carried out as a cotransformation by which a nonselectable plasmid is cotransformed into 7. reesei with high frequency using a selectable marker inserted in another plasmid (e.g., amdS in p3SR2 or argB in pSal43).
  • a great proportion of the transformants contain the nonselectable plasmid integrated into the genome if equi olar amounts of DNA are used for transformation.
  • an argB 7.
  • reesei strain is used in transformation with equimolar amounts of plasmids p3SR2 and pSa!43, transformants can be obtained on double selection medium (minimal medium, acetamide, CsCl). Alternatively, a single plasmid may be used for transformation in which also the selectable marker is inserted.
  • Transformants are then cultured in a suitable medium.
  • a glucose inducible gene or the glucose inducible promoter thereof
  • Trichoderma is grown on a medium containing 2% glucose, secretion of all extracellular proteins, including proteases is strongly suppressed.
  • the gene coding for the desired protein for example, an immunoglobulin
  • the desired protein is preferably secreted to the growth medium. Since other proteins are not produced under these conditions, the desired protein is the major protein secreted into the medium.
  • the fungal mycelium is removed from such a culture, the desired protein is present in the resulting solution in a very pure form. If required, such protein can be very easily further purified, since it is almost the only protein present.
  • solid media permits rapid screening of thousands of colonies arising .from transformed Trichoderma conidia for the presence or absence of specific proteins and allows qu ⁇ titative estimation of the amount of such proteins produced.
  • solid media for detection of such proteins are known in the art.
  • fungi form large diffuse colonies when grown on solid media. Addition of chemical agents restrictive to colony growth may therefore be desired to allow development of more than one (up to 100) colony per one plate.
  • agents used for the purpose are rose bengal, oxgall and phosphon D, Triton X-100 and saponin.
  • replica plating technique analogous to that developed for bacteria can, in certain cases, be used to test the properties of fungal colonies on different growth media. Screening on plates is usually followed by cultivation of the selected colonies in shake flasks in a liquid production medium for measurement of protein production. Trichoderma is well adapted to fer enter cultivations and can utilize cheap raw materials for growth.
  • the best isolates showing enhanced recombinant protein production in shake flask scale may be utilized in a second round of transformation if it is desired to express more than one recombinant protein in a host.
  • a desired Trichoderma host may be cotransformed with more than one gene of interest at the same time.
  • the recombinant hosts described above having the desired properties are cultivated under suitable conditions.
  • the desired proteins are secreted from the Trichoderma hosts and into the culture medium, and the proteins are recovered from said culture medium by methods known in the art.
  • the protein preparation can be produced by cultivating the Trichoderma strain in a fermentor having the desired properties for example in a liquid cultivation medium, which may comprise for example 6% Solka Floe cellulose (BW40, James River Corporation, Ralphensack, NJ), 3% distiller's spent grain (waste after alcohol distillation, ALKO, Ltd., Koskenkorva, Finland), 0.5% KH 2 P0 , 0.5% (NH 4 ) 2 S0 4 , and 0.1% struktol as an antifoa ing agent (struktol SB 2023, Schill & Seilacher, Hamburg, FRG).
  • a liquid cultivation medium which may comprise for example 6% Solka Floe cellulose (BW40, James River Corporation, Ralphensack, NJ), 3% distiller's spent grain (waste after alcohol distillation, ALKO, Ltd., Koskenkorva, Finland), 0.5% KH 2 P0 , 0.5% (NH 4 ) 2 S0 4 , and 0.1% struktol as an antifo
  • the Trichoderma strain will be sensitive to glucose repression and expression of such protein will require an inducer (cellulose, lactose or sophorose) (Allen et a7., Biotechnology and Bioengineering 33:650-656 (1989)).
  • the pH should preferably be kept at approximately pH 5 by the addition of phosphoric acid or ammonia and the temperature at 30°C during the cultivation. However, the temperature and pH may be adjusted according to the strain and according to the protein to be produced (Merivuori et a7., Biotechnology Letters 12(2):117- 120 (1990)).
  • the recombinant protein preparation is recovered from the culture medium by using methods well known in the art. If the hosts of the invention are partially or completely deficient in cellulase activity or if the product is produced by a promoter which functions in the presence of glucose, the recombinant protein may be pure enough to be utilized directly from the culture medium with no further purification. If desired, such preparations may be lyophilized or the immunological activity otherwise concentrated and/or stabilized for storage.
  • the recombinant protein preparations of the invention are very economical to provide and use because the proteins are secreted into the culture medium, only the culture medium need be recovered to obtain the desired protein preparation; there is no need to extract such protein from the Trichoderma hosts.
  • an expressed protein may be further purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chro atography, electrophoresis, or the like.
  • the proteins of the invention may be conjugated, either chemically or by genetic engineering, to fragments of other agents which provide a targeting of such proteins to a desired site of action.
  • other compounds may be conjugated, either chemically or by genetic engineering, to the recombinant protein or active fragment thereof, so as to enhance or provide additional properties to such protein, especially properties which enhance the protein's function, or its ability to treat or alleviate an undesired condition or physiological state.
  • Amounts and regimens for the administration of the proteins, and especially immunoglobulins of the invention can be determined readily by those with ordinary skill in the clinical or industrial art. Generally, the amount of protein and especially immunoglobulin will vary depending upon considerations such as: type of application, type of monoclonal antibody employed; age, health, conditions of a patient being treated; kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired; counterindications, if any, and other variables to be adjusted by the individual physician or scientist.
  • Dosage can be administered in one or more applications to obtain the desired results.
  • the recombinant protein of the invention can be administered in any appropriate agricultural or pharmacological carrier for administration.
  • the recombinant protein can be administered in any form that effects prophylactic, palliative, preventative or curing conditions of a desired condition, or in any form which retains a desired function, for example, the immunological function of an immunoglobulin preparation.
  • Preparations of the recombinant proteins of the invention for parenteral administration includes sterile aqueous or non-aqueous solvents, suspensions and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, fish oil, and injectable organic esters.
  • Aqueous carriers include water, water-alcohol solutions, emulsions or suspensions, including saline and buffered medical parenteral vehicles including sodium chloride solution, Ringer's dextrose solution, dextrose plus sodium chloride solution, Ringer's solution containing lactose, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based upon Ringer's dextrose and the like.
  • the recombinant proteins of the invention may also be administered by means of pumps, sprays, and in sustained- release form, especially, when it is desired to prolong the effect of a given application or dose.
  • the proteins of the invention may also be delivered to specific organs in high concentration by means of suitably inserted catheters, or by providing such molecules as a part of a chimeric molecule (or complex) which is designed to target specific organs.
  • Administration in a sustained-release form is more convenient for a patient when repeated injections for prolonged periods of time are indicated.
  • the recombinant proteins of the invention can be employed in dosage forms such as tablets, capsules, powder packets, or liquid solutions for oral administration if the biological activity of the protein is not destroyed by the digestive process and if the characteristics of the compound allow it to be absorbed across the intestinal tissue.
  • compositions providing the recombinant proteins of the present invention are manufactured in a manner which is in itself known, for example, by means of conventional mixing, granulating, dragee-making, dissolving, iyophilizing or similar processes.
  • the compositions of the present invention in and of themselves, find utility in the diagnosis and control of many physiological and agricultural conditions, in the industrial treatment of wastes, sewage, and in the industrial preparation of biologicals, among other uses.
  • compositions of the present invention have a sufficiently rapid onset of action to be useful in the acute management of a physiological condition needing immediate amelioration.
  • a low potency version is useful in the management of mild or chronic physiological disorders.
  • compositions are conveniently provided in kit form, such kits containing appropriate buffers and mixing solutions for use of the recombinant proteins in a given application.
  • compositions of the present invention may provide requisite reagents for diagnostic kits or for the laboratory assay of any antigen recognized by the recombinant proteins of the invention.
  • Trichoderma is an especially useful and practical host for the synthesis of recombinant proteins and especially for the synthesis of immunoglobulins because Trichoderma is capable of secreting large amounts of protein, for example, concentrations as much as 40 g/L culture fluid have been reported;
  • the homologous Trichoderma cbhl promoter provides a very convenient promoter for expression of a desired gene because it is a strong, single copy promoter which normally directs the synthesis of up to 60% of the secreted protein from the Trichoderma host;
  • the transformation system is highly versatile and can be adapted for any gene of interest;
  • the Trichoderma host provides an "animal cell type" high mannose glycosylation pattern; and culture of Trichoderma is supported by previous extensive experience in industrial scale fermentation techniques.
  • Proteins, and especially immunoglobulins and their derivatives produced by the methods of the invention have applications in, for example, the agricultural, foodstuffs and pharmaceutical industries.
  • Applications of immunoglobulins include their use as im unotoxins, and as immunological contrast media for the location of tumors, milk processing, large-scale purification of enzymes, purification of stereospecific drug substances and other molecules, purification of sewage and the development of sensitive and specific biosensors.
  • Plasmids 1. p285 is ATCC No. 20681.
  • pCAMG91 is described in Boel, E., et a7., 7 ⁇ e EMBO Journal 3:1581-1585 (1984).
  • piC19R is described in Marsh, J.L., et a7., Gene 32:481-485 (1984).
  • pSal43 is described in John, M.A., et al. , Enzyme Microb. Technol. 5:386-389 (1984); Berse, B., et a7., Gene 25:109-117 (1983).
  • p3SR2 is described in Tilburn, J., et a7., Gene 25:205-221 (1983); Kelly, J.M., et a7., EMBO Journal 4:475-479 (1985).
  • pDJB2 is described in Ballance, D.J., et al . , Gene 35:321-331 (1985).
  • pMC1871 is described in Casadaban, M.J., et al . , Methods Enzymol . 1005:293-308 (1983).
  • pTTOl and pTTll is described in Patent Application WO 85/04672.
  • pTZ18R and pTZ19R are available from Pharmacia.
  • pAN5-41B is described in Van Gorcom, R.F.M., et a7., Gene 45:211-217 (1986).
  • pAMH102 is described in Harkki, A., Uusitalo J., Bailey, M., Penttila, M. & Knowles, J.K.C, A novel fungal expression system: secretion of active calf chymosin from the filamentous fungus Trichoderma reesei . Bio/Technology 7(1989), 596-603.
  • pAMHlll is described in Harkki, A, Mantyla, A, Penttila, M., Muttilainen, S., Buhler, R., Suominen P., Knowles, J. & Nevalainen, H., Genetic engineering of Trichoderma to produce strains with novel cellulase profiles. Enzyme Microb. Technol . (1990, in press).
  • p3SR2 is described in Kelly, J.M. & Hynes, M.J., Transformation of Aspergillus niger by amdS gene of Aspergil lus niger EMBO Journal 4 (1985): 475-479; Tilburn, J., Schazzocchio, C, Taylor, G.T., Zabjckyzissman, J.H., Lockington, R.A & Davies, R.W., Transformation by integration in Aspergil lus nidulans. Gene 26 (1983), 205-221.
  • pML5 is described in Takkinen, K., Laukkanen, M-L., Sizmann, D., Alfthan, K. , Immonen, T., Vanne, L., Kaartinen, M., Knowles, J.K.C, and Teeri, T.T., An active single chain antibody containing a cellulase linker domain is secreted by Escherichia coli . Protein Engineering, submitted.
  • E. coli strains JM101, JM103, JM109 and DH1 (Yanish- Perron, C et a7., Gene 33:103-119 (1980)) and DH5 ⁇ (BRL) are used as hosts in E. coli cloning.
  • Trichoderma reesei strains QM9414 (ATCC 26921) and Trichoderma reesei strain RUT-C-30 (ATCC 56765) (Montenecourt, B.S. & Eveleigh, D.E., Selective screening methods for isolation of high yielding cellulase mutants of Trichoderma reesei . Adv. Chem. Ser. (1979), 289- 301) are used in fungal transformation and expression studies.
  • Trichoderma minimal medium Trichoderma minimal medium
  • mice which were immunized against 2- phenyloxazolone (Kaartinen, M., Griffiths, G.M., Markham, A.F. & Milstein, C, mRNA sequences define an unusually restricted IgG response to 2-phenyloxazolone and its early diversification. Nature 304 (1983), 320-324. Exampl e 1
  • Auxotrophic mutants requiring tryptophan or arginine for growth were isolated using filtration enrichment as described by Nevalainen (Nevalainen, H., Technical Research Center of Finland, Publication 26 (1985)).
  • the mutagenic agent used was UV-light.
  • mutants deficient in the argB gene were identified by using a series of minimal plates supplied by different intermediates in the arginine biosynthesis (Nevalainen, H., Technical Research Center of Finland, Publication 26 (1985)).
  • Mutants requiring citrulline for growth were considered as possible argB' mutants.
  • the argB ' character of isolated mutants was confirmed by transforming them into prototrophy using the plasmid pSa!43 containing the A. nidulans argB gene (example 4).
  • Tryptophan requiring mutants were characterized on a series of minimal medium plates supplied by anthranilic acid, indole, or tryptophan (Sanchez, F. et a7., Gene 51:97-102 (1987)). Colonies requiring indole for growth were taken as putative trpC mutants and their trpC character was confirmed by, for example, transformation with the pHYlOl plasmid of A. nidulans (Van Gorcom, R.F.M. et a7., Gene 40:99-106 (1985)).
  • Mycelium was grown on cellophane disks on Potato Dextrose
  • Agar plates (Difco). Mycelium from 5 cellophane cultures was suspended in 15 ml of a 5 mg/ml solution of Novozym ⁇ 234 in
  • the viability of the protoplasts was estimated on Trichoderma minimal medium with 1 M sorbitol as osmotic stabilizer.
  • the protoplasts were plated in 3% agar overlay.
  • the regeneration frequency was 40-80% for the strain QM 9414 (ATCC 26921).
  • Plasmid p3SR2 was used in transformation. It contained
  • A. nidulans DNA carrying the whole acetamidase structural gene amdS and its regulatory region amdl (Hynes, M.J., et al . ,
  • the selective medium was Trichoderma minimal medium with 10 mM acetamide as the sole nitrogen source instead of (NH 4 ) 2 S0 , and supplemented with 1 M sorbitol as osmotic stabilizer and 12.5 M CsCl to repress the background growth.
  • Transformation frequencies from 40 to 600 transformants per ⁇ g DNA were obtained for the strain QM 9414 (ATCC 26921). The highest frequencies were obtained when the DNA was purified with two cycles of CsCl/ethidium-bromide centrifuga- tion.
  • the mitotic stability of the transformants wasomme- gated.
  • Ten large transformants of varying size were subcul- tured on acetamide-CsCl plates, sporulated on potato dextrose (PD) and replated on PD.
  • PD potato dextrose
  • One positive clone from each original transfor ant was subjected to successive platings (5 growth cycles) on non-selective medium and phenotype was tested after each generation on the selective medium.
  • the presence of the plasmid DNA in the transformants was analyzed by Southern blots of total DNA, isolated from transformants (Raeder, U. et al . , Lett . Appl . Microbiol . 1 : 17- 20 (1985)), cut with Xhol or 5a7I and fcoRI.
  • the vector pUC18 and the 5a7I-fcoRI fragment containing the amdS gene of plasmid p3SR2 were used as probes.
  • the transformation was shown to have occurred by recombination at a number of different sites in the Trichoderma genome DNA, one to several copies per genome.
  • Chromosomal DNA was isolated from the transformants and used for Southern hybridization experiments to verify the proper integration of plasmid DNA into the chromosomal DNA of 7. reesei . Multiple tandem copies of pSall43 were integrated in the genome. Southern and dot blot hybridizations showed that in transformants analyzed the copy number of argB varies from appr. 2 to over 100. The ArgB + transformants were shown to be phenotypically 100% stable through at least 3 genera ⁇ tions. This was tested by successive platings of conidia from five transformants onto complete medium and thereafter testing the Arg + phenotype on minimal medium (50-80 colonies/transfor ant).
  • argB " 7. reesei strain was transformed with equal molar amounts of A. nidulans plasmids pSa!43 (argB) and p3SR2 (amdS) , transformants were selected for Arg + phenotype and tested for acquisition of amdS by streaking on acetamide- CsCl-plates. Of the ArgB transformants, 86% were also AmdS + . Southern analysis of cotransfor ants indicated that both plasmids were integrated as variable amounts of copies in several different locations in the genome. Double selection on minimal acetamide-CsCl-medium resulted in -100 big Amd + Arg + transformants per ⁇ g of DNA and a number of small colonies, characteristic of amdS transforma ⁇ tion.
  • the plasmid pAN5-4IB which contains the f. co7/ 7acZ gene coupled in phase to the promoter and N-terminal protein coding region of the A. nidulans glyceroT phosphate dehydrogenase gene (gpd) (Van Gorcom, R.F.M., et a7., Gene 45:211-217 (1988)) was also used for cotransformation.
  • this plasmid contains the A. nidulans argB gene and pBR322 sequences. Prototrophic 7.
  • reesei (QM 9414) was cotransformed with the plasmid p3SR2 (amdS) and pAN5-4IB, transformants were selected for Amd + phenotype and screened for J-gal expression on acetamide-CsCl-plates containing Xgal . No endogenous 7. reesei ⁇ -galactosidase activity could be detected when glucose was present in the medium and pH of the Xgal plates was neutral .
  • Trichoderma reesei strain QM 9414 (ATCC 26921) was shown to contain only one chromosomal copy of cbhl gene by Southern hybridization using cbhl specific probes and so one recombination event should inactivate the gene.
  • An inactive gene was constructed as follows.
  • the plasmid pTTOl (W0 85/04672) containing the full length cDNA clone of the cbhl gene in the vector pUC8 was cut with restriction enzymes Bgll and 5o7II.
  • the 0.8 kb fragment from the 5' terminal region of the cbhl gene was isolated from agarose gel by conventional techniques. The fragment was made blunt-ended using Sl nuclease and it was ligated to an EcoRI cut, blunt-ended pUCl ⁇ vector and transformed to E. coli JM109.
  • DNA from the clone containing the cbhl gene fragment was isolated and digested with restriction enzyme fcoRI which cuts in the middle of the Bgll-Bglll fragment of cbhl .
  • the fcoRI-generated termini were filled in and back-ligated.
  • a plasmid pMS4 containing a frameshift mutation in the middle of the truncated chbl gene fragment was generated (fig. 2).
  • Trichoderma was cotransformed with the plasmid pMS4 and the A. nidulans amdS containing plasmid p3SR2 with 3-4 times molar excess of plasmid pMS4.
  • Transformants were selected on the basis of the amdS + phenotype as described (example 3) and purified on selective medium containing acetamide as a sole carbon source. Purified transformants were grown on microtiter plates in 200 ⁇ l of Trichoderma minimal medium with 1% Solka floe cellulose as carbon source and 0.2% proteose peptone as nitrogen source. The cellulase phenotype was tested by the Ouchterlony immuno- diffusion by using undiluted growth media against the CBH I specific antiserum (sheep) as described (Nummi, M. et a7., Biochem. J. 215:677-683 (1983)). A number of transformants were identified which produced normal amount of CBH II but no detectable CBH I.
  • pUC9 was cut with 5a71 and filled out with Klenow polymerase and ligated with T4 ligase.
  • the obtained plasmid was cut with BamHl-EcoRl and the 2.7 kb large fragment was ligated with a 0.47 kb 5a/nHI-EcoRI fragment from pR26 to create pUC9' containing a HindlU site N-terminally of the prochymosin gene.
  • pUC13 was cut with BamWl-Narl and Narl-Xmal and the large respective small fragment was ligated with a 0.64 kb Xmal-Bcll fragment of pR26 to obtain plasmid pUC13' containing a Xbal-site C-terminally of the prochymosin gene.
  • a 0.65 kb X al-Xbal fragment of pUC13' was ligated with 0.46 kb Hindlll- X al fragment of pUC9' and a 11 kb Xbal-Hindlll fragment of p285 to create plasmid p285' proC containing the prochymosin gene as illustrated in fig.3.
  • pCAMG91 was digested with 5a7I and PstI restriction endonucleases. From such a digests a 698 bp fragment was isolated on an agarose gel. This Sall-Pstl fragment contains the region encoding the 140 bp 3' untranslated part of the glucoamylase mRNA plus 540 bp 3' to the poly(A)-addition site. This 3' fragment was treated with T4-DNA polymerase to "blunt end" the restriction sites before the addition of Xbal linkers and digestion with Xbal restriction enzyme. This 3' end of the glucoamylase gene was ligated to pUC13 linearized with Xbal to create plasmid pAMG/Term containing the glucoamylase gene poly(A) addition region.
  • a 0.5 kb 5/ ⁇ aII-5ssHII fragment of pCAMG91 containing the promoter and the sequences coding for the glucoamylase (AMG) signal and leader peptide was ligated to Smal-BamHl digested pUC13 and a synthetic SssHII-fiamHI adaptor encoding the first 6 amino acids of prochymosin.
  • pMT626 From the resulting plasmid, pMT626, a 0.8 kb ⁇ t ⁇ el- ⁇ amHI fragment was isolated and ligated to a 0.5 kb 5amHI-EcoRI fragment from p285' proC containing the sequence for the N-terminal half of proC and to a 3.0 kb EcoRl-Ndel fragment of pMT622 containing the C-terminal part of the sequence for proC. (pMT622 is simply an EcoRl-Xbal subclone of p285' proC in pUC13).
  • the resulting plasmid pMT648 see fig.
  • pMT648 contains the entire prochymosin gene preceded by AMG promoter and signal/leader encoding se- quences.
  • pMT648 was further modified to contain the argB gene of A. nidulans (John, M.A., et a7., Enzyme Microb. Technol . 5:386-389 (1984)).
  • a 1.8 kb ⁇ arl-filled in Xbal fragment of pMT648 was ligated to a 6.8 kb Clal filled-in EcoRI fragment of pSa!43, to give pMT651. Sequences further upstream of the AMG promoter (upstream activating sequences; UAS's) were furthermore inserted.
  • pMT830 The resulting plasmid, " pMT830, has got an expression unit containing the UAS's, promoter, signal and leader sequences from AMG and the entire gene for proC. Finally, the terminator sequence of AMG is taken as a 0.6 kb Xbal-Xbal fragment from plasmid pAMG/Term and inserted into the Xbal cut and dephosphorylated pMT830 to give pMT837.
  • the construction of pMT837 is illustrated in fig. 5.
  • Plasmid pMT837 contains the prochymosin gene preceded by the AMG promoter and signal/- leader sequence.
  • the construction of plasmid pMT837 was described in Example 7 (see also figs. 3-5). Cotransformation and selection was carried out as in Example 5.
  • transformants were cultured on minimal medium containing 1% Solka floe cellulose and 0.2% proteose peptone.
  • the mycelia were collected and the supernatant was concentrated, when necessary, by TCA precipitation and diluted with 2 M NaOH 10 mM Tris (pH 8.5).
  • the samples were frac- tioned on SDS-page (7.5%-15% polyacrylamide gradient) and electroblotted to a nitrocellulose filter.
  • the filters were incubated with rabbit prochymosin antiserum and stained with 4-chloro-l-naftol using ⁇ -rabbit-IgG-peroxidase conjugate purchased from Sigma. Chymosin was shown to be secreted into the medium approximately at the level of 1 ⁇ g/1. Since the AMG promoter clearly functions inefficiently in 7. reesei homologous 7. reesei promoter-terminator vectors were constructed to improve the production level of chymosin.
  • heterologous expression vectors for production of calf prochymosin in 7. reesei using the cbhl promoter
  • the calf prochymosin gene was obtained from plasmid pR27 (fig. 6).
  • the Pstl-Pstl fragment (-1110 bp) containing almost the whole gene was isolated from agarose gel by conventional techniques and ligated to the PstI site of pUC19.
  • This plasmid was partially digested with PstI, ends made blunt with Sl nuclease and an Avail terminator fragment (-750 bp) of cbhl gene (blunt ends with Klenow fragment) was ligated into this plasmid.
  • the terminator region of cbhl gene was obtained from the terminal 1.8 kb BamHI fragment of cbhl cDNA subcloned in pBR322 (Teeri, T., et a7., Bio/Technology 1:696- 699 (1983)).
  • pAMHIOO This plasmid containing the proC fragment coupled with the terminator region of cbhl gene in pUC19 was called pAMHIOO (fig. 7).
  • the SacII-PstI fragment (80 bp) coding for the N- terminal region of prochymosin was isolated from plasmid pR27 (see fig. 6).
  • the cbhl promoter region was first subcloned from the genomic clone X44A as a 2.8 kb long EcoRI-EcoRI fragment into pUC18 (plasmid pUAOl, fig. 8), and then a -2.2 kb long EcoRl-Bgl l fragment was isolated from this subclone.
  • the Bgl l site is located in the middle of the signal sequence of cbhl gene.
  • amdS gene of A. nidulans Hynes, M.J. et a7., Ao7. Ce77. 5/o7. 3:1430-1439 (1983)
  • argB gene of A. nidulans John, M.A. et a7., Enz. Microbiol. Technol. 5:386-389 (1984)
  • the amdS gene was isolated as a Pvul-Sall fragment from p3SR2 and ligated to the 6 kb long Pvul-Sall fragment of pAMH 102. This selectable vector was named pAMH104 (Fig. 11).
  • the aminoterminal PstI fragment (-150 bp) of prepro- chymosin was isolated from pR26 (fig. 3) which includes a complete preprochy osin cDNA clone, starting 12 bp upstream from ATG, inserted into PstI site in pBR322. This fragment was subcloned into the polylinker of pTZ19R together with the cbhl EcoRl-Sacl (-2.6 kb) promoter fragment which also includes the signal sequence coding region of cbhl (fig. 8).
  • pTZ19R is a pUC19 based plasmid including the FI origin of replication and the T7 promoter enabling the use of ss-te plate (single-stranded) for oligonucleotide mutagenesis.
  • the construction-of the resulting plasmid (pAMH 105) is illustrated in Figure 8.
  • oligonucleotide directed mutagenesis was illustrated in fig. 9.
  • the oligonucleotide in question was phosphorylated, annealed to ss pAMH 105 DNA (Marsh, J.L., et a7., Gene 32:481-485 (1984), ss-DNA was isolated from JM103/pAMH105 (as described by Pharmacia) in a 20:1 molar ratio, respectively.
  • the oligonucleotide primer was elongated using Klenow polymerase and T4 ligase as described in Eghtedarzadeh, M.K. et a7., Nucl . Acids Res. 14:5115 (1986).
  • the elongated mixture was digested with S al (resides in the polylinker of pTZ19R, see fig. 9) prior to transformation into the mutl mismatch repair deficient strain of E. co7/, BMH71.18 (Vieira, J., et al. , Gene 19:259-268 (1982)).
  • the pool of transformants was grown overnight in 5 ml liquid culture (Luria broth as described in Miller, J.H., in Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, New York, (1972), with 100 ⁇ g/ml ampicillin). Plasmid DNA was isolated from pool of transformants and it was redigested with Smal and retransformed into E. co7/ JM109 strain. The screening of potential deletion clones was performed by digestion using different restriction enzymes and the specificity of deletion was further confirmed by sequencing. The resulting plasmid was called pAMHllOl (fig. 12). This plasmid was further digested with EcoRI and PstI and the resulting pcbhl-preproC fragment was isolated (fig.
  • pAMHIOl contains the cbhl promoter area fused to the signal sequence of prochymosin and the whole prochymosin coding region fused to the terminator area of cbhl gene (fig. 14) .
  • plasmid pAMH1103 contains a signal sequence fusion including amino acids (aa) 1-12 from the cbhl signal sequence fused to aa 12-16 from the preproC signal sequence preceding the aminoter inal part (-140 bp) of the coding region of proC gene (fig. 12).
  • the resulting plasmid pAMH 103 includes the promoter area of cbhl and signal sequence fusion of cbhl and preproC preceding the proC coding region fused to the cbhl terminator area.
  • plasmid pAMH 1106 (fig. 13) was performed essentially as pAMH 1101 described in details in the preceding section IV with the exception that the specific oligonucleotide used for this construction , was 0AMH2 (fig. 10 and 9). From the resulting plasmid pAMH 1106 including the promoter area of cbhl , signal sequence of cbhl and the coding region of 1-20 first aa of mature CBH I fused to the coding region of proC the fragment containing pc ⁇ l-mature o-proC was subcloned into pAMHlOO essentially as described in the preceding section IV (Fig. 12). The resulting plasmid pAMHIO ⁇ includes the promoter area of cbhl , signal sequence of cbhl , coding region of aa 1-20 of mature CBHI fused to the coding region of proC.
  • reesei strains QM 9414 and RUT-C-30 were cotransformed with plasmids pAMH102 and p3SR2 in molar ratio 5:1, respec ⁇ tively.
  • the construction of plasmid pAMH102 was described in Example 9 section II. Cotransformation and selection was carried out as in Example 5. The transformants were purified on selective acetamide plates as described in Example 3.
  • chymosin production transformants were cultured on minimal medium (10 to 50 ml) containing 1 % Solka floe cellulose and 0.2 % proteose peptone.
  • the mycelia were collected and the supernatant was concentrated by TCA precipi- tation and diluted into 2M NaOH 10 mM Tris (pH 8.5).
  • the mycelia were broken in the presence of liquid nitrogen, broken cells were pelleted and the supernatant treated in the same way as the culture media.
  • the samples were fractioned on SDS-page (7.5 % - 15 % polyacrylamide gradient) and electro- blotted to a nitrocellulose filter.
  • the filters were incu ⁇ bated with rabbit prochymosin antiserum and ⁇ -rabbit-IgG-AP (alkaline phosphatase) conjugate and stained with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate purchased from Promega Biotec.
  • the amount of chymosin inside and outside the mycelium was compared (fig. 17). It was shown by Southern hybridization that the clones containing higher number of copies of plasmid pAMH102 integrated into the fungal chromosomal DNA also produced more chymosin. When using a one-copy transformant the amount of secreted chymosin was 100 ⁇ g/1 of culture medium when approximated from Western gels (fig. 17).
  • the secreted prochymosin was shown to be processed to an active chymosin by Western gels and by the ilk clotting activity (chymosin clots milk by cleavage of ⁇ - casein determin. :ion) (Bailey, M.J., et al . , Biotechnol . Lett. 10:161-166 (1988)).
  • the amount of various forms of chymosin (preproC, proC, C and chymosin derived degradation products inside the cell) inside the mycelium was determined to be 0.04 % of mycelial total protein (fig. 17) and the amount of secreted chymosin was 60 % of the total chymosin produced. This shows the efficiency of 7.
  • Transformants with several copies of plasmid pAMH104 (fig. 11), able to secrete larger amounts of prochymosin were screened for by determining the milk clotting activity of the growth media. The best transformant out of the 40 studied was found to secrete 500 ⁇ g/1 of culture medium as determined on Western gels and by milk clotting activity.
  • the PstI terminator fragment was isolated from pAMHUO', the cbhl promoter area was isolated from pAMH102 as an EcoRI-PstI fragment including the amino terminus of proC gene and these fragments were subcloned into an EcoRI-PstI digested pUC19* from which the single ⁇ t ⁇ el site had been made blunt with Klenow prior to this subcloning step.
  • the resulting plasmid pAMHUO includes the promoter and terminator of cbhl gene and between these sequences a stuffer fragment which can be removed by digestion with SacII and Ndel . After the ends are made blunt any cDNAs or chromosomal copies of genes can be inserted between the promoter and terminator (fig. 15).
  • Construction was made in four steps, resulting in vectors, pEN301, pEN302, pEN303, pEN304 and pEN305 (Fig. 22).
  • a chymosin expression cassette (- 4200 bp) was cut out from plasmid pAMH102 by Sad and partial Sp ⁇ I digestion.
  • Plasmid pTZ19R (2860 bp; Pharmacia) was linearized by PstI digestion. Ends of both fragments were made blunt by mung bean nuclease treatment. The blunt ended Sad - Sp ⁇ I-fragment was ligated to the blunt ended PstI opened vector. The resulting plasmid was termed pEN301.
  • Plasmid pEN301 was cut with PstI and 5c7I to give a 5.9 kb vector.
  • the light chain cDNA insert was cut from pLTl
  • Fig. 18 by Xhol - Sacl digestion (896 bp). Fragment ends were made blunt (mung bean nuclease) and ligated to form plasmid pEN302 (6.9 kb).
  • the other fragment (88 bp) was created by using one oligo (29 nt) from the junction area, with an Apal site and another oligo (18 nt) having a PstI site (within the light chain cDNA) .
  • the 83 bp fragment was digested with Kspl and Apal
  • the 88 bp fragment was digested with Apal and PstI.
  • the fragments were ligated at their -4pal sites to create a 132 bp fragment, which was then ligated to the Kspl-Pstl opened vector pEN302.
  • the new joint was checked by sequencing.
  • the plasmid is referred as pEN303.
  • Plasmid pAN8-l (Mattern, I.E., Punt, P.J., Unkles, S. Pouwels, P.H. and van den Hondel , C A. M. J. J., Transforma- tion of Aspergil lus oryzae. In: Abstracts of the 19th Lunteren lectures on Molecular Genetics of Yeasts and Fi lamentous Fungi and its Impact on Biotechnology, Lunteren, the Netherlands (1987), 34) was cut with Ecofll - Ndel to release a 3.5 kb phleomycin resistance cassette from the vector. Plasmid pEN303 was cut with EcoPI and the 6.8 kb fragment was iso ⁇ lated.
  • a gene coding the heavy chain (IgG3) was cut from plasmid pLT2 (Fig. 19) by Hindl l l and EcoPI, resulting a 870 bp insert, which was ligated to Hindl l l - EcoRI opened plasmid pTZ18R (2.8 kb; Pharmacia). The resulting 3.7 kb plasmid is referred as pEN201.
  • Plasmid pEN202 (5.8 kb) was build by cutting the chromosomal cbhl gene from pENlOl (Fig. 21) with Aval (2.1 kb).
  • Plasmid pENlOl is a subclone, where the chromosomal cbhl gene was removed from ⁇ 44A clone (Teeri, T., Salovuori I. & Knowles, J., The molecular cloning of the major cellulase gene from Trichoderma reesei BIO/TECHNOLOGY 1 : 696-699(1983)) to plasmid pTZ18R.
  • Lambda DNA was cut with Apal and pTZ18R opened by Bgl l - fragments were treated with mung bean nuclease before ligation).
  • Plasmid pEN201 was opened with Hindl l l (3.7 kb). Both fragments were blunt ended by mung bean nuclease before ligation.
  • the aim was to fuse a large part of the efficiently secreted CBHI protein (corresponding to amino acids 1 - 484) to the heavy chain in order to facilitate the secretion of the heavy chain from the Trichoderma host cell.
  • CBHI protein corresponding to amino acids 1 - 484
  • this CBHI fragment was meant to serve only as a secretion "carrier,” that is, a secretion signal, and its secretion-promoting functions are not needed in the final product, a Factor Xa recognition site was added between the two regions to allow the CBHI part to be easily proteolytically removed after secretion by methods known in the art.
  • An exact joint was made by deleting 265 bp.
  • a single stranded DNA preparation of pEN202 was made with help of phage: M13K07.
  • the plasmid was made double stranded with dNTPs, Klenow polymerase and ligase. Double stranded DNA mixture was digested with Sp ⁇ I (site in the loop; single site in the plasmid), to enrich the deleted plasmids. Digested DNA mixture was transformed to JM109, and the whole pool of transformants was grown in liquid medium. A DNA preparation was made from the pool, and the mixture was digested with Sp ⁇ I - again to enrich the correct plasmids. Digested mixture was transformed to JM109 Correct clones were identified by sequencing the joint area. The resulting plasmid is referred as pEN203 (5.5 kb).
  • Plasmid pEN204 (5.5 kb) was opened by Kpnl . Fragment ends were blunt ended by mung bean nuclease treatment before ligation.
  • the plasmid is referred as pEN205 (6.0 kb) .
  • the heavy chain cDNA in the construction thus far consists of V, Cl, hinge and part of C2 region.
  • the translation stop signal has to be placed after Cl region.
  • the primers used are depicted in Fig. 23 on plasmid pEN206 and are identified by their size in nucleo- tides.
  • a 890 bp long sequence was amplified by PCR by using a 30-mer hybridizing to the cbhl gene and having a 5stEII site in it and another 30-mer hybridizing to the end of heavy Cl and having a 5c7I site in it.
  • Another -240 bp fragment was made, where a 47-mer hybridizing to the cbhl terminator region and having a 5c7I site in it and one 30-mer hybridizing further down in the terminator and having a Nsil site in it.
  • the first amplified fragment was cut with BstEll and 5c7I, the other with 5c7I and Nsil .
  • the fragments were ligated at their 5c7I sites, between which the deleted sequence is located, and the formed -1100 bp fragment was ligated to ⁇ stEII - Nsil opened plasmid pEN205.
  • the PCR made region in the resulting plasmid was sequenced and the plasmid is referred as pEN206.
  • IgG3 heavy chain cDNA To make the exchange of the IgG3 heavy chain cDNA to the IgGl heavy chain cDNA, a 640 bp long IgGl heavy chain cDNA was cut out from plasmid pTI8 (Fig. 20) by Narl - Ndel digestion. Plasmid pEN206 was cut with Narl and 5c7I (5.2 kb). The fragments were ligated at their Narl sites. The fragment ends were made blunt (mung bean nuclease) and ligated to form a 5.8 kb plasmid pEN207.
  • the cbhl promoter was cut from plasmid pUJIO (Fig.
  • the amdS expression cassette was cut from plasmid p3SR2 (Cabilly, S., et a7., EP 125,023; Kaiser, C.A., et al . , Science 235:312-317 (1987)) with Sspl, resulting a 2.7 kb insert, which was ligated to the Sspl opened plasmid pEN208 (7.5 kb).
  • the resulting 10.2 kb plasmid was referred as pEN209.
  • the cbhl gene (1533 bp) was deleted from plasmid pEN207 by using PCR.
  • the primers are depicted in Fig. 25 on plasmid pAJ201 and were identified by their size in nucleotides.
  • An 83 bp fragment was amplified by using an oligo (19 nt) with a Kspl site that hybridized to the cbhl promoter region and another oligo (28 nt) with an Apal site in the junction between signal sequence and heavy chain gene.
  • the Apal site was created by changing the sequence CGTGCT to CGGGCC at the end of the cbhl signal sequence, so that the codons for Arg and Ala were retained.
  • the other fragment (943 bp) was created by using one oligo (29 nt) from the junction area, with an Apal site and another oligo (19 nt) having a Nsil site (within the cbhl terminator).
  • the 83 bp fragment was digested with Kspl and Apal , the other (943 bp) fragment was digested with Apal and Nsil .
  • the fragments were ligated at their Apal sites to create an approximately 1000 bp fragment, which was then ligated to Kspl - Nsil opened vector pUJIO (Fig. 26).
  • the new joint was checked by sequencing.
  • the plasmid is referred as pAJ201 (6.0 kb).
  • Plasmid paJ201 (6.0 kb) was opened by Sall-Xhol digestion.
  • the amdS expression cassette from p3SR2 was cut with Xbal- Sail (3.8 kb) . Fragments were ligated at their Sa71 sites. Ends of the 9.8 kb product were filled in with Klenow and the plasmid was circularized by ligation to result plasmid pAJ202 (9.8 kb).
  • Trichoderma reesei strain RUT-C-30 (ATCC 56765) was transformed with expression vectors for the light chain, pEN304 and pEN305 (both have phleomycin ⁇ marker). A cotransformation was also made, where plasmid pEN303 (no marker) and plasmid pAN8-l (containing the phleomycin resistance gene) were both transformed into RUT-C-30 at the same time. Transformation in Trichoderma may be made using a standard 7. reesei transformation method (Penttila, M., Nevalainen, H., Ratto, M., Salminen E.
  • Two different kinds of plate/overlay media were used. One was based on 7. reesei minimal medium, where KH 2 P0 4 , was substituted by K HP0 4 , and 0.44 M sucrose was added as osmotic stabilizer.
  • the other, rich medium (MnR) consisted of 0.3 g K-phtalate, 2.5 g yeast extract, 2.5 g glucose and 150 g sucrose/liter. In both media pH was adjusted to 7 to avoid breakage of phleomycin. In bottom medium 1.5% agar and in overlays 0.6% agarose was used as a solidifying agent.
  • the protoplasts were plated and incubated 2 - 3 h at room temperature, after which 5 ml of phleomycin (Cayla, France) containing overlay was added to the plates. Two phleomycin concentrations were used, 35 ⁇ g/ml or 50 ⁇ g/ml total plate medium. A fresh stock (30 mg/ml) of phleomycin was made in sterile water.
  • Transformation plates were incubated at +28°C for 5 - 7 days, after which the transformants were picked and grown on slants in MnR + 100 ⁇ g/ml phleomycin either for three cycles, one week each (pEN304 and pEN305), or just for one cycle/ one week (pEN303 - pAN81) to select for stable transformants.
  • PD slants Paneto Dextrose Agar
  • Spores were suspended to 5 ml 0.9 % NaCl per slant and transferred to 50 ml of liquid medium (7. reesei minimal medium supplemented with 2 % Solca Floe cellulose and 1 % spent grain, pH 5.0).
  • Transformants were grown in shake flasks for four days at +28 ⁇ C shaking in 160 rpm. The best producers were cultivated in 500 ml medium (inoculum from two slants) for five days and 250 rpm. Culture medium was separated from mycelia by either filtration or centrifugation (3000 g). From filtrations the mycelia were collected for Southern analysis.
  • Culture supernatant samples (100 ⁇ l) were added to microtiter wells, both undiluted and in 1:20 dilution in 0.5 % BSA in PBS.
  • the substrate used for color reaction was 2 mg di-Na-salt of p- nitrophenylphosphate (Orion company, Helsinki) per one ml of diethanolamine-MgCl 2 -buffer (Orion).
  • the wavelength used for absorbance measurement was 405 nm.
  • the amount of secreted light chain (pEN304/RUT-C-30) was 200 ⁇ g/1 of culture medium (500 ml cultivation), when approximated from ELISA test.
  • Light-chain producing 7. reesei strain was deposited as accession number CBS 252.90 on June 25, 1990 at the Centraalbureau voor Schimmelcultures, P.O. Box 273; Oosterstraat 1, 3740 AG Baarn, The Netherlands.
  • the filter was blocked with 0.5 % milk in TBS (20 M Tris, pH
  • the heavy chain construction pEN209 (amdS marker), is transformed to Trichoderma reesei strain RUT-C-30. Plasmids pEN208 (no marker) and p3SR2 are cotransformed to RUT-C-30. Transformants are selected by their ability to use acetamide as their sole nitrogen source.
  • Plate and overlay medium used in transformation is 7. reese minimal medium, in which 0.5 % ammonium sulfate is substituted by 12 mM acetamide. 15 mM CsCl is added to inhibit background growth.
  • the osmotic stabilizer used is 1 M sorbitol . From transformation plates, after 8-10 days from plating, transformants are removed to PD slants for sporulation.
  • Transformants are grown in shake cultivations and tested by ELISA test and western analysis as described in example 4.
  • the heavy chain construction pAJ202 is transformed to strain RUT-C-30, and selected for AmdS + phenotype.
  • Heavy chain constructions pEN209 and pAJ202, were transformed to the light chain expressing parent strain. Transformants were screened for AmdS + phenotype for their ability to use acetamide as their sole nitrogen source. 7. reesei transformants, pEN304/pEN209 and pEN304/pAJ202, were deposited as accession numbers CBS 287.91 (on June 3, 1991) and CBS 288.91 (on June 3, 1991), respectively, at the Centraalbureau voor Schimmelcultures P.O. Box 273, Oosterstraat 1, 3740 AG Baarn, The Netherlands.
  • Transformants were cultivated in 50 ml of liquid medium (7. reesei minimal medium supplemented with 3% whey and 1.5% spent grain, pH 5.0). Transformants were grown in shake flasks for four days at 28°C, 200 rpm.
  • ELISA was also used for measuring the i munoreactivity of the Fabs produced.
  • Microtiter plates were coated with the hapten i.e., phenyloxazolone conjugated to BSA (Makela, 0., Kaartinen, M., Pelkonen, J.L., and Karjalainen, K., Inheritance of Antibody Specificity V. Anti -2-Phenyl oxazol one in the Mouse, J. Exp. Med. 145:1644-1660 (1978)).
  • Ox-BSA was used in 50 ⁇ g/ml concentration in 0.1 M bicarbonate buffer, pH 9.6. Otherwise, the ELISA test was carried out as described in Example 16.
  • the yield of secreted Fab and CBHI-Fab of the best transformants was about 0.8 mg/1 and about 45 mg/1, respectively, when approximated for immunoreactivity from ELISA test.
  • the medium contained, in g/1: Whey 60; spent grain 30; (NH ) 2 S0 4 6; KH 2 P0 2 3. Initial pH was 4.8 without adjustment.
  • the inculum was grown on half-strength medium (buffered with 15 g/1 KH 2 P0 4 ) in two stages of 1 x 200 ml for two days and 5 x 200 ml for one day.
  • the fermenter used was a Chemap CF 2000 bioreactor, working volume 15 1 + 1 1 inoculum and with automatic control of temperature (33 ⁇ C for 48 h, subsequently 29 ⁇ C), pH (between 4.0 and 5.5 was controlled by NH 4 0H and H 3 P0 4 ) and p0 2 .
  • Fermentations were continued for four and five days for Fab and CBHI-Fab producing strains, respectively.
  • the growth medium and mycelia were separated by centrifugation and the growth medium was clarified by adding 50 g/1 silica, mixing and filtering through GF/B micro fiber filters (Whatman) before storing at -20°C
  • the supernatants were screened for antibody production by ELISA and western analysis. According to ELISA, the amount of produced, immunoreactive Fabs and CBHI-Fabs were 0.5 mg/1 (the fourth day) and 150 mg/1 (the fifth day, level was slightly better on the fourth day), respectively. The amount of total protein (Lowry) in Fab/RUT-C-30 fermentation was 9.3 g/1 and in CBHI-Fab/RUT-C-306.5 g/1.
  • Protein purification was made only from the fusion protein growth medium, because of the better antibody yield. Purification was performed in the three major steps. An anion exchange separation was used as a crude purification step. Affinity chromatography, in which hapten was used as a ligand, resulted in a pure antibody preparation. Gel filtration was used as a last step to separate autoproteolytically cleaved, Fab resembling antibody from the original fusion antibody. CBHI-Fab protein containing fermented growth medium was transferred to the starting buffer, 10 mM sodium phosphate buffer, pH 7.2, through Sephadex G-25 (coarse) for subsequent -82
  • CBHI-Fab preparation was contaminated by autoproteolytically cleaved, Fab resembling antibody.
  • the two antibody forms were separated by gel filtration using
  • Protein concentrations of the fusion and cleaved forms of antibodies were determined from the absorbance at 280 nm using calculated molar extinction coefficients at 280 nm based on the Trp and Tyr content of each protein sequence (167 760 M ⁇ * c ⁇ r* and 86 670m " l c " * for the fusion antibody and the cleaved antibody, respectively) (Wetlaufer, D.E., Adv. Prot. Chem. 17:303-390 (1962)). Sizes of the antibody chains were verified by SDS-PAGE electrophoresis and western analysis. Protein samples were tested against anti-IgGl (Southern Biotechnology Associates, Inc.) and anti-CBHI core (CH5, provided by C.P.
  • the size of the fusion heavy chain is about 86 kD, its cleaved counterpart is about 24 kD, the same as the size of the nonfused Fab heavy chain. Size of light chain is 23 kD.
  • Immunoreactivity of the fusion and cleaved antibodies were tested on the ELISA. The autoproteolytically cleaved antibody and the authentic proteolytically digested Fab derived from ascites were 27 times more active on binding oxazolone than the original fusion antibody.
  • the N-terminal amino acid sequence of the purified fusion and cleaved antibodies was determined on an Applied Biosystems 477A on line 120 A modified pulsed liquid phase/gas phase sequencer (Baumann, M., Comparative Gas Phase and Pulsed Liquid Phase Sequencing on a Modif ied Appl ied Biosystems 477 A Sequencer, Ana l . Biochem. 190: 198-208 (1990)).
  • the N-termini of the chains have been correctly processed.
  • the cleavage of the fusion has occurred two amino acid residues before the authentic N-terminus of the heavy chain.
  • the cleavage after tyrosine residue implies the existence of low chymotrypsin like protease activity in Trichoderma growth medium. Only about 0.02% of the fusion was cleaved immediately after fermentation (fifth day) according to the ELISA results from ion exchange fractions, in which cleaved antibody was eluted in the first fractions.
  • SCA single chain antibody
  • Construction of the functional gene was as described in Teeri, T., Takkinen, K., Laukkanen, M.-L., Alfthan, K., Sizmann, D., and Knowles, J.K.C, Recombinant secretable fusion proteins .
  • the structure of the functional gene is shown in Figure 29. The regulatory regions were rebuilt for Trichoderma expression.
  • the SCA encoding gene was amplified from plasmid pML5 (Fig.
  • a 5.4 kb fragment of pAJ202 was ligated with a 730 bp PCR fragment to create a plasmid pEN401 (6.2 kb).
  • SCA encoding gene is regulated by cbhl promoter and terminator regions in plasmid pEN401.
  • the SCA encoding gene was also cloned under a promoter expressed on glucose, i.e., Aspergillus gpd promoter.
  • the gpd promoter (1.2 kb) was amplified from plasmid pAN8-l.
  • a 39 mer oligo was used from the 5' end of the published gpd promoter (Punt, J.P., 3,manse, M.A., Kuyvenhoven, A., Soede, R.D.M., Pouwels, P.H., and van den Hondel, C.A.M.J.J., Functional elements in the promoter region of the Aspergillus nidul ans gpd A gene encoding glyceraldehyde-3- phosphate dehydrogenase, Gene 93:101-109 (1990)) with created EcoRI and Xbal sites in the extreme 5' end.
  • Transformants were grown in shake flasks in 50 ml 7. reesei minimal medium supplemented with 3% whey and 1.5% spent grain, pH 5.0. pEN402/RUT-C30 transformants were also cultivated in 50 ml 7. reesei minimal medium supplemented with 2% glucose and 0.5% peptone, pH 5.0.
  • Immunoreactive SCAs was tested from culture supernatants by ELISA using Ox-BSA coated microtiter wells as described in Example 19. Purified IgG fraction of rabbit anti-Ox IgG3 Fab antiserum was used as a first detection antibody (1:300). Second detection antibody layer was goat anti-rabbit IgG (H+L) alkaline phosphatase conjugate (Bio-Rad Laboratories).
  • the mRNAs of the light and heavy chain Ox IgG3 subclass antibody were isolated from hybridoma cell clone DHK-2, 6 (received from M. Kaartinen, Department of Bacteriology and Immunology, University of Helsinki, 00290 Helsinki 29, Finland) .
  • the first strand of the cDNA was synthesized using specific primers.
  • the light chain primer was complementary to the 3' noncoding region of the light chain mRNA containing a Xbal restriction site used in the cloning of the cDNA fragment.
  • the heavy chain primer was complementary to the region of constant two domain containing a Xbal restriction site.
  • the first strand cDNA synthesis was carried out as described by Maniatis et al . , J.
  • the mRNA of the IgGl subclass antibody was isolated from hybridoma cell clone NQ-17.4.1 (Kaartinen et a7., Nature 304 :320 (1983)).
  • the first strand of the heavy chain cDNA was synthesized using oligo(dT) primer (Maniatis et a7., 1982).
  • the cDNA:mRNA hybrid was directly subjected to PCR amplification.
  • the 5' end PCR-primer was identical with the sequence of the 5' end of the variable domain containing an EcoRI restriction site.
  • the 3' end PCR primer was complementary to the heavy chain constant two domain coding region containing a Hindlll restriction site.
  • the amplified IgGl heavy chain region cDNA was cloned as an EcoRI -Hindi I I restriction fragment into the pSP73 vector (Promega).
  • the heavy chain cDNA was modified with PCR before cloning it into the E. co7 expression vector.
  • the very 3' end of the coding region of the heavy constant one domain was modified by changing the three cysteine residues involved in disulfide linkages with the other heavy chain in an intact IgGl molecule ( Figure 20).
  • a ribosome binding site sequence for the light chain translation and a Ndel restriction site used in the frame cloning with the light chain coding region were introduced in the modified 3' sequence of the heavy chain ( Figure 20).
  • the modified heavy chain cDNA of the Ox IgGl subclass was cloned into E. co7/ expression vector by replacing the Narl-Ndel restriction fragment of the heavy chain coding region in the Ox IgG3 subclass Fab-fragment expression vector with that of the modified IgGl subclass cDNA clone.
  • the E. co7/ expression vector of the Ox IgGl subclass Fab-fragment was designated pTI8.

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Abstract

L'invention décrit des procédés permettant de produire des immunoglobulines recombinantes dans un organisme hôte de Trichodermas.
PCT/FI1991/000218 1990-07-16 1991-07-15 PRODUCTION D'IMMUNOGLOBULINES PAR LES $i(TRICHODERMAS) WO1992001797A1 (fr)

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JP91511365A JPH05508768A (ja) 1990-07-16 1991-07-15 トリコデルマによる免疫グロブリン製造
AU80938/91A AU657911B2 (en) 1990-07-16 1991-07-15 Immunoglobulin production by (Trichoderma)
FI925724A FI925724A (fi) 1990-07-16 1992-12-16 Producering av immunoglobuliner i trichoderma

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WO1994003612A1 (fr) * 1992-07-31 1994-02-17 Primalco Oy PRODUCTION D'ENZYMES DE DEGRADATION DU PHYTATE DANS LE $i(TRICHODERMA)
WO1994029457A2 (fr) * 1993-06-09 1994-12-22 Unilever N.V. Procede de production de proteines de fusion comprenant des fragments de scfv a l'aide d'un moule transforme
WO1998031821A2 (fr) * 1997-01-17 1998-07-23 Genencor International, Inc. Sequences d'adn, vecteurs et polypeptides de fusion pour la secretion de polypeptides dans des champignons filamenteux
US5834286A (en) * 1992-07-31 1998-11-10 Rohm Enzyme Finland Oy Recombinant cells that express phytate degrading enzymes in desired ratios
WO2005093073A1 (fr) * 2004-03-25 2005-10-06 Genencor International, Inc. Proteine de fusion de cellulase exo-endo
WO2005093050A2 (fr) * 2004-03-25 2005-10-06 Genencor International, Inc. Proteine de fusion cellulase et construction de fusion cellulase heterologue codant ladite proteine
US7816129B2 (en) 1994-07-29 2010-10-19 Ab Enzymes Gmbh Production and secretion of proteins of bacterial origin in filamentous fungi

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EP1545217B1 (fr) * 2002-09-10 2013-02-27 Genencor International, Inc. Induction de l'expression genique au moyen d'un melange de sucres a haute concentration
JP2012235767A (ja) * 2011-04-27 2012-12-06 Toyota Motor Corp 変異トリコデルマ属微生物及びこれを用いたタンパク質の製造方法

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EP0125023A1 (fr) * 1983-04-08 1984-11-14 Genentech, Inc. Préparations d'immunoglobuline recombinante, méthodes pour leur préparation, séquences d'ADN, vecteurs d'expression et cellules d'hôtes recombinantes
EP0215594A2 (fr) * 1985-08-29 1987-03-25 Genencor International, Inc. Polypeptides hétérologues exprimés dans les champignons filamenteux, leur procédé de préparation ainsi que vecteurs pour leur préparation
EP0234592A1 (fr) * 1986-02-28 1987-09-02 Teijin Limited Plasmide contenant un fragment d'ADN codant pour la région Fc de protéine immunoglobuline G humaine, et son utilisation pour la production de ladite protéine
EP0244234A2 (fr) * 1986-04-30 1987-11-04 Alko Group Ltd. Transformation de trichoderma
EP0314161A1 (fr) * 1987-10-28 1989-05-03 Bristol-Myers Squibb Company Immunoglobulines humaines produites par voie de génie génétique

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EP0125023A1 (fr) * 1983-04-08 1984-11-14 Genentech, Inc. Préparations d'immunoglobuline recombinante, méthodes pour leur préparation, séquences d'ADN, vecteurs d'expression et cellules d'hôtes recombinantes
EP0215594A2 (fr) * 1985-08-29 1987-03-25 Genencor International, Inc. Polypeptides hétérologues exprimés dans les champignons filamenteux, leur procédé de préparation ainsi que vecteurs pour leur préparation
EP0234592A1 (fr) * 1986-02-28 1987-09-02 Teijin Limited Plasmide contenant un fragment d'ADN codant pour la région Fc de protéine immunoglobuline G humaine, et son utilisation pour la production de ladite protéine
EP0244234A2 (fr) * 1986-04-30 1987-11-04 Alko Group Ltd. Transformation de trichoderma
EP0314161A1 (fr) * 1987-10-28 1989-05-03 Bristol-Myers Squibb Company Immunoglobulines humaines produites par voie de génie génétique

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Dialog Information Services, File 5: Biosis, Accession No. 6500289, Biosis Accession No. 85100810, PENTTILA M et al.: "A versatile transformation system for the cellulolytic filamentous fungus trichoderma-reesei", & Gene (AMST) 61 (2), 1987, 155-164. *

Cited By (20)

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Publication number Priority date Publication date Assignee Title
US5830733A (en) * 1986-04-30 1998-11-03 Rohm Enzyme Finland Oy Nucleic acid molecules encoding phytase and pH2.5 acid phosphatase
US5780292A (en) * 1987-04-29 1998-07-14 Alko Group Ltd. Production of phytate degrading enzymes in trichoderma
US5834286A (en) * 1992-07-31 1998-11-10 Rohm Enzyme Finland Oy Recombinant cells that express phytate degrading enzymes in desired ratios
WO1994003612A1 (fr) * 1992-07-31 1994-02-17 Primalco Oy PRODUCTION D'ENZYMES DE DEGRADATION DU PHYTATE DANS LE $i(TRICHODERMA)
WO1994029457A2 (fr) * 1993-06-09 1994-12-22 Unilever N.V. Procede de production de proteines de fusion comprenant des fragments de scfv a l'aide d'un moule transforme
WO1994029457A3 (fr) * 1993-06-09 1995-03-09 Unilever Plc Procede de production de proteines de fusion comprenant des fragments de scfv a l'aide d'un moule transforme
US7816129B2 (en) 1994-07-29 2010-10-19 Ab Enzymes Gmbh Production and secretion of proteins of bacterial origin in filamentous fungi
WO1998031821A2 (fr) * 1997-01-17 1998-07-23 Genencor International, Inc. Sequences d'adn, vecteurs et polypeptides de fusion pour la secretion de polypeptides dans des champignons filamenteux
WO1998031821A3 (fr) * 1997-01-17 1998-11-05 Genencor Int Sequences d'adn, vecteurs et polypeptides de fusion pour la secretion de polypeptides dans des champignons filamenteux
US6265204B1 (en) 1997-01-17 2001-07-24 Genencor International, Inc. DNA sequences, vectors, and fusion polypeptides for secretion of polypeptides in filamentous fungi
US6590078B2 (en) 1997-01-17 2003-07-08 Genencor International, Inc. DNA sequences, vectors, and fusion polypeptides for secretion of polypeptides in filamentous fungi
WO2005093073A1 (fr) * 2004-03-25 2005-10-06 Genencor International, Inc. Proteine de fusion de cellulase exo-endo
WO2005093050A3 (fr) * 2004-03-25 2006-08-24 Genencor Int Proteine de fusion cellulase et construction de fusion cellulase heterologue codant ladite proteine
JP2007530054A (ja) * 2004-03-25 2007-11-01 ジェネンコー・インターナショナル・インク セルラーゼ融合タンパク質及びこれをコードする異種セルラーゼ融合構築体
JP2007530050A (ja) * 2004-03-25 2007-11-01 ジェネンコー・インターナショナル・インク エキソ−エンドセルラーゼ融合構築体
EP2034024A1 (fr) * 2004-03-25 2009-03-11 Genencor International, Inc. Protéine de fusion de cellulase exo-endo
WO2005093050A2 (fr) * 2004-03-25 2005-10-06 Genencor International, Inc. Proteine de fusion cellulase et construction de fusion cellulase heterologue codant ladite proteine
EP2354222A1 (fr) * 2004-03-25 2011-08-10 Genencor International, Inc. Protéine de fusion de cellulase et construction de fusion de cellulose hétérologue la codant
CN1934258B (zh) * 2004-03-25 2011-12-14 金克克国际有限公司 外切-内切纤维素酶融合蛋白
US8097445B2 (en) 2004-03-25 2012-01-17 Danisco Us Inc. Exo-endo cellulase fusion protein

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