# Patent

## Title
KEX2 cleavage regions of recombinant fusion proteins

## Abstract

The invention relates to a fusion DNA construct comprising a KEX2 region comprising a KEX2 site and a KEX2 site pre-sequence immediately 5′ to the KEX2 site, a fusion polypeptide, vectors and cells comprising the fusion DNA construct, methods for producing desired proteins from filamentous fungal cells and methods for enhancing the secretion and/or cleavage of a desired protein from a cell.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 12/373,121, filed Aug. 31, 2009, now issued as U.S. Pat. No. 8,198,046, issued on Jun. 12, 2012, which is a National Stage entry of PCT/US07/14476, filed Jun. 21, 2007, and is a continuation-in-part of U.S. Ser. No. 11/484,814, filed Jul. 11, 2006 now abandoned, which is hereby incorporated by reference.


STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
Portions of this work were funded by Contract No. W911NF-05-C-0072 by the Defense Advanced Research Projects Agency (DARPA) of the U.S. Accordingly, the United States Government may have certain rights in this invention.


SEQUENCE LISTING
The sequence listing submitted via EFS, in compliance with 37 C.F.R. §1.52(e), is incorporated herein by reference. The sequence listing text file submitted via EFS contains the file “GC908US_CorrectedSequenceListing.text”, created on Aug. 30, 2012, which is 42,267 bytes in size.
FIELD OF THE INVENTION
The present invention relates to increased secretion and cleavage of desired proteins, such as functional antibody proteins and industrial enzymes from filamentous fungi. The invention discloses fusion DNA constructs, vectors and fusion polypeptides incorporating KEX2 regions for protein cleavage and methods of producing desired proteins.
BACKGROUND
During protein secretion in a fungal cell, certain proteins are cleaved by KEX2, a member of the KEX2 or “kexin” family of serine peptidase (EC 3.4.21.61). KEX2 is a highly specific calcium-dependent endopeptidase that cleaves the peptide bond that is immediately C-terminal to a pair of basic amino acids (i.e., the “KEX2 site”) in a protein substrate during secretion of that protein. KEX2 proteins generally contain a cysteine residue near the histidine residue of its active site and are inhibited by p-mercuribenzoate. The founding member of this group, the KEX2 peptidase of S. cerevisiae (Fuller et al., 1989, Proc. Natl. Acad. Sci. USA 86:1434-1438), cleaves the α-factor pheromone and killer toxin precursors during their secretion.
Production of fusion polypeptides has been reported in a number of organisms including E. coli, yeast and filamentous fungi. For example, bovine chymosin has been produced in Aspergillus niger as a fusion to full length glucoamylase (GAI) (Ward et al., (1990) Bio/technology 8:435-440; U.S. Pat. No. 6,265,204 and U.S. Pat. No. 6,590,078); human interleukin 6 (hIL6) has been produced in Aspergillus nidulans as a fusion to full-length A. niger glucoamylase (GAI) (Contreras et al., (1991) Biotechnology 9:378-381); hen egg white lysozyme (Jeenes et al., (1993) FEMS Microbiol. Lett. 107:267-273) and human lactoferrin (Ward et al., (1995) Bio/Technology 13:498-503) have been produced in Aspergillus niger as a fusion to residues 1-498 of glucoamylase; and bovine chymosin has been produced in Aspergillus niger as a fusion with full length native alpha amylase (Korman et al., (1990) Curr. Genet. 17: 203-212) and in Aspergillus oryzae as a fusion with truncated forms of A. oryzae glucoamylase (Tsuchiya et al., (1994) Biosci. Biotech. Biochem. 58: 895-899). Reference is also made to Shoemaker et al., 1981 Bio/Technology 1: 691-696; Nunberg et al., (1984) Mol. Cell. Biol. 4:2306-2315 and Boel et al., (1984) EMBO J. 3:1097-1102. In some of these fusion proteins, a KEX2 protease recognition site (Lys-Arg) has been inserted between a glucoamylase and a desired protein (e.g., Contreras et al., 1991 and Ward et al., 1995). The inventors of the present invention have found that protein secretion and/or protein cleavage may be enhanced in a fusion protein when the KEX2 recognition site has been manipulated to include an amino acid KEX2 site pre-sequence.
Specific literature of interest includes: Ward et al., (2004) Appl. Environ. Microbiol. 70:2567-2576; Goller et al., (1998) Appl. Environ. Microbiol. 64:3202-3208; La Grange et al., (1996) Appl. Environ. Microbiol. 62:1036-1044; Bergquist et al., (2002) Biochem. Biotechnol. 100:165-176; Spencer et al., (1998) Eur. J. Biochem. 258:107-112; Jalving et al., (2000) Appl. Environ. Microbiol. 66:363-368); Brenner and Fuller (1992) Proc. Natl. Acad. Sci. 89:922-926; Durand et al., (1999) Appl. Microbiol. Biotechnol. 52: 208-214; Ahn et al., (2004) Appl. Microbiol. Biotechnol. 64:833-839; Gouka et al., (1997) Appl Microbiol Biotechnol. 47:1-11 Broekhuijsen et al., (1993) J. Biotechnol. 31:135-145; MacKenzie et al., (1998) J. Biotechnol. 63:137-146 and published patent applications 20040018573 and 20050158825. Also U.S. Pat. No. 4,816,567 and U.S. Pat. No. 6,331,415 disclose processes for producing immunoglobulin molecules in recombinant host cells. The above cited literature is incorporated by reference herein for all purposes.
While numerous methods are available for the production of industrial enzymes and therapeutic proteins, there remains a need for alternative methods of protein production and particularly for therapeutic protein production, such as antibody production, which will result in relatively quick scale up time and high levels of produced protein with limited risk of contamination by viral or other adventitious agents. The present invention answers this need.
SUMMARY OF THE INVENTION
A fusion DNA construct, vectors, a fusion polypeptide, a cell comprising the fusion DNA construct, and methods for enhancing the secretion and/or cleavage of a desired protein from a cell are provided. More specifically, a KEX2 region encompassed by the invention has been included in a fusion polypeptide to provide for cleavage of a desired protein from the fusion polypeptide. Accordingly, the invention pertains to a KEX2 region for protein cleavage.
In some embodiments, the invention relates to a fusion DNA construct encoding a fusion polypeptide, comprising in operable linkage from the 5′ end of said construct, a promoter; a first DNA molecule encoding a signal sequence; a second DNA molecule encoding a carrier protein; a third DNA molecule encoding a KEX2 region, said KEX2 region comprising a KEX2 site and a KEX2 site pre-sequence immediately 5′ to the KEX2 site; and a fourth DNA molecule encoding a desired protein. In some aspects of this embodiment, the invention relates to a vector, such as an expression vector, which comprises the fusion DNA construct, and in other aspects the invention relates to host cells transformed with the vector or comprising the fusion DNA construct.
In other embodiments, the invention relates to a fusion polypeptide comprising from an amino terminus of said fusion polypeptide a first amino acid sequence comprising a signal sequence functional as a secretory sequence; a second amino acid sequence comprising a carrier protein; a third amino acid sequence comprising a KEX2 region, said KEX2 region comprising a KEX2 site and a KEX2 site pre-sequence immediately N-terminal to said KEX2 site; and a fourth amino acid sequence comprising a desired protein.
In further embodiments, the invention relates to a KEX2 region (X4X3X2X1B1B2) comprising a KEX2 site (B1B2) and a KEX2 site pre-sequence (X4X3X2X1) immediately N-terminal to said KEX2 site.
In yet other embodiments, the invention relates to a process of producing a desired protein in a filamentous fungal host cell and particularly in a Trichoderma cell, comprising obtaining a filamentous fungal host cell comprising a fusion DNA construct according to the invention and culturing the filamentous fungal host cell under suitable conditions which allow the expression and secretion of the desired protein. In some aspects of this embodiment, the desired protein will be recovered. In other aspects of this embodiment, the cleavage of the desired protein from the fusion polypeptide will be greater than the cleavage of the same desired protein from an equivalent fusion polypeptide that lacks the KEX2 site pre-sequence. In other aspects of this embodiment, the secretion of the desired protein from the fusion polypeptide will be greater than the secretion of the same desired protein from an equivalent fusion polypeptide, which lacks the KEX2 site pre-sequence.
In an additional embodiment, the invention relates to a method for identifying enhanced secretion and/or cleavage of a desired protein comprising a) altering a KEX2 site pre-sequence of a parental fusion polypeptide, said parental fusion polypeptide comprising a signal sequence; a KEX2 region comprising a KEX2 site and the KEX2 site pre-sequence which is located immediately N-terminal to said KEX2 site, and an amino acid sequence comprising a desired protein to produce a set of test fusion polypeptides that are identical to said parental fusion polypeptide except for said KEX2 site pre-sequence; b) evaluating secretion of said test fusion polypeptides and said parental fusion polypeptide by a filamentous fungal cell; c) identifying a test fusion polypeptide that has enhanced secretion and/or cleavage as compared to said parental fusion polypeptide.
In further aspects of this embodiment, the invention relates to a method of identifying an optimized KEX2 site pre-sequence which comprises, testing a plurality of different test fusion polypeptides obtained as described above; and determining which of said different test fusion polypeptides has greater secretion and/or protein cleavage, wherein said optimized KEX2 site pre-sequence is the altered KEX2 site pre-sequence of the test fusion polypeptide that has the greatest secretion and/or protein cleavage.



BRIEF DESCRIPTION OF THE FIGURES
Certain aspects of the following detailed description are best understood when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures:
FIG. 1 schematically illustrates an embodiment of a fusion polypeptide according to the invention, including a carrier protein, a KEX2 region and a desired protein, wherein the carrier protein is illustrated as a cellobiohydrolase I (CBH1) core/linker, which comprises the catalytic domain and part of the linker region of the CBH1 protein and the desired protein is illustrated as an antibody light chain or heavy chain.
FIG. 2 depicts a map of the pTrex4-her2 light chain DNA2.0 plasmid used for the expression of a fusion polypeptide. The plasmid includes a Trichoderma reesei cbh1 promoter; a polynucleotide encoding a CBH1 signal sequence and carrier protein; a KEX2 region inserted immediately after the SpeI site, a polynucleotide encoding the desired protein illustrated as an antibody (trastuzumab) light chain; a Trichoderma reesei cellobiohydrolase (cbh1) terminator; an amdS Aspergillus nidulans acetamidase marker.
FIG. 3A-E provide the nucleotide sequence (SEQ ID NO: 103) (10885 bp) of the pTrex4-her2 light chain DNA2.0 plasmid of FIG. 2.
FIG. 4 shows a Western blot of supernatants of cultured Trichoderma reesei cells comprising KEX2 region sequences as further described in examples 1, 2, 3 and 4. Lane 1 represents a molecular weight marker (See Blue Plus 2, Invitrogen). Lanes 2 and 3 represent a GGGKR variant (SEQ ID NO: 5); lane 4 represents a GGGKRGGG variant (SEQ ID NO: 7); lane 5 represents a VAVEKR variant (SEQ ID NO: 9) KEX2 region encompassed by the invention; and lanes 6 and 7 represent a KRGGG variant (SEQ ID NO: 2).
FIG. 5 shows a Western blot of supernatants of cultured Trichoderma reesei cells comprising KEX2 regions encompassed by the invention as further described in example 5. Lane 1 represents a molecular weight marker as described above. Lanes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 correspondingly represent VAVEKR (SEQ ID NO: 9), VAVWKR (SEQ ID NO: 25), VAVGKR (SEQ ID NO: 26), VAVRKR (SEQ ID NO: 27), VAVTKR (SEQ ID NO: 28), VAVVKR (SEQ ID NO: 29), VAVAKR (SEQ ID NO: 30), VAVLKR (SEQ ID NO: 31), VAVDKR (SEQ ID NO: 32), VAVNKR (SEQ ID NO: 33), VAVYKR (SEQ ID NO: 34), VAVHKR (SEQ ID NO: 35) KEX2 regions.
FIG. 6 shows a Western blot of supernatants of cultured Trichoderma reesei cells containing KEX2 region sequences as further described in examples 5, 6, and 7. Lanes 1 and 10 represent a molecular weight marker, as described above. Lanes 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15 and 16 correspondingly represent AAVEKR (SEQ ID NO: 38), GAVEKR (SEQ ID NO: 37), MAVEKR (SEQ ID NO: 36), LAVEKR (SEQ ID NO: 39), WAVEKR (SEQ ID NO: 40), KAVEKR (SEQ ID NO: 41), PAVEKR (SEQ ID NO: 42), DAVEKR (SEQ ID NO: 51), VAVEKR (SEQ ID NO: 9), HAVEKR (SEQ ID NO: 52), QAVEKR (SEQ ID NO: 47), SAVEKR (SEQ ID NO: 46), NVISKR (SEQ ID NO: 22), and SDVTKR (SEQ ID NO: 24) KEX2 regions.
FIG. 7 shows a Western blot of supernatants of cultured Trichoderma reesei cells containing KEX2 region sequences as further described in example 5. Lane 1 represents a molecular weight marker, as described above. Lanes 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 correspondingly represent VAVEKR (SEQ ID NO: 9), VGVEKR (SEQ ID NO: 56), VTVEKR (SEQ ID NO: 65), VEVEKR (SEQ ID NO: 55), VPVEKR (SEQ ID NO: 62), VWVEKR (SEQ ID NO: 67), VKVEKR (SEQ ID NO: 58), VRVEKR (SEQ ID NO: 63), VVVEKR (SEQ ID NO: 66), and VIVEKR (SEQ ID NO: 57) KEX2 regions.
FIG. 8 shows a Western blot of supernatants of cultured Trichoderma reesei cells containing KEX2 region sequences as further described in example 5. Lanes 1-11 correspondingly represent VADEKR (SEQ ID NO: 70), VAAEKR (SEQ ID NO: 69), VAFEKR (SEQ ID NO: 72), VAGEKR (SEQ ID NO: 73), VAIEKR (SEQ ID NO: 74), VANEKR (SEQ ID NO: 76), VALEKR (SEQ ID NO: 75), VASEKR (SEQ ID NO: 79), VAREKR (SEQ ID NO: 78) and VAPEKR (SEQ ID NO: 83) KEX2 regions.
FIG. 9 shows an SDS-PAGE gel of supernatants of cultured A. niger cells containing a VAVEKR (SEQ ID NO: 9) KEX2 region as further described in example 8. Lane 1 represents a molecular weight marker, Marker 12 MW standard (Invitrogen). Lanes 2, 3, and 4 represent 3 transformants and correspond respectively to transformants A 10, A 11 and A 12.



DEFINITIONS
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with the general meaning of many of the terms used herein. Still, certain terms are defined below for the sake of clarity and ease of reference.
The term “recombinant” when used in reference to a cell, nucleic acid, protein or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express nucleic acids or polypeptides that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed, over expressed or not expressed at all.
A “gene” refers to a DNA segment that is involved in producing a polypeptide and includes regions preceding and following the coding regions, e.g., the promoter and terminator, as well as intervening sequences (introns) between individual coding segments (exons).
The term “nucleic acid” encompasses DNA, RNA, single stranded or double stranded and chemical modifications thereof. The terms “nucleic acid” and “polynucleotide” may be used interchangeably herein. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present invention encompasses polynucleotides, which encode a particular amino acid sequence.
The term “DNA construct” means a DNA sequence which is operably linked to a suitable control sequence capable of effecting expression of a protein in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.
The term “fusion DNA construct” or “fusion nucleic acid” refers to a nucleic acid which comprises from 5′ to 3′ a number of polynucleotide sequences (e.g. a DNA molecule encoding a signal sequence, a DNA molecule encoding a carrier protein, a DNA molecule coding for a KEX2 site and a DNA molecule encoding a desired protein) operably linked together and which encode a fusion polypeptide.
A “vector” refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.
An “expression vector” refers to a vector that has the ability to incorporate and express heterologous DNA fragment in a foreign cell. Many prokaryotic and eukaryotic expression vectors are commercially available.
A “promoter” is a regulatory sequence that is involved in binding RNA polymerase to initiate transcription of a gene.
The term “signal sequence” refers to a sequence of amino acids at the amino terminus of a protein that directs the protein to the secretion system for secretion from a cell. The signal sequence is cleaved from the protein prior to secretion of the protein. In certain cases, a signal sequence may be referred to as a “signal peptide” or “leader peptide”. The definition of a signal sequence is a functional one. The mature form of the extracellular protein lacks the signal sequence which is cleaved off during the secretion process.
“Under transcriptional control” is a term well understood in the art that indicates that transcription of a polynucleotide sequence, usually a DNA sequence, depends on its being operably linked to an element which contributes to the initiation of, or promotes transcription.
“Under translational control” is a term well understood in the art that indicates a regulatory process which occurs after mRNA has been formed.
The term “operably linked” refers to juxtaposition wherein the elements are in an arrangement allowing them to be functionally related. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence.
The term “selective marker” refers to a protein capable of expression in a host that allows for ease of selection of those hosts containing an introduced nucleic acid or vector. Examples of selectable markers include but are not limited to antimicrobials (e.g., hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage, such as a nutritional advantage on the host cell.
The terms “protein” and “polypeptide” are used interchangeably herein. The conventional one-letter or three-letter code for amino acid residues is used herein.
The term “carrier protein” refers to a polypeptide sequence or functional portion thereof from a naturally secreted fungal polypeptide.
The term “antibody protein” used interchangeably with immunoglobulins (Igs), refers to a protein containing one or more polypeptides that specifically binds an antigen. Included by this term are antibodies of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, Fd, Fab′, Fv, F(ab′)2 antibodies, antibody fragments that retain specific binding to antigen, monoclonal antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies, bi-functional (i.e. bi-specific) hybrid antibodies and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein.
The monomeric form of an antibody comprises four polypeptide chains of two different types, one heavy and one light. Different types of heavy and light chains are recognized. The light chains are structurally divided into two domains, a variable region (VL) and a constant region (CL). The heavy chain is also divided into distinct structural domains. For example, a γ heavy chain comprises, from the amino terminus, a variable region (VH), a constant region (CH1), a hinge region, a second constant region (CH2) and a third constant region (CH3).
The term “equivalent fusion polypeptide” refers to a fusion polypeptide which has an identical amino acid sequence compared to a reference fusion polypeptide, except for a KEX2 site pre-sequence. A first fusion polypeptide having a first KEX2 site pre-sequence is equivalent to a second fusion polypeptide having a different KEX2 site pre-sequence if the polypeptides have identical amino acid sequences, except for the difference in the KEX2 site pre-sequence.
The term “derived” encompasses the terms “originated from”, “obtained” or “obtainable from”, and “isolated from”.
“Host strain” or “host cell” means a suitable host for an expression vector or DNA construct comprising a polynucleotide encoding a polypeptide and particularly a recombinant fusion polypeptide encompassed by the invention. In specific embodiments, the host strains may be a filamentous fungal cell. The term “host cell” includes both cells and protoplasts.
The term “filamentous fungi” refers to all filamentous forms of the subdivision Eumycotina (See, Alexopoulos, C. J. (1962), INTRODUCTORY MYCOLOGY, Wiley, New York). These fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, glucans, and other complex polysaccharides. The filamentous fungi of the present invention are morphologically, physiologically, and genetically distinct from yeasts. Vegetative growth by filamentous fungi is by hyphal elongation and carbon catabolism is obligatory aerobic.
The term “culturing” refers to growing a population of microbial cells under suitable conditions in a liquid or solid medium.
The term “heterologous” with reference to a polynucleotide or polypeptide refers to a polynucleotide or polypeptide that does not naturally occur in a host cell. In some embodiments, the protein is a commercially important industrial protein and in some embodiments, the heterologous protein is a therapeutic protein. It is intended that the term encompass proteins that are encoded by naturally occurring genes, mutated genes, and/or synthetic genes.
The term “homologous” with reference to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in the host cell.
The terms “recovered”, “isolated”, and “separated” as used herein refer to a protein, cell, nucleic acid or amino acid that is removed from at least one component with which it is naturally associated.
As used herein, the terms “transformed”, “stably transformed” and “transgenic” used in reference to a cell means the cell has a non-native (e.g., heterologous) nucleic acid sequence or additional copy of a native (e.g., homologous) nucleic acid sequence integrated into its genome or has an episomal plasmid that is maintained through multiple generations.
As used herein, the term “expression” refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation.
The term “glycosylated” protein means a protein that has oligosaccharide molecules added to particular amino acid residue on the protein.
The term “non-glycosylated” protein is a protein that does not have oligosaccharide molecules attached to the protein.
The term “introduced” in the context of inserting a nucleic acid sequence into a cell, means “transfection”, or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell wherein the nucleic acid sequence may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
The term “KEX2” refers to a calcium-dependent endopeptidase having an activity defined as EC 3.4.21.61, according to IUBMB Enzyme Nomenclature. KEX2 cleaves a peptide bond (the KEX2 cleavage site) that is immediately C-terminal to a pair of basic amino acids during protein secretion.
The term “KEX2 region” refers to a contiguous eight to four amino acid residue region which is located between the C-terminus end of a carrier protein and the N-terminal end of a desired protein in a fusion polypeptide. The KEX2 region is comprised of a KEX2 site and a KEX2 site pre-sequence.
The term “KEX2 site” refers to a two amino acid KEX2 cleavage motif in a protein. A KEX2 site contains two contiguous basic amino acids (e.g., lysine, histidine and/or arginine) in any order, (e.g., KK, RR, KR or RK).
The term “KEX2 site pre-sequence” refers to the two to six contiguous amino acids [(X)n where n is 2 to 6] immediately preceding (i.e., immediately N-terminal to) the KEX2 site. For example, if a KEX2 region is defined as VAVEKR, the “KR” motif is the KEX2 site of the region; n is 4 and the “VAVE” motif corresponds to the KEX2 site pre-sequence of the region.
The term “variant” refers to a region of a protein that contains one or more different amino acids as compared to a reference protein.
The term “secreted protein” refers to a region of a polypeptide that is released from a cell during protein secretion. In some embodiments, the secreted protein is the protein that is released or cleaved from a recombinant fusion polypeptide of the invention.
The term “secretion” refers to the selective movement of a protein across a membrane in a host cell to the extracellular space and surrounding media.
The terms “determining”, “measuring”, “evaluating”, “assessing” and “assaying” are used interchangeably herein to refer to any form of measurement, and include determining if an element is present or not. These terms include both quantitative and/or qualitative determinations. Assessing may be relative or absolute.
“Assessing the presence of” includes determining the amount of something present, as well as determining whether it is present or absent.
Other definitions of terms may appear throughout the specification.
DETAILED DESCRIPTION
Before the exemplary embodiments are described in more detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a gene” includes a plurality of such candidate agents and reference to “the cell” includes reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.
Fusion Polypeptides—
As noted above, the subject fusion polypeptide comprises: a) a signal sequence; b) a carrier protein; c) a KEX2 region comprising: i) a KEX2 site and ii) a KEX2 site pre-sequence immediately N-terminal to the KEX2 site; and d) a desired protein.
FIG. 1 illustrates a subject fusion polypeptide of the invention. The various parts of a subject polypeptide (i.e., “signal sequence”, carrier protein, “KEX2 region” and “desired protein”) are so labeled solely for clarity and convenience. It is recognized that the subject fusion polypeptide may also be referred to as a “pro-protein” or “precursor protein” because it generally contains an N-terminal region that is cleaved off during secretion and a C-terminal region that is secreted.
Signal Sequences and Carrier Proteins—
The signal sequence of a subject fusion polypeptide may be any signal sequence that facilitates protein secretion from a filamentous fungal cell. In particular embodiments, the subject fusion polypeptide may comprise a signal sequence for a protein that is known to be highly secreted from the filamentous cell in which the fusion protein is to be produced. The signal sequence employed may be endogenous or non-endogenous to the cell in which the fusion polypeptide is to be produced. In particular embodiments, the signal sequence may comprise a “carrier” that contains the signal sequence at its N-terminus, where the carrier is at least an N-terminal portion of a protein that is endogenous to the cell and efficiently secreted by a cell.
Suitable signal sequences and carriers are known in the art (see, e.g., Ward et al, Bio/Technology 1990 8:435-440; and Paloheimo et al, Applied and Environmental Microbiology 2003 69: 7073-7082). Examples of suitable signal sequences and carrier proteins include those of cellobiohydrolase I, cellobiohydrolase II, endoglucanases I, II and III, α-amylase, aspartyl proteases, glucoamylase, phytase, mannanase, α and β glucosidases, bovine chymosin, human interferon and human tissue plasminogen activator and synthetic consensus eukaryotic signal sequences such as those described by Gwynne et al., (1987) Bio/Technology 5:713-719.
In some embodiments, if Trichoderma (e.g. T. reesei) is employed as a host cell, the signal sequence or carrier of T. reesei mannanase I (Man5A, or MANI), T. reesei cellobiohydrolase II (Cel6A or CBHII), endoglucanase I (Cel7b or EGI), endoglucanase II (Cel5a or EGII), endoglucanase III (Cel12A or EGIII), xylanases I or II (XynIIa or XynIIb) or T. reesei cellobiohydrolase I (Cel7a or CBHI) may be employed in the fusion polypeptide.
In other embodiments, if an Aspergillus (e.g. A. niger) is employed as a host cell, the signal sequence or carrier of A. niger glucoamylase (GlaA) or alpha amylase may be employed in the fusion polypeptide. Aspergillus niger and Aspergillus awamori glucoamylases have identical amino acid sequences. Two forms of the enzyme are generally recognized in culture supernatants. GAI is the full length form (amino acid residues 1-616) and GAII is a natural proteolytic fragment comprising amino acid residues 1-512. GAI is known to fold as two separate domains joined by an extended linker region. The two domains are the 471 residue catalytic domain (amino acids 1-471) and the 108 residue starch binding domain (amino acids 509-616), the linker region between the two domains being 36 residues (amino acids 472-508). GAII lacks the starch binding domain. Reference is made to Libby et al., (1994) Protein Engineering 7:1109-1114. In some embodiments, the glucoamylase which is used as a carrier protein and including a signal sequence will have greater than 95%, 96%, 97%, 98% and 99% sequence identity with a catalytic domain of an Aspergillus or Trichoderma glucoamylase. The term “catalytic domain” refers to a structural portion or region of the amino acid sequence of a protein which posses the catalytic activity of the protein.
In certain embodiments, the signal sequence and the carrier protein are obtained from the same gene. In some embodiments, the signal sequence and the carrier protein are obtained from different genes.
The carrier protein may include all or part of the mature sequence of a secreted polypeptide. In some embodiments, full length secreted polypeptides are used. However, functional portions of secreted polypeptides may be employed. As used herein “portion” of a secreted polypeptide or grammatical equivalents means a truncated secreted polypeptide that retains its ability to fold into a normal, albeit truncated, configuration.
In some cases, the truncation of the secreted polypeptide means that the functional protein retains a biological function. In some embodiments, the catalytic domain of the secreted polypeptide is used, although other functional domains could be used, for example the substrate binding domain. In one embodiment, when glucoamylase is used as the carrier protein (i.e. glucoamylase from Aspergillus niger), preferred functional portions retain the catalytic domain of the enzyme and include amino acids 1-471 (see, WO 03089614, e.g., example 10). In another embodiment, when CBH I is used as the carrier protein (i.e. CBH I from Trichoderma reesei) preferred functional portions retain the catalytic domain of the enzyme. Reference is made to SEQ ID NO:1 of FIG. 2 of WO 05093073, wherein the sequence encoding a Trichoderma reesei CBH1 signal sequence, T. reesei CBH1 catalytic domain (also referred to as catalytic core or core domain) and T. reesei CBH1 linker is disclosed. In some embodiments, a CBH1 carrier protein and including a signal sequence will have greater than 95%, 96%, 97%, 98% and 99% sequence identity with SEQ ID NO:1 of FIG. 2 of WO 05093073.
In general, if the carrier protein is a truncated protein, it is C-terminally truncated (i.e., contains an intact N-terminus). Alternatively, the carrier protein may be N-terminally truncated, or optionally truncated at both ends to leave a functional portion. Generally such portions of a secreted protein which comprise a carrier protein comprise greater than 50%, greater than 70%, greater than 80% and greater than 90% of the secreted protein and preferably the N-terminal portion of the secreted protein. In some embodiments, the carrier protein will include a linker region in addition to the catalytic domain. In the fusion constructs of the examples herein, part of the linker region of the CBHI protein was used in the carrier protein.
As used herein, the first amino acid sequence comprising a signal sequence functional as a secretory sequence is encoded by a first DNA molecule. The second amino acid sequence comprising the carrier protein is encoded by a second DNA sequence. However, as described above the signal sequence and the carrier protein may be obtained from the same gene.
KEX2 Region—
The KEX2 region comprises a KEX2 site (B1B2) and a KEX2 site pre-sequence ((X)n=2-6) immediately N-terminal to said KEX2 site. In some embodiments the KEX2 region provides means for cleavage (separation) at the amino terminus of the desired protein from the fusion polypeptide in vivo. The KEX2 region of a fusion polypeptide of the encompassed by the invention is not a naturally occurring region between the carrier protein and the desired protein.
The KEX2 cleavage site, which occurs at the C-terminal end of the KEX2 region, may be cleaved by a native filamentous fungal protease (e.g. a native Aspergillus KEXB-like protease or native Trichoderma KEX2 protease). The desired protein is cleaved from a fusion polypeptide according to the invention immediately downstream of the KEX2 site.
The KEX2 site contains amino acid sequence “B1B2” wherein B1 and B2, are independently, basic amino acids. Preferably the KEX2 site includes any one of KK, KR, RK or RR and more preferably is KR.
The KEX2 site pre-sequence comprises amino acid sequence (X)n=2-6 wherein X is any amino acid and n is 2 to 6 and preferably 4. The KEX2 region as defined herein is not found naturally in the carrier protein at the C-terminus of the carrier protein, which comprises the fusion polypeptide according to the invention. In some embodiments, the KEX2 site pre-sequence is an amino acid sequence that is different from the naturally occurring contiguous (X)n=2-6 amino acid residues on the C-terminus of the carrier protein. However, the contiguous (X)n=2-6 amino acid residues may be found in other parts of the carrier protein and may be linked with a KEX2 site (B1B2) but the KEX2 region will not be attached to the N-terminus of the desired protein.
In some embodiments, when the KEX2 site pre-sequence is defined as X4X3X2X1B1B2,
a) X1, X2 and X3 are not G;
b) X1 is not S, if X2 and X3 are G, X4 is A, or X3 is S;
C) X4 is not T, if X3 is A and X2 is S; or
d) X1 is not D.
In some preferred embodiments, the KEX2 region is X4X3X2X1B1B2 wherein B1B2 is KR and
a) X1, X2 and X3 are not G;
a) X1 is not S, if X2 and X3 are G, X4 is A, or X3 is S;
b) X4 is not T, if X3 is A and X2 is S; or
c) d) X1 is not D.
In other embodiments, the KEX2 site pre-sequence is defined as X4X3X2X1 wherein,
a) X4 is V, S, N, L, or K;
a) X3 is A, V, D, W, E or P;
b) X2 is V, I, L or F; and
c) X1 is E, S, T or Y.
In yet other embodiments the KEX2 site pre-sequence is defined as X4X3X2X1 wherein,
a) X4 is V, N, or L;
a) X3 is A, V, D, W, E or P;
b) X2 is V, I, L or F; and
c) X1 is E or Y.
In yet further embodiments the X4X3X2X1KR KEX2 region may be selected from the group of X4 is V; X3 is A; X2 is V; X1 is E or Y and combinations thereof.
In some embodiments, the KEX2 site pre-sequence is selected from the group consisting of VAVE (SEQ ID NO: 84); NVIS (SEQ ID NO: 85); SDVT (SEQ ID NO: 86); VAVY (SEQ ID NO: 87); LAVE (SEQ ID NO: 88); KAVE (SEQ ID NO: 89); VAIE (SEQ ID NO: 90); VALE (SEQ ID NO: 91); VAFE (SEQ ID NO: 92); VWVE (SEQ ID NO: 93); VEVE (SEQ ID NO: 94); and VPVE (SEQ ID NO: 95).
In some embodiments, the KEX2 site pre-sequence is not KSRS (SEQ ID NO: 109); SRIS (SEQ ID NO: 111); GGGS (SEQ ID NO: 110); TSTY (SEQ ID NO: 96); ASIS (SEQ ID NO: 97); ATAS (SEQ ID NO: 98); TASQ (SEQ ID NO: 99); TASL (SEQ ID NO: 100), SVIS (SEQ ID NO: 101); NVIS (SEQ ID NO: 85); GGG; TSRD (SEQ ID NO: 102); SPMD (SEQ ID NO: 106); DLGE (SEQ ID NO: 107); or TPTA (SEQ ID NO: 108).
While the preferred KEX2 region is defined as X4X3X2X1B1B2, as indicated above, the KEX2 site pre-sequence can include 6 amino acid residues, in some embodiments, the KEX2 region may include one or two more amino acid residues In other embodiments, the KEX2 site pre-sequence may include only 2 or 3 amino acid residues (X3X2X1B1B2 or X2X1B1B2). In this embodiment,
a) X1, X2 and X3 are not G (e.g. GGGB1B2 or GGB1B2),
a) X1 is not S, if X2 and X3 are G or X3 is S (e.g. SX2S or SGS); and
b) X1 is not D.
In some embodiments, the KEX2 site pre-sequence provides for enhanced cleavage and/or secretion of a desired protein from a host cell as compared to the cleavage and/or secretion of the desired protein from an equivalent fusion polypeptide lacking a KEX2 site pre-sequence.
In some embodiments, the KEX2 site pre-sequence is an optimized KEX2 site pre-sequence. An optimized KEX2 pre-sequence is a KEX2 pre-sequence encompassed by the invention but which provides greater or more efficient cleavage or secretion from a host cell as compared to other variant KEX2 site pre-sequences.
In some embodiments, the fusion polypeptide encompassed by the invention will include an optimized KEX2 pre-sequence as the KEX2 pre-sequence. The optimized KEX2 pre-sequence may be employed with any signal sequence, any carrier region from a secreted protein, any KEX2 site, or any desired protein. A subject KEX2 region containing an optimized KEX2 site pre-sequence may be non-naturally occurring. In certain embodiments, a subject KEX2 region containing an optimized KEX2 site pre-sequence is not found in any protein that is secreted from a filamentous fungal cell.
Desired Proteins—
The desired protein (or the carrier protein) may be any portion of a protein that can be secreted from a filamentous fungal cell, which proteins include, so called industrial enzymes, therapeutic proteins, hormones, structural proteins, plasma proteins, food additives and foodstuffs and the like. The desired protein may be a heterologous or homologous protein and may include hybrid polypeptides that comprise a combination of partial or complete polypeptides each of which may be homologous or heterologous with regard to the fungal expression host. The desired secreted protein may be derived from bacterial (e.g. Bacillus species and Pseudomonas species) fungal (e.g. Aspergillus, Trichoderma, Humicola, or Mucor species), viral (e.g. Hepatitis A or B or Adenovirus), mammalian (e.g. human or mouse), and plant sources. Desired proteins include naturally occurring allelic variations of proteins as well as engineered variations.
In one embodiment, the desired protein may be an enzyme such as a carbohydrase, such as a starch hydrolyzing α-amylase, an alkaline α-amylase, a β-amylase, a cellulase; a dextranase, an α-glucosidase, an α-galactosidase, a glucoamylase, a hemicellulase, a pentosanase, a xylanase, an invertase, a lactase, a naringanase, a pectinase or a pullulanase; a protease such as an acid protease, an alkali protease, bromelain, ficin, a neutral protease, papain, pepsin, a peptidase, rennet, rennin, chymosin, subtilisin, thermolysin, an aspartic proteinase, or trypsin; a granular starch hydrolyzing enzyme, such as a glucoamylase or an alpha amylase; a lipase or esterase, such as a triglyceridase, a phospholipase, a pregastric esterase, a phosphatase, a phytase, an amidase, an iminoacylase, a glutaminase, a lysozyme, or a penicillin acylase; an isomerase such as glucose isomerase; a phenol oxidizing enzyme, e.g., a laccase; an oxidoreductases, e.g., an amino acid oxidase, a catalase, a chloroperoxidase, a glucose oxidase, a hydroxysteroid dehydrogenase or a peroxidase; a lyase such as a acetolactate decarboxylase, a aspartic β-decarboxylase, a fumarese or a histadase; a transferase such as cyclodextrin glycosyltranferase or an acyl transferase; or a ligase, for example. In particular embodiments, the protein may be an aminopeptidase, a carboxypeptidase, a chitinase, a glucoamylase, an alpha amylase, a cutinase, a phytase, a deoxyribonuclease, an α-galactosidase, a β-galactosidase, a β-glucosidase, a laccase, a mannosidase, a mutanase, a pectinolytic enzyme, a polyphenoloxidase, ribonuclease or transglutaminase.
In other embodiments, the desired protein may be a therapeutic protein (i.e., a protein having a therapeutic biological activity). Examples of suitable therapeutic proteins include: erythropoietin, cytokines such as interferon-α, interferon-β, interferon-γ, interferon-o, and granulocyte-CSF, GM-CSF, coagulation factors such as factor VIII, factor IX, and human protein C, antithrombin III, thrombin, soluble IgE receptor α-chain, immunoglobulin, such as immunoglobulin G (IgG), IgG fragments, IgG fusions, IgM or IgA; interleukins, urokinase, chymase, and urea trypsin inhibitor, IGF-binding protein, epidermal growth factor, growth hormone-releasing factor, annexin V fusion protein, angiostatin, vascular endothelial growth factor-2, myeloid progenitor inhibitory factor-1, osteoprotegerin, α-1-antitrypsin, α-feto proteins, DNase II, kringle 3 of human plasminogen, glucocerebrosidase, TNF binding protein 1, follicle stimulating hormone, cytotoxic T lymphocyte associated antigen 4-Ig, transmembrane activator and calcium modulator and cyclophilin ligand, soluble TNF receptor Fc fusion, glucagon like protein 1 and IL-2 receptor agonist.
In some preferred embodiments, the desired protein is an immunoglobulin from any class, G, A, M, E or D. (See, U.S. Pat. No. 4,816,567 and references cited therein for a discussion of immunoglobulin structure). In other preferred embodiments, the antibody proteins such as monoclonal antibodies including heavy or light chains and fragments thereof. In further embodiments, humanized antibodies are of particular interest as a desired protein (e.g. trastuzumab (herceptin)). Some specific examples of preferred monoclonal antibody fragments are truncated forms of the heavy chain to remove part of the constant region such as Fab fragments in which the heavy chain (Fd) lacks the hinge region and the CH2 and CH3 domains; Fab′ fragments in which the heavy chain includes the hinge region but lacks the CH2 and CH3 domains; and F(ab′)2 fragments which includes the Fab portion connected by the hinge region. (Verma et al., (1998) J. Immunological Methods 216:165-181 and Pennell and Eldin (1998) Res. Immunol. 149:599-603). Also of interest are single chain antibodies (ScFv) and single domain antibodies (e.g., camelid antibodies).
In some particularly preferred embodiments a fusion polypeptide according to the invention will comprise in operable linkage a signal sequence; a carrier protein; a KEX2 region and a desired protein as indicated below:
Fusion DNA Constructs and Vectors—
In some embodiments, the invention provides a fusion DNA construct encoding a fusion polypeptide as disclosed above, comprising in operable linkage from the 5′ end of said construct, a promoter; a first DNA molecule encoding a signal sequence; a second DNA molecule encoding a carrier protein; a third DNA molecule encoding a KEX2 region, said KEX2 region comprising a KEX2 site and a KEX2 site pre-sequence immediately 5′ to the KEX2 site; and a fourth DNA molecule encoding a desired protein. Since the genetic code is known, the design and production of these nucleic acids is well within the skill of an artisan, given the description of the subject fusion polypeptide. In certain embodiments, the nucleic acids may be codon optimized for expression of the fusion polypeptide in a particular host cell. Since codon usage tables are available for many species of filamentous fungi, the design and production of codon-optimized nucleic acids that encodes a subject fusion polypeptide would be well within the skill of one of skill in the art.
Promoters—
Examples of suitable promoters for directing the transcription of a subject nucleic acid in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase (Korman et al (1990) Curr. Genet. 17:203-212; Gines et al., (1989) Gene 79: 107-117), Aspergillus niger or Aspergillus awamori glucoamylase (glaA) (Nunberg et al., (1984) Mol. Cell. Biol. 4:2306-2315; Boel E. et al., (1984) EMBO J. 3: 1581-1585), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase (Hyner et al., (1983) Mol. Cell. Biol. 3:1430-1439), Fusarium venenatum amyloglucosidase, Fusarium oxysporum trypsin-like protease (WO 96/00787), Trichoderma reesei cellobiohydrolase I (Shoemaker et al. (1984) EPA EPO 0137280), Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase); and mutant, truncated, and hybrid promoters thereof. Reference is also made to Yelton et al., (1984) Proc. Natl. Acad. Sci. USA 81:1470-1474; Mullaney et al., (1985) Mol. Gen. Genet. 199:37-45; Lockington et al., (1986) Gene 33: 137-149; Macknight et al., (1986) Cell 46: 143-147; Hynes et al., (1983) Mol. Cell. Biol. 3: 1430-1439. Higher eukaryotic promoters such as SV40 early promoter (Barclay et al (1983) Molecular and Cellular Biology 3:2117-2130) may also be useful. Promoters may be constitutive or inducible promoters. Some preferred promoters include a Trichoderma reesei cellobiohydrolase I or II, a Trichoderma reesei endoglucanase I, II or III, and a Trichoderma reesei xylanase H.
Vectors—
A subject polynucleotide may be present in a vector, for example, a phage, plasmid, viral, or retroviral vector. In certain embodiments, the vector may be an expression vector for expressing a subject fusion polypeptide in a filamentous fungal cell.
Vectors for expression of recombinant proteins are well known in the art (Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.).
A fusion DNA construct according to the invention may be constructed using well known techniques as is generally described for example in EPO publication 0 215 594.
Natural or synthetic polynucleotide fragments encoding for the desired protein (e.g. an immunoglobulin) may be incorporated into heterologous nucleic acid constructs or vectors, capable of introduction into and replication in a filamentous fungal cell.
Once a DNA construct or more specifically a fusion DNA construct encompassed by the invention is made it may be incorporated into any number of vectors as is known in the art. While the DNA construct will preferably include a promoter sequence, in some embodiments the vector will include other regulatory sequences functional in the host to be transformed, such as ribosomal binding sites, transcription start and stop sequences, terminator sequences, polyadenylation signals, enhancers and or activators. In some embodiments, a polynucleotide encoding the desired protein and KEX2 region will be inserted into a vector which comprises a promoter, signal sequence and carrier protein at an appropriate restriction endonuclease site by standard procedures. Such procedures and related sub-cloning procedures are deemed to be within the scope of knowledge of those skilled in the art.
Terminator sequences which are recognized by the expression host to terminate transcription may be operably linked to the 3′ end of the fusion DNA construct encoding the fusion protein to be expressed. Those of general skill in the art are well aware of various terminator sequences that may be used with filamentous fungi. Non-limiting examples include the terminator from the Aspergillus nidulans trpC gene (Yelton M. et al., (1984) Proc. Natl. Acad. Sci. USA 81: 1470-1474) or the terminator from the Aspergillus niger glucoamylase genes (Nunberg et al. (1984) Mol. Cell. Biol. 4: 2306-2353) or the terminator from the Trichoderma reesei cellobiohydrolase I gene.
Polyadenylation sequences are DNA sequences which when transcribed are recognized by the expression host to add polyadenosine residues to transcribed mRNA. Examples include polyadenylation sequences from A. nidulans trpC gene (Yelton et al (1984) Proc. Natl. Acad. Sci. USA 81; 1470-1474); from A. niger glucoamylase gene (Nunberg et al. (1984) Mol. Cell. Biol. 4:2306-2315); the A. oryzae or A. niger alpha amylase gene and the Rhizomucor miehei Carboxyl protease gene. Any fungal polyadenylation sequence is likely to be functional in the present invention.
In further embodiments, the fusion DNA construct or the vector comprising the fusion DNA construct will contain a selectable marker gene to allow the selection of transformed host cells. Selection marker genes are well known in the art and will vary with the host cell used. Examples of selectable markers include but are not limited to ones that confer antimicrobial resistance (e.g. hygromycin, bleomycin, chloroamphenicol and phleomycin). Genes that confer metabolic advantage, such as nutritional selective markers also find use in the invention. Some of these markers include amdS. Also sequences encoding genes which complement an auxotrophic defect may be used as selection markers (e.g. pyr4 complementation of a pyr4 deficient A. nidulans, A. awamori or Trichoderma reesei and argB complementation of an argB deficient strain). Reference is made to Kelley et al., (1985) EMBO J. 4: 475-479; Penttila et al., (1987) Gene 61:155-164 and Kinghorn et al (1992) Applied Molecular Genetics of Filamentous Fungi, Blackie Academic and Professional, Chapman and Hall, London.
Host Cells—
A host cell comprising a fusion DNA construct according to the invention is also provided. In certain embodiments, the host cell may be a filamentous fungal host cell. In some embodiments, the cells may be filamentous fungal cells of a strain that has a history of use for production of proteins that have GRAS status, i.e., a Generally Recognized as Safe, by the FDA.
In particular embodiments, the subject fungal cell may be a cell of the following species: Trichoderma, (e.g., Trichoderma reesei (previously classified as T. longibrachiatum and currently also known as Hypocrea jecorina), Trichoderma viride, Trichoderma koningii, and Trichoderma harzianum)); Penicillium sp.: Humicola sp. (e.g., Humicola insolens and Humicola grisea); Chrysosporium sp. (e.g., C. lucknowense); Gliocladium sp.; Aspergillus sp. (e.g., Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans, Aspergillus kawachi, Aspergillus aculeatus, Aspergillus japonicus, Aspergillus sojae, and Aspergillus awamori), Fusarium sp.; Mucor sp.; Neurospora sp.; Hypocrea sp.; or Emericella sp. (See also, Innis et al., (1985) Sci. 228:21-26), among others. In some embodiments, subject fungal cells may be strains of Aspergillus oryzae, ATCC 11490, Aspergillus niger which include ATCC 22342, ATCC 44733, ATCC 14331, NRRL 3112, and strains derived therefrom. In some embodiments, subject fungal cells may be strains of Trichoderma which include functional equivalents of RL-P37 (Sheir-Neiss et al. (1984) Appl. Microbiol. Biotechnology 20:46-53). Useful Trichoderma host strains include; NRRL 15709, ATCC 13631, ATCC 26921 (QM 9414) ATCC 32098, ATCC 32086, and ATCC 56765 (RUTC-30).
In some embodiments, a host cell may be one wherein native genes have been deleted or inactivated. In some embodiments, preferred host cells have inactivated protease genes (e.g. aspartyl protease) and reference is made to Berka et al. (1990) Gene 86:153-162 and U.S. Pat. No. 6,509,171. In some embodiments, preferred host cells have inactivated cellulase genes (e.g. cbh1, cbh2 and egl1, and egl2) and reference is made to the quad deleted strain of T. reesei disclosed in WO 05/001036.
The above described fusion DNA construct may be present in the nuclear genome of the host cell or may be present in a plasmid that replicates in the host cell, for example.
Transformation
Introduction of a DNA construct or vector into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction; transfection, (e.g., lipofection mediated and DEAE-Dextrin mediated transfection); incubation with calcium phosphate DNA precipitate; high velocity bombardment with DNA-coated microprojectiles; and protoplast fusion. General transformation techniques are known in the art (See, e.g., Ausubel et al., (1987), supra, chapter 9; and Sambrook (1989) supra, and Campbell et al., (1989) Curr. Genet. 16:53-56). Reference is also made to WO 05/001036; U.S. Pat. No. 6,022,725; U.S. Pat. No. 6,103,490; U.S. Pat. No. 6,268,328; [and published U.S. patent applications 20060041113, 20060040353, 20060040353 and 20050208623], which publications are incorporated herein by reference.
The expression of recombinantly introduced proteins in Trichoderma is described in U.S. Pat. No. 6,022,725; U.S. Pat. No. 6,268,328; Harkki et al. (1991); Enzyme Microb. Technol. 13:227-233; Harkki et al., (1989) Bio Technol. 7:596-603; EP 244,234; EP 215,594; and Nevalainen et al., “The Molecular Biology of Trichoderma and its Application to the Expression of Both Homologous and Heterologous Genes”, in MOLECULAR INDUSTRIAL MYCOLOGY, Eds. Leong and Berka, Marcel Dekker Inc., NY (1992) pp. 129-148). Reference is also made to Cao et al., (2000) Protein Sci. 9:991-1001; Yelton et al., (1984) Proc. Natl. Acad. Sci. 81:1470-1471; U.S. Pat. No. 6,590,078; and Berka, et al., (1991) in: Applications of Enzyme Biotechnology, Eds. Kelly and Baldwin, Plenum Press, NY) for transformation of Aspergillus strains.
In one embodiment, the preparation of Trichoderma sp. for transformation involves the preparation of protoplasts from fungal mycelia. (See, Penttila et al., (1987) Gene 61:155-164). In some embodiments, the mycelia are obtained from germinated vegetative spores.
Generally, cells are cultured in a standard medium containing physiological salts and nutrients (See, e.g., Pourquie, J. et al., BIOCHEMISTRY AND GENETICS OF CELLULOSE DEGRADATION, eds. Aubert, J. P. et al., Academic Press, pp. 71-86, 1988 and Ilmen, M. et al., (1997) Appl. Environ. Microbiol. 63:1298-1306). Common commercially prepared media (e.g., Yeast Malt Extract (YM) broth, Luria Bertani (LB) broth and Sabouraud Dextrose (SD) broth also find use in the present invention. Preferred culture conditions for a given filamentous fungus are known in the art and may be found in the scientific literature and/or from the source of the fungi such as the American Type Culture Collection (ATCC) and Fungal Genetics Stock Center.
In some embodiments, when an immunoglobulin is the desired protein immunoglobulin expressing cells will be cultured under conditions typically employed to culture the parental cell line. Generally, cells will be cultured in standard medium containing physiological salts and nutrients such as that described by Ilmen et al., (1997) supra. Culture conditions will also be standard (e.g. incubation at 25-30° C. in shake flasks on a rotary shaker) until desired levels of immunoglobulin expression is achieved.
Protein Production Methods
Methods of producing a desired protein in a filamentous fungal cell are also encompassed by the invention. In some embodiments these methods include, obtaining a filamentous host cell comprising a fusion DNA construct or vector according to the invention and culturing the filamentous host cell under suitable conditions which allow the expression and secretion of the desired protein. While a culture of host cells (i.e., a composition containing subject host cells and growth media) may contain the secreted protein of the fusion polypeptide described above, in some embodiments the desired protein is recovered from the culture media. In other embodiments, the desired protein is purified. Protein may be recovered from growth media by any convenient method.
In some embodiments, a subject fungal cell may be cultured under batch or continuous fermentation conditions. A classical batch fermentation is a closed system, wherein the composition of the medium is set at the beginning of the fermentation and is not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the medium is inoculated with the desired organism(s). In this method, fermentation is permitted to occur without the addition of any components to the system. Typically, a batch fermentation qualifies as a “batch” with respect to the addition of the carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. The metabolite and biomass compositions of the batch system change constantly up to the time the fermentation is stopped. Within batch cultures, cells progress through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase eventually die. In general, cells in log phase are responsible for the bulk of production of end product.
A variation on the standard batch system is the “fed-batch fermentation” system, which also finds use with the present invention. In this variation of a typical batch system, the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Measurement of the actual substrate concentration in fed-batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as CO2. Batch and fed-batch fermentations are common and known in the art.
Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.
Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth and/or end product concentration. For example, in one embodiment, a limiting nutrient such as the carbon source or nitrogen source is maintained at a fixed rate and all other parameters are allowed to moderate. In other systems, a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions. Thus, cell loss due to medium being drawn off must be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are known.
Expression and Secretion—
The production of a desired protein in a filamentous fungal cell comprising a fusion DNA construct encoding a fusion polypeptide results in the secretion of the desired protein of the fusion polypeptide. During the secretion process in fungi, sugar chains may be attached to a protein to be secreted to produce a glycosylated protein. In the present invention, the production of the desired protein, (e.g. an antibody), may include glycosylated or non-glycosylated protein.
In some embodiments, the secreted protein of the subject fusion polypeptide is generally present in the culture medium of the filamentous fungal cell at an amount that is higher than the amount of the desired secreted protein of an equivalent fusion polypeptide that lacks the KEX2 site pre-sequence, produced by an equivalent filamentous fungal cell (i.e., the same cell type, grown under the same conditions). A culture of the subject cells producing a desired protein from a fusion polypeptide according to the invention may contain more than 5%, more than 10%, more than 20%, more than 40%, more than 60%, more than 80%, more than 100%, more than 150%, more than 200%, more than 300%, more than 500%, and more than 1000% desired protein in the growth medium, as compared to an equivalent cell culture that expresses an otherwise equivalent protein that does not have a KEX2 site pre-sequence as encompassed by the invention.
In some embodiments, the level of expression and secretion for a desired protein (e.g. a full-length antibody) will be greater than 0.5 g/L. Routinely greater than 1.0 g/L of the desired protein may be recovered from a culture media. Reproducible levels of greater than 1.5, 2.0 and 3.0 g/L may be attained. In some embodiments, the level of expression and secretion of the desired protein will be greater than 10 g/L and even greater than 20 g/L.
In some embodiments of the invention, the cleavage of the desired protein from the recombinant fusion polypeptide will be greater than the cleavage of the same desired protein from an equivalent recombinant fusion polypeptide which lacks the KEX2 site pre-sequence. In some embodiments, the KEX2 site pre-sequence may result in a fusion protein that is cleaved to at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% efficiency, wherein 100% efficiency results in a completely cleaved desired secretion protein from the fusion polypeptide.
In certain embodiments, the efficiency of protein cleavage may be calculated by determining amount of cleavage that has occurred, e.g., by determining the amount of cleaved versus the amount of uncleaved protein. In one embodiment, the amount of protein cleavage may be calculated by determining the ratio of the amount of cleaved protein in the growth medium to the amount of non-cleaved fusion protein in the growth medium per volume of cell culture.
A fusion polypeptide containing a KEX2 site pre-sequence or an optimized KEX2 site pre-sequence may, in certain embodiments, result in a fusion polypeptide that is cleaved to at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% efficiency, wherein 100% efficiency is a completely cleaved desired protein.
In other embodiments, the efficiency of secretion of a subject fusion polypeptide may be calculated by determining the amount of the secreted portion of that fusion polypeptide in the growth medium of a cell secreting that protein. This determination may be quantitative, qualitative, relative or absolute. In one embodiment, the amount of secreted protein in the growth medium of a cell secreting a subject fusion may be at least 10%, at least 30%, at least 50%, at least 70%, at least 90%, at least twice, at least five times, or at least ten times greater than the amount of the secreted protein secreted by a cell producing an equivalent fusion polypeptide that does not contain an optimized KEX2 pre-sequence.
In some embodiments the increase in secretion and/or cleavage may be measured against a standard KEX2 region defined as GGGB1B2, wherein B1B2 is KK, KR, RK or RR and preferably KR. In an embodiment, the amount of secreted protein or desired protein in the growth medium of a cell secreting a subject fusion may be at least 10%, at least 30%, at least 50%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2×, at least 3×, at least 5×, and at least 10× greater than the amount of the secreted protein or desired protein secreted by an equivalent fusion polypeptide in an equivalent host under essentially the same conditions.
Screening Methods—
Screening methods for identifying optimized KEX2 site pre-sequences are also provided. These methods may include: a) altering a KEX2 site pre-sequence of a parental fusion polypeptide to produce a test polypeptide and b) evaluating secretion of the test fusion polypeptide by a filamentous fungal cell. In certain embodiments, the secretion and/or cleavage of the desired protein from test fusion polypeptides is compared to the secretion and/or cleavage of the parental fusion protein. In particular embodiments, the method includes evaluating the amount of a secreted protein of the fusion polypeptide in a growth medium relative to the amount of a secreted portion of the fusion polypeptide in a cell, per volume of culture, or assessing the amount of a secreted protein of a recombinant fusion polypeptide in a growth medium. In another embodiments the method includes evaluating the amount of a secreted protein (desired protein) released or cleaved from a fusion polypeptide in a growth medium relative to the amount of the secreted protein that remains in the form of the fusion polypetide (e.g. attached to the carrier protein).
In these screening assays, the parental fusion protein has an amino acid sequence that is schematically illustrated in FIG. 1, where X is any amino acid. In certain embodiments, the parental fusion protein and the test fusion protein may be identical except for their KEX2 site pre-sequences. A parental recombinant fusion protein and a test recombinant fusion protein may differ in one, two, three or four amino acids in the KEX-2 site pre-sequence. An alteration may be an amino acid substitution, insertion or deletion, and if there are two or three alterations, the alterations may be in contiguous amino acids, non-contiguous amino acids, or a combination of contiguous and non-contiguous amino acids.
In one embodiment, the KEX2 site pre-sequence of a parental fusion polypeptide may be altered to produce a plurality of different test fusion polypeptides that each contains different KEX2 site pre-sequences, and then evaluating secretion and/or cleavage of the test fusion polypeptides and the parental fusion polypeptides by a filamentous fungal cell.
These methods may be performed using protocols that are generally known (see, e.g., Ward et al (1990) Bio/Technology 8:435-440 and Spencer (1998) Eur. J. Biochem 258: 107-112, among others), in which a vector is introduced into a cell, the cell is cultured, and the cell culture is assayed for the presence of the cellular protein. In one embodiment, a recombinant nucleic acid encoding a parent fusion (the structure of which is shown in FIG. 1) is altered to produce a nucleic acid encoding a test polypeptide, and the two nucleic acids are used to transform identical filamentous fugal cells (which may be any of the host cells listed above). The two cell lines are cultured under identical conditions, and the efficiency of secretion and/or cleavage of the secreted portion of the protein is evaluated. The signal sequence, secreted protein, the KEX2 site and the KEX2 site pre-sequence of the parental fusion protein may be any known signal sequence, secreted protein, KEX2 site or KEX2 site pre-sequence, including those listed above.
As noted above, the efficiency of protein secretion or cleavage may be evaluated in many different ways, for example, by comparing the absolute or normalized amounts of secreted portion in growth media between the different cultures, or by comparing the amount of the secreted portion of the protein to the amount of the unsecreted portion of the protein. This evaluation may be quantitative, qualitative, relative or absolute, for example.
An optimized KEX2 site pre-sequence may be identified by testing a plurality of different test fusion proteins according to the above methods; and determining which of the different test fusion proteins is secreted and/or cleaved most efficiently; wherein the optimized KEX2 site pre-sequence is the KEX2 site pre-sequence of the test recombinant fusion protein that is secreted most efficiently.
A culture of cells that contains at least 10%, at least 20%, at least 30%, at least 50%, at least 70% and at least 95% more secreted or more desired protein than a control culture indicates that KEX2 site pre-sequence increases protein secretion and/or cleavage from those cells.
EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade (° C.), pressure is at or near atmospheric and the following abbreviations apply, M (Molar); μM (micromolar); N (Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); g (grams); mg (milligrams) kg (kilograms); μg (micrograms); L (liters); ml (milliliters); h (hours); min (minutes); PAGE (polyacrylamide gel electrophoresis); kDa (kilodaltons); and by (base pairs). The following assays and methods are used in the examples provided below:
A. Construction of the pTrex4 Vector:
Synthetic DNA was cloned into a Trichoderma expression vector (pTrex4) to generate appropriate expression plasmids for use in the examples described below.
PTrex4 is a modified version of pTrex2 and derived from a pTrex3g expression vector. The construction of pTrex3g is described in detail in Example 6 of WO 05/001036. In brief, the pTrex3g is based on the E. coli vector pSL1180 (Pharmacia, Inc., Piscataway, N.J.) which is a pUC118 plasmid based vector with an extended multiple cloning site containing 64 hexamer restriction enzyme recognition sequences. It was designed as a Gateway destination vector (Hartley, J. L. et al., (2000) Genome Research 10:1788-1795) to allow insertion using Gateway Technology (Invitrogen) of any desired open reading frame between the promoter and terminator regions of the T. reesei cbh1 gene.
The details of pTrex4-her2 light chain DNA2.0 are as follows (FIG. 2 and FIG. 3): The plasmid is 10885 kb in size (SEQ ID NO: 103). Inserted into the polylinker region of pSL1180 are the following segments of DNA:
A 2.2 bp segment of DNA from the promoter region of the T. reesei cbh1;
DNA sequence of T. reesei cbh1 signal sequence (underlined); catalytic domain; linker (italics) (1570 bases) (SEQ ID NO: 104)








ATGTATCGGAAGTTGGCCGTCATCTCGGCCTTCTTGGCCACAGCTCGTGCTCAGTCGGCCTG



 


CACTCTCCAATCGGAGACTCACCCGCCTCTGACATGGCAGAAATGCTCGTCTGGTGGCACTT


 


GCACTCAACAGACAGGCTCCGTGGTCATCGACGCCAACTGGCGCTGGACTCACGCTACGAA


 


CAGCAGCACGAACTGCTACGATGGCAACACTTGGAGCTCGACCCTATGTCCTGACAACGAG


 


ACCTGCGCGAAGAACTGCTGTCTGGACGGTGCCGCCTACGCGTCCACGTACGGAGTTACCA


 


CGAGCGGTAACAGCCTCTCCATTGGCTTTGTCACCCAGTCTGCGCAGAAGAACGTTGGCGCT


 


CGCCTTTACCTTATGGCGAGCGACACGACCTACCAGGAATTCACCCTGCTTGGCAACGAGTT


 


CTCTTTCGATGTTGATGTTTCGCAGCTGCCGTAAGTGACTTACCATGAACCCCTGACGTATCT


 


TCTTGTGGGCTCCCAGCTGACTGGCCAATTTAAGGTGCGGCTTGAACGGAGCTCTCTACTTC


 


GTGTCCATGGACGCGGATGGTGGCGTGAGCAAGTATCCCACCAACACCGCTGGCGCCAAGT


 


ACGGCACGGGGTACTGTGACAGCCAGTGTCCCCGCGATCTGAAGTTCATCAATGGCCAGGC


 


CAACGTTGAGGGCTGGGAGCCGTCATCCAACAACGCAAACACGGGCATTGGAGGACACGG


 


AAGCTGCTGCTCTGAGATGGATATCTGGGAGGCCAACTCCATCTCCGAGGCTCTTACCCCCC


 


ACCCTTGCACGACTGTCGGCCAGGAGATCTGCGAGGGTGATGGGTGCGGCGGAACTTACTC


 


CGATAACAGATATGGCGGCACTTGCGATCCCGATGGCTGCGACTGGAACCCATACCGCCTG


 


GGCAACACCAGCTTCTACGGCCCTGGCTCAAGCTTTACCCTCGATACCACCAAGAAATTGAC


 


CGTTGTCACCCAGTTCGAGACGTCGGGTGCCATCAACCGATACTATGTCCAGAATGGCGTCA


 


CTTTCCAGCAGCCCAACGCCGAGCTTGGTAGTTACTCTGGCAACGAGCTCAACGATGATTAC


 


TGCACAGCTGAGGAGGCAGAATTCGGCGGATCCTCTTTCTCAGACAAGGGCGGCCTGACTC


 


AGTTCAAGAAGGCTACCTCTGGCGGCATGGTTCTGGTCATGAGTCTGTGGGATGATGTGAGT


 


TTGATGGACAAACATGCGCGTTGACAAAGAGTCAAGCAGCTGACTGAGATGTTACAGTACT


 


ACGCCAACATGCTGTGGCTGGACTCCACCTACCCGACAAACGAGACCTCCTCCACACCCGGT


 


GCCGTGCGCGGAAGCTGCTCCACCAGCTCCGGTGTCCCTGCTCAGGTCGAATCTCAGTCTCC


 


CAACGCCAAGGTCACCTTCTCCAACATCAAGTTCGGACCCATTGGCAGCACCGGCAACCCTA


 


GCGGCGGCAACCCTCCCGGCGGAAACCCGCCTGGCACCACCACCACCCGCCGCCCAGCCACTA


 


CCACTGGAAGCTCTCCCGGACCTACTAGT






The amino acid sequence of the T. reesei cbh1 signal sequence; catalytic domain; linker (480 amino acids) is represented below (SEQ ID NO: 105)








MYRKLAVISAFLATARAQSACTLQSETHPPLTWQKCSSGGTCTQQTGSVVIDANWRWT



 


HATNSSTNCYDGNTWSSTLCPDNETCAKNCCLDGAAYASTYGVTTSGNSLSIGFVTQSAQKNV


 


GARLYLMASDTTYQEFTLLGNEFSFDVDVSQLPCGLNGALYFVSMDADGGVSKYPTNTAGAKY


 


GTGYCDSQCPRDLKFINGQANVEGWEPSSNNANTGIGGHGSCCSEMDIWEANSISEALTPHPCTT


 


VGQEICEGDGCGGTYSDNRYGGTCDPDGCDWNPYRLGNTSFYGPGSSFTLDTTKKLTVVTQFE


 


TSGAINRYYVQNGVTFQQPNAELGSYSGNELNDDYCTAEEAEFGGSSFSDKGGLTQFKKATSGG


 


MVLVMSLWDDYYANMLWLDSTYPTNETSSTPGAVRGSCSTSSGVPAQVESQSPNAKVTFSNIK


 


FGPIGSTGNPSGGNPPGGNPPGTTTTRRPATTTGSSPGPTS






The plasmid also contains a cbh1 terminator, an A. nidulans amdS selectable marker and nucleotides encoding the antibody light chain.
B. Biolistic Transformation of T. Reesei: 
In all examples below transformation was performed on a derivative of the quad deleted (Δchb1, Δcbh2, Δegl1, and Δegl2) T. reesei strain (WO 05/001036) originally derived from RL-P37 (Sheir-Neiss et al., (1984) Appl. Microbiol. Biotechnol. 20:46-53; U.S. Pat. No. 4,797,361) with the appropriate pTrex4 vector using the protocol outlined below.
A suspension of spores (approximately 5×108 spores/ml) from the Trichoderma strain was prepared. 100 ul-200 ul of spore suspension was spread onto the center of plates of MM acetamide medium. MM acetamide medium had the following composition: 0.6 g/L acetamide; 1.68 g/L CsCl; 20 g/L glucose; 20 g/L KH2PO4; 0.6 g/L CaCl2.2H2O; 1 ml/L 1000× trace elements solution; 20 g/L Noble agar; pH 5.5. 1000× trace elements solution contained 5.0 g/l FeSO4.7H2O, 1.6 g/lMnSO4.H2O, 1.4 g/lZnSO4.7H2O and 1.0 g/l CoCl2.6H2O. The spore suspension was allowed to dry on the surface of the MM acetamide medium.
Transformation of the Trichoderma strain by the biolistic transformation method was accomplished using a Biolistic® PDS-1000/He Particle Delivery System from Bio-Rad (Hercules, Calif.) following the manufacturers instructions (see, WO 05/001036 and US 2006/0003408).
C. Transformation of Aspergillus—
The Aspergillus transformation protocol was a modification of the Campbell method (Campbell et at. (1989). Curr. Genet. 16:53-56). Also details of the transformation method for Aspergillus niger are disclosed in WO 03089614 and US Pat. Pub. 20050153399. Transformants were assayed for protein production on SDS gel and Western blot to select the transformants based on the amount of protein produced.
D. Fermentation of T. Reesei and Aspergillus Niger Strains Transformed with the Expression Vector:
In general the fermentation protocol as described in Foreman et al., (2003) J. Biol. Chem. 278:31988-31997 was followed.
E. Proflo Media
Contains: 30 g/L α-lactose; 6.5 g/L (NH4)2SO4; 2 g/L KH2PO4; 0.3 g/L MgSO4.7H2O; 0.2 g/L CaCl2; 1 ml/L 1000× trace element salt solution; 2 ml/L 10% Tween 80; 22.25 g/L Proflo cottonseed flour (Traders Protein, Memphis, Tenn.); 0.72 g/L CaCO3.
F. Defined Media
Contains: 5 g/L (NH4)2SO4; 33 g/L PIPPS buffer; 9 g/L casamino acids; 4.5 g/L KH2PO4; 1 g/L CaCl2; 1 g/L MgSO4.7H2O; 5 ml/L Mazu DF60-P antifoam (Mazur Chemicals, Gurnee, Ill.); 1 ml/L 1000× trace elements solution. After autoclaving 40 ml of 40% lactose was added.
G. 1000× Trace Elements Solution
Contains: 5.0 g/l FeSO4.7H2O, 1.6 g/l MnSO4.H2O, 1.4 g/l ZnSO4.7H2O and 1.0 g/l CoCl2.6H2O
H. Protein Analysis was accomplished by standard SDS gel and Western blot analysis.
Example 1
Construction of a Trastuzumab (Light Chain Expression Strain Containing a KRGGG (SEQ ID NO: 2) KEX2 Cleavage Site
DNA (SEQ ID NO:1) encoding the light chain of trastuzumab according to the published amino acid sequence of antibody 4D5-8 (Carter et al, Proc. Natl. Acad. Sci. 1992 89: 4285-4289) was synthesized by DNA2.0 Inc. (1455 Adams Drive, Menlo Park, Calif. 94025).








(SEQ ID NO: 1)



ACTAGTAAACGCGGTGGCGGTGATATTCAAATGACACAATCTCCTTCTTCTCTGTCA



 


GCCTCAGTGGGCGACCGTGTGACGATTACTTGCCGCGCCTCTCAGGACGTTAACACT


 


GCCGTCGCATGGTACCAGCAGAAGCCAGGCAAGGCGCCCAAGCTTCTGATTTACAG


 


CGCTTCGTTCCTGTACTCTGGCGTGCCATCCCGCTTCTCTGGCAGCCGAAGCGGCAC


 


GGATTTCACCCTGACCATTTCGTCCCTGCAGCCCGAGGATTTCGCCACGTATTACTG


 


CCAGCAGCACTACACCACTCCACCCACCTTTGGCCAAGGAACGAGAGTCGAAATCA


 


CTCGCACGGTCGCTGCCCCTTCAGTCTTCATCTTCCCCCCCAGCGACGAACAGCTGA


 


AGTCTGGTACGGCCAGCGTCGTTTGCTTGCTTAATAACTTCTATCCGCGAGAGGCGA


 


AGGTCCAATGGAAGGTTGATAACGTTCTGCAGTCCGGCAATTCGCAGGAGAGCGTG


 


ACCGAGCAGGATTCAAAGGATAGCACCTACTCACTCAGCAGCACCCTGACGTTGTC


 


CAAGGCCGATTACGAGAAGCATAAGTTGTATGCATGCGAGGTCACCCACCAGGGAC


 


TGTCAAGCCCAGTTACCAAGTCGTTCAATCGAGGCGAGTGCTAAGGCGCGCC.






The light chain encoded by the DNA contains a KRGGG (SEQ ID NO:2) KEX2 cleavage site at its N-terminal end. The restriction sites SpeI and AscI were included for cloning proposes. The synthetic DNA was cloned into Trichoderma expression vector (pTrex4) to generate an expression plasmid named pTrex4-her2 light chain DNA2.0 (FIG. 2). The resultant plasmid encodes a fusion protein containing a Trichoderma CBHI core/linker region and the antibody light chain, separated by a KEX2 site. The plasmid was digested with XbaI restriction enzyme and transformed biolistically into a Trichoderma reesei strain derived from the quad deleted strain described in WO 05001036, example 5). More than 20 transformants were obtained and transferred to new plates. Twenty stable transformants were selected to grow in Proflo media for 2 days at 30° C. 5 mls of 2 days old culture from Proflo were transferred to 50 mls of Define media. The cultures were grown for 5 days at 28° C. Culture broths were centrifuged and supernatants were used for protein analysis. Western blot data (FIG. 4) indicated that more than 90% of the fusion protein was cleaved in the best light chain producing strain (transformant 1010-18, KRGGG variant). However, GGG will remain at the N-terminus of the cleaved antibody light chain which is undesirable. A band of about 50 kd was also detected in Western blot, which may result from dimerization of two light chain molecules.
Example 2
Construction of a Trastuzumab Light Chain Expression Strain Containing the GGGKR (SEQ ID NO: 5) KEX2 Cleavage Site
Two primers (GGACTAGTGGTGGCGGTAAACGCGATATTCAAATGACACAATCT C; SEQ ID NO:3 and AAGGCGCGCCTTAGCACTCGCCTCGATTG; SEQ ID NO:4) were synthesized by Invitrogen (1600 Faraday Avenue. Carlsbad, Calif. 92008) and used to amplify trastuzumab light chain DNA.
The resulting PCR fragment encodes the antibody light chain containing a GGGKR (SEQ ID NO:5) sequence kex2 site at its N-terminal end. The PCR fragment was digested with restriction enzymes SpeI and AscI and cloned to expression Vector pTrex4 to generate a plasmid named as pTrex4-GGGKR-her2 DNA2.0. Fidelity of the PCR fragment was analyzed by DNA sequencing. The plasmid was digested with XbaI restriction enzyme and transformed biolistically using standard techniques into the T. reesei strain described above. More than 20 transformants were obtained and transferred to new plates. A total of 21 stable transformants were selected to grow in Proflo media for 2 days at 30° C. 5 mls of 2 days old culture from Proflo were transferred to 50 mls of Define media. The cultures were grown for 5 days at 28° C. Culture broths were centrifuged and supernatants were used. Western blot indicated that, more than 95% of the protein from transformant 1010-B5 (GGGKR variant) and transformant 1010-B6 (GGGKR variant), was an uncleaved fusion protein (FIG. 4). A band of about 150 kd was also detected in Western blot. It may result from dimerization of two CBHI core-light chain fusion molecules.
Example 3
Construction of a Trastuzumab Light Chain Expression Strain Containing a GGGKRGGG (SEQ ID NO: 7) KEX2 Cleavage Site
Two oligos, GGACTAGTGGCGGTGGCAAACGCGGTGGCGGTGATATTC (SEQ ID NO. 6) and AAGGCGCGCCTTAGCACTCGCCTCGATTG (SEQ ID NO. 4), were synthesized by Invitrogen and used to amplify light chain DNA. The resulting PCR fragment encodes light chain and GGGKRGGG (SEQ ID NO:7) sequence for kex2 cleavage. The PCR fragment was digested with restriction enzymes SpeI and AscI and cloned to expression Vector pTrex4 to generate a plasmid named as pTrex4-GGGKRGGG-her2 light chain DNA2.0. Fidelity of the PCR fragment was analyzed by DNA sequencing. The plasmid was digested with XbaI restriction enzyme and transformed biolistically into the T. reesei strain as described above. More than 10 transformants were obtained and transferred to new plates. 3 stable transformants were selected to grow in Proflo media for 2 days at 30° C. 5 mls of 2 days old culture from Proflo were transferred to 50 mls of Define media. The cultures were grown for 5 days at 28° C. Culture broths were centrifuged and supernatants were used for protein analysis. Western gel data indicated that, in transformant 1011-1 (GGGKRGGG variant), more than 90% of the fusion protein was cleaved (FIG. 4). However, GGG remained at the N-terminus of the cleaved antibody light chain which is undesirable.
Example 4
Construction of a Trastuzumab Light Chain Expression Strain Containing a VAVEKR (SEQ ID NO: 9) KEX2 Region
A VAVEKR (SEQ ID NO: 9) KEX2 region is found naturally in the proregion of the T. reesei high pI xylanase, Xyn2 (Torronen et al., (1992) Biotechnol. 10:1461-1465). To construct a fusion polypeptide according to the invention, two oligos, GGACTAGTGTCGCCGTTGAGAAACGCGATATTCAAATGACACAAT CTCC (SEQ ID NO. 8) and AAGGCGCGCCTTAGCACTCGCCTCGATTG (SEQ ID NO. 4), were synthesized by Invitrogen and used to amplify light chain DNA.
The resulting PCR fragment encodes light chain and VAVEKR (SEQ ID NO:9) sequence for kex2 cleavage. The PCR fragment was digested with restriction enzymes SpeI and AscI and cloned to expression Vector pTrex4 to generate a plasmid named as pTrex4-VAVE-her2 light chain DNA2.0. Fidelity of the PCR fragment was analyzed by DNA sequencing. The plasmid was digested with XbaI restriction enzyme and transformed biolistically into the T. reesei strain as described above. More than 20 transformants were obtained and transferred to new plates. 6 stable transformants were selected to grow in Proflo media for 2 days at 30° C. 5 mls of 2 days old culture from Proflo were transferred to 50 mls of Define media. The cultures were grown for 5 days at 28° C. Culture broths were centrifuged and supernatants were used for protein analysis. Western gel data indicated that, in transformant 1012-2 (VAVEKR variant, SEQ ID NO: 9), more than 95% of the fusion proteins were cleaved (FIG. 4).
Example 5
Construction of a Trastuzumab Light Chain Expression Strain Containing Variants of the VAVEKR (SEQ ID NO: 9) KEX2 Region
DNA (SEQ ID NO: 10) encoding the trastuzumab antibody light chain was synthesized by Geneart (Josef-Engert-Strasse 11, 93053 Regesburg, Germany).








(SEQ ID NO: 10)



ACTAGTAAGCGCGGCGGCGGCGAGGTCCAGCTCGTCGAGAGCGGCGGCGGCCTCGT



 


CCAGCCCGGCGGCAGCCTCCGCCTCAGCTGCGCCGCCAGCGGCTTCAACATCAAGG


 


ACACCTACATCCACTGGGTCCGCCAGGCCCCCGGCAAGGGCCTCGAGTGGGTCGCC


 


CGCATCTACCCCACCAACGGCTACACCCGCTACGCCGACAGCGTCAAGGGCCGCTT


 


CACCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTCCAGATGAACAGCCTCC


 


GCGCCGAGGACACCGCCGTCTACTACTGCAGCCGCTGGGGCGGCGACGGCTTCTAC


 


GCCATGGACTACTGGGGCCAGGGCACCCTCGTCACGGTCTCCAGCGCCAGCACCAA


 


GGGCCCAAGCGTCTTTCCCCTCGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACCG


 


CCGCCCTCGGCTGCCTCGTCAAGGACTACTTCCCCGAGCCCGTCACTGTCAGCTGGA


 


ACAGCGGCGCTCTCACCAGCGGCGTCCACACCTTCCCCGCCGTCCTCCAGAGCAGC


 


GGCCTCTACAGCCTCAGCAGCGTCGTCACCGTCCCCAGCAGCAGCCTCGGCACCCA


 


GACCTACATCTGCAACGTCAACCACAAGCCCAGCAACACCAAGGTCGACAAGCGCG


 


TCGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCCGCCCCCGAG


 


CTGCTCGGCGGCCCCTCCGTCTTTCTCTTCCCCCCCAAGCCCAAGGACACCCTCATG


 


ATCAGCCGCACCCCCGAGGTCACCTGCGTCGTCGTCGATGTCAGCCACGAGGACCC


 


CGAGGTCAAGTTCAACTGGTACGTCGACGGCGTCGAGGTCCACAACGCCAAGACCA


 


AGCCCCGCGAGGAGCAGTACAACAGCACCTACCGCGTCGTCAGCGTCCTGACCGTC


 


CTCCACCAGGACTGGCTCAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAGGC


 


CCTCCCCGCCCCCATCGAAAAGACCATCAG


 


CAAGGCCAAGGGCCAGCCCCGCGAGCCCCAGGTCTACACCCTCCCCCCCAGCCGCG


 


AGGAGATGACCAAGAACCAGGTCTCCCTCACCTGCCTGGTCAAGGGCTTCTACCCC


 


AGCGACATCGCCGTCGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGA


 


CCACCCCCCCCGTCCTCGACAGCGACGGCAGCTTCTTCCTCTACAGCAAGCTCACCG


 


TCGACAAGAGCCGCTGGCAGCAGGGCAACGTCTTTAGCTGCAGCGTCATGCACGAG


 


GCCCTCCACAACCACTACACCCAGAAGAGCCTCAGCCTCAGCCCCGGCAAGTAAGG


 


CGCG






This DNA encodes KRGGG (SEQ ID NO: 2) and the human antibody light chain. Two restriction sites SpeI and AscI were included for cloning proposes. The nucleotide sequence was mutated to remove an internal kex2 site by site-direct mutagenesis (Stratagene, 11011 North Torrey Pines Road, La Jolla, Calif. 92037) and two primers used for the mutagenesis in the PCR reaction are TCGAGATCACCCGCACCGTCGCG
GCGCCAAG (SEQ ID NO: 11) and
CGACGGTGCGGGTGATCTCGACCTTGGTGCCCTGG
CCG (SEQ ID NO: 12). The resulting light chain encoding DNA contained two substituted nucleotides at the DNA sequence which changed amino acid K to T.
Two oligos, GGACTAGTGTCGCCGTTGAGAAACGCGACATCCAGATGACCCAGAGC (SEQ ID NO: 13)
and CTAAAGGGAACAAAAGCTGGAGC (SEQ ID NO: 14), were synthesized by Invitrogen and used to amplify light chain DNA. The resulting PCR fragment encodes light chain and VAVEKR (SEQ ID NO: 9). The PCR fragment was digested with restriction enzymes SpeI and AscI and cloned to expression Vector pTrex4 to generate a plasmid named as pTrex4-VAVE-her2 light chain Geneart (KR-TR). Fidelity of the PCR fragment was analyzed by DNA sequencing. The plasmid was digested with XbaI restriction enzyme and co-transformed biolistically into the T. reesei strain with heavy chain expression plasmid. More than 40 transformants were obtained and transferred to new plates. More than 20 stable transformants were selected to grow in Proflo media for 2 days at 30° C. 5 mls of 2 days old culture from Proflo were transferred to 50 mls of Define media. The cultures were grown for 4 days at 28° C. Culture broths were centrifuged and supernatants were used for protein analysis. Western blot data indicated that in the VAVEKR variant (transformant 17-43), more than 90% of the fusion protein was cleaved (FIG. 5).
To generate amino acid changes at the glutamine residue of the KEX2 site pre-sequence of VAVEKR (SEQ ID NO: 9), a degenerate primer (GGACTAGTGTCGCCGTTNNSAAACGCGACATCC AGATGACCCAGAG (SEQ ID NO:15) was synthesized and used in a PCR reaction with reverse primer (SEQ ID NO:14) to amplify DNA to generate a pool of PCR fragments. The mixed PCR fragments were cloned into Trichoderma expression vector (pTrex4). 13 clones were sequenced and 7 variants were produced (table 1). All 7 plasmids were transformed biolistically into the T. reesei strain. More than 40 transformants were obtained for each variant and transferred to new plates. For the first set of three variants (VAVWKR (SEQ ID NO: 25), VAVGKR (SEQ ID NO: 26) and VAVRKR (SEQ ID NO: 27)), 15 stable transformants for each variant were selected. For the second set of four variants (VAVTKR (SEQ ID NO: 28), VAVVKR (SEQ ID NO: 29), VAVAKR (SEQ ID NO: 30) and VAVLKR (SEQ ID NO: 31)), 11 stable transformants for each variant were selected. The selected transformants were grown in Proflo media for 2 days at 28° C. 5 mls of 2 days old culture from Proflo were transferred to 50 mls of Define media. The cultures were grown for 4 days at 28° C. Culture broths were centrifuged and supernatants were used for protein analysis.
A new primer (GGACTAGTGTCGCCGTTNACAAACGCGACATCCAGATGAC CCAGAG SEQ ID NO: 16) was synthesized and used in a PCR reaction with reverse primer (SEQ ID NO: 14) to amplify DNA to generate PCR fragments with multiple sequences. The mixed PCR fragments were cloned into Trichoderma expression vector (pTrex4). 10 clones were sequenced and 4 more variants (VAVDKR (SEQ ID NO: 32), VAVNKR (SEQ ID NO: 33), VAVYKR (SEQ ID NO: 34) and VAVHKR (SEQ ID NO: 35)) were produced. The plasmids were transformed biolistically into the Trichoderma strain described above. More than 40 transformants for each variant were obtained and transferred to new plates. 10 stable transformants for each variant were selected and grown in Proflo media for 2 days at 28° C. 5 mls of 2 days old culture from Proflo were transferred to 50 mls of Define media. The cultures were grown for 4 days at 28° C. Culture broths were centrifuged and supernatants were used for protein analysis.
One transformant (the best light chain producing transformant) from each variant at the glutamine residue was selected to be compared. Western analysis indicated that the variant VAVYKR (SEQ ID NO: 34) produced more light chain than any other variant. VAVTKR (SEQ ID NO: 28) and VAVDKR (SEQ ID NO: 32) variants had more fusion protein indicating less efficient cleavage. (FIG. 5).
To generate amino acid changes at the first Valine residue of the KEX2 pre-sequence site (VAVEKR, SEQ ID NO: 9), a degenerate primer (GGACTAGTNNSGCCGTCGAGAAGCGCGACATCCAGATGACCCAG AG; SEQ ID NO:17) was synthesized which was used in a PCR reaction with reverse primer CTAAAGGGAACAAAAGCTGGAGC (SEQ ID NO:14) to amplify DNA to generate PCR fragment with multiple sequences. The mixed PCR fragments were cloned into Trichoderma expression vector (pTrex4). 30 clones were sequenced and 13 variants (MAVEKR (SEQ ID NO: 36), GAVEKR (SEQ ID NO: 37), AAVEKR (SEQ ID NO:38), LAVEKR (SEQ ID NO: 39), WAVEKR (SEQ ID NO: 40), KAVEKR (SEQ ID NO: 41), PAVEKR (SEQ ID NO: 42), RAVEKR (SEQ ID NO: 43), NAVEKR (SEQ ID NO: 44), TAVEKR (SEQ ID NO: 45), SAVEKR (SEQ ID NO: 46), QAVEKR (SEQ ID NO: 47) and EAVEKR (SEQ ID NO: 48)) were produced A new primer was designed, synthesized (GGACTAGTNWCGCCGTCGAGAAGCGCGACATCCAGATGACCCAGAG SEQ ID NO:18) and used in a PCR reaction with reverse primer CTAAAGGGAACAAAAGCTGGAGC (SEQ ID NO:14) to amplify DNA to generate PCR fragment with multiple sequences. The mixed PCR fragments were cloned into Trichoderma expression vector (pTrex4). 19 clones were sequenced and 5 more variants (YAVEKR (SEQ ID NO: 49), FAVEKR (SEQ ID NO: 50), DAVEKR (SEQ ID NO: 51), HAVEKR (SEQ ID NO: 52) and IAVEKR (SEQ ID NO: 53)) were produced. The plasmids containing the following 11 variants (MAVEKR (SEQ ID NO: 36), GAVEKR (SEQ ID NO: 37), AAVEKR (SEQ ID NO: 38), LAVEKR (SEQ ID NO:39), WAVEKR (SEQ ID NO: 40), KAVEKR (SEQ ID NO: 41), PAVEKR (SEQ ID NO: 42), HAVEKR (SEQ ID NO: 52), DAVEKR (SEQ ID NO: 51), SAVEKR (SEQ ID NO: 46) and QAVEKR (SEQ ID NO: 47)) were transformed biolistically into the T. reesei strain.
More than 20 transformants were obtained for each variant and transferred to new plates. More than 8 stable transformants for each variant were selected and grown in Proflo media for 2 days at 28° C. 5 mls of 2 days old culture from Proflo were transferred to 50 mls of Define media. The cultures were grown for 4 days at 28° C. Culture broths were centrifuged and supernatants were analyzed by protein SDS-PAGE. One transformant (the best producing transformant) from each variant was selected. Western analysis indicated (FIG. 6) that all variants produced light chain. All showed less than 95% cleavage except LAVEKR (SEQ ID NO: 39). This variant showed more efficient KEX2 cleavage than the VAVEKR (SEQ ID NO: 9) variant.
To generate amino acid changes at the Alanine residue of the KEX2 region (VAVEKR, (SEQ ID NO: 9)), a degenerate primer (GGACTAGTGTCNNSGTTGAGAAAGGCGACATCCAGATGACCCA GAGC; SEQ ID NO:19) was synthesized which was used in a PCR reaction with reverse primer (SEQ ID NO:14) to amplify DNA to generate PCR fragment with multiple sequences. The mixed PCR fragments were cloned into Trichoderma expression vector (pTrex4). 96 clones were sequenced and 15 variants (VDVEKR (SEQ ID NO: 54), VEVEKR (SEQ ID NO: 55), VGVEKR (SEQ ID NO: 56), VIVEKR (SEQ ID NO: 57), VKVEKR (SEQ ID NO: 58), VLVEKR (SEQ ID NO: 59), VMVEKR (SEQ ID NO: 60), VNVEKR (SEQ ID NO: 61), VPVEKR (SEQ ID NO:62), VRVEKR (SEQ ID NO: 63), VSVEKR (SEQ ID NO: 64), VTVEKR (SEQ ID NO: 65), VVVEKR (SEQ ID NO: 66), VWVEKR (SEQ ID NO: 67) and VYVEKR (SEQ ID NO: 68)) were produced. 5 plasmids were transformed biolistically into the T. reesei strain. More than 20 transformants for each variant were obtained and transferred to new plates. For this first set of 5 variants (VGVEKR (SEQ ID NO: 56), VTVEKR (SEQ ID NO: 65), VWVEKR (SEQ ID NO: 67), VEVEKR (SEQ ID NO: 55) and VPVEKR (SEQ ID NO: 62)), 10 stable transformants were selected. For the second set of 4 variants (VKVEKR (SEQ ID NO: 58), VRVEKR (SEQ ID NO: 63), VVVEKR (SEQ ID NO: 66) and VIVEKR (SEQ ID NO: 57)), 10 stable transformants were selected. The selected transformants were grown in Proflo media for 2 days at 28° C. 5 mls of 2 days old culture from Proflo were transferred to 50 mls of Define media. The cultures were grown for 4 days at 28° C. Culture broths were centrifuged and supernatants are analyzed by protein SDS gel. One transformant (the best producing transformant) from each variant was selected to be compared (table 1). Western analysis (FIG. 7) indicated that only the free light chain could be detected in the three variants: VGVEKR (SEQ ID NO: 56); VEVEKR (SEQ ID NO: 55) and VWVEKR (SEQ ID NO: 67). The variant VPVEKR (SEQ ID NO: 62) produced less free light chain and some uncleaved CBHI-light fusion.
To generate amino acid changes at the second valine residue of the KEX2 site (VAVEKR, SEQ ID NO: 9), a degenerate primer (GGACTAGTGTCGCCNNSGAGAAACGCGACATCCAGATGACCCAG AG; SEQ ID NO:20) was synthesized which was used in a PCR reaction with reverse primer (SEQ ID NO:14) to amplify DNA to generate PCR fragment with multiple sequences. The mixed PCR fragments were cloned into Trichoderma expression vector (pTrex4). 36 clones were sequenced and 15 variants (VAAEKR (SEQ ID NO: 69), VADEKR (SEQ ID NO: 70), VAEEKR (SEQ ID NO: 71), VAFEKR (SEQ ID NO: 72), VAGEKR (SEQ ID NO: 73), VAIEKR (SEQ ID NO: 74), VALEKR (SEQ ID NO: 75), VANEKR (SEQ ID NO: 76), VAQEKR (SEQ ID NO: 77), VAREKR (SEQ ID NO: 78), VASEKR (SEQ ID NO: 79), VATEKR (SEQ ID NO: 80), VAWEKR (SEQ ID NO: 81), VAYEKR (SEQ ID NO: 82) and VAPEKR (SEQ ID NO: 83)) were produced. Plasmids were transformed biolistically into the T. reesei strain. More than 20 transformants for each variant were obtained and transferred to new plates. For the first set of 8 variants (VAAEKR (SEQ ID NO: 69), VADEKR (SEQ ID NO: 70), VAEEKR (SEQ ID NO: 71), VAFEKR (SEQ ID NO: 72), VAGEKR (SEQ ID NO: 73), VANEKR (SEQ ID NO: 76), VALEKR (SEQ ID NO: 75) and VAIEKR (SEQ ID NO: 74)), 10 stable transformants were selected. For the second set of 2 variants (VASEKR (SEQ ID NO: 79) and VAREKR (SEQ ID NO: 78), 8 stable transformants were selected. Only 4 transformants were selected for VAPEKR (SEQ ID NO: 83) variant. The selected transformants were grown in Proflo media for 2 days at 28° C. 5 mls of 2 days old culture from Proflo were transferred to 50 mls of Define media. The cultures were grown for 4 days at 28° C. Culture broths were centrifuged and supernatants were analyzed. One transformant (the best producing transformant) from each variant was selected to be compared (Table 1). Western analysis (FIG. 8) indicated that VAIEKR (SEQ ID NO: 74) and VALEKR (SEQ ID NO: 75) generated complete cleavage of the fusion polypeptide since a fusion band was not observed in the gel. Western blot (FIG. 8) indicated that VAFEKR (SEQ ID NO: 72) produced the highest amount of antibody light chain even though the cleavage was not 100%.










TABLE 1







MAVEKR
VKVEKR
VAAEKR
VAVWKR


(SEQ ID NO: 36)
(SEQ ID NO: 58)
(SEQ ID NO: 69)
(SEQ ID NO: 25)


 


GAVEKR
VRVEKR
VADEKR
VAVGKR


(SEQ ID NO: 37)
(SEQ ID NO: 63)
(SEQ ID NO: 70)
(SEQ ID NO:26)


 


AAVEKR
VVVEKR
VAEEKR
VAVRKR


(SEQ ID NO: 38)
(SEQ ID NO: 66)
(SEQ ID NO: 71)
(SEQ ID NO:27)


 


LAVEKR
VIVEKR
VAFEKR
VAVTKR


(SEQ ID NO: 39)
(SEQ ID NO: 57)
(SEQ ID NO: 72)
(SEQ ID NO:28)


 


WAVEKR
VEVE
VAGEKR
VAVVKR


(SEQ ID NO: 40)
(SEQ ID NO: 55)
(SEQ ID NO: 73)
(SEQ ID NO: 29)


 


KAVEKR
VGVEKR
VAIEKR
VAVAKR


(SEQ ID NO: 41)
(SEQ ID NO: 56)
(SEQ ID NO: 74)
(SEQ ID NO: 30)


 


PAVEKR
VPVEKR
VALEKR
VAVLKR


(SEQ ID NO: 42)
(SEQ ID NO: 62)
(SEQ ID NO: 75)
(SEQ ID NO: 31)


 


SAVEKR
VTVEKR
VANEKR
VAVDKR


(SEQ ID NO: 46)
(SEQ ID NO: 65)
(SEQ ID NO: 76)
(SEQ ID NO: 32)


 


QAVEKR
VWVEKR
VASEKR
VAVNKR


(SEQ DI NO: 47)
(SEQ ID NO: 67)
(SEQ ID NO: 79)
(SEQ ID NO: 33)


 


DAVEKR

VAREKR
VAVYKR


(SEQ ID NO: 51)

(SEQ ID NO: 78)
(SEQ ID NO: 34)


 


HAVEKR

VAPEKR
VAVHKR


(SEQ ID NO: 52)

(SEQ ID NO: 83)
(SEQ ID NO: 35)









Example 6
Construction of a Trastuzumab Light Chain Expression Strain Containing the NVISKR (SEQ ID NO: 22) KEX2 Region
A NVISKR KEX2 region is found naturally in the prosequence of the A. niger glucoamylase (glaA). To construct a fusion polypeptide an oligo, GGACTAGTAACGTCATCAGCAAGCGCGACATCCAGATGACCCAGAGC (SEQ ID NO. 21) was synthesized by Invitrogen and used to amplify light chain DNA with reverse primer (SEQ ID NO. 14), The resulting PCR fragment encodes light chain and NVISKR (SEQ ID NO:22) sequence for kex2 cleavage. The PCR fragment was digested with restriction enzymes SpeI and AscI and cloned to expression Vector pTrex4 to generate a plasmid named as pTrex4-NVIS-her2 light chain geneart (KR-TR). Fidelity of the PCR fragment was analyzed by DNA sequencing. The plasmid was transformed biolistically into the Trichoderma reesei strain. More than 20 transformants were obtained and transferred to new plates. 10 stable transformants were selected to grow in Proflo media for 2 days at 30° C. 5 mls of 2 days old culture from Proflo were transferred to 50 mls of Define media. The cultures were grown for 5 days at 28° C. Culture broths were centrifuged and supernatants were used for protein analysis. Western analysis indicated that more than 95% of the fusion proteins were cleaved (FIG. 6).
Example 7
Construction of a Trastuzumab Light Chain Expression Strain Containing the SDVTKR (SEQ ID NO: 24) KEX2 Region
An oligo, GGACTAGTAGCGACGTCACCAAGCGCGACATCCAGATGACCCAGAGC (SEQ ID NO: 23) was synthesized by Invitrogen and used to amplify light chain DNA with reverse primer (SEQ ID NO: 14), The resulting PCR fragment encodes light chain and SDVTKR (SEQ ID NO: 24) sequence for kex2 cleavage. The PCR fragment was digested with restriction enzymes SpeI and AscI and cloned to expression Vector pTrex4 to generate a plasmid named as pTrex4-SDVT-her2 light chain geneart (KR-TR). Fidelity of the PCR fragment was analyzed by DNA sequencing. The plasmid was transformed biolistically into the Trichoderma reesei strain. More than 20 transformants were obtained and transferred to new plates. 10 stable transformants were selected to grow in Proflo media for 2 days at 30° C. 5 mls of 2 days old culture from Proflo were transferred to 50 mls of Define media. The cultures were grown for 5 days at 28° C. Culture broths were centrifuged and supernatants were used for protein analysis. Western analysis indicated that more than 50% of the fusion proteins were cleaved (FIG. 6).
Example 8
Construction of a Trastuzumab Light Chain Expression Strain Containing the VAVEKR (SEQ ID NO: 9) KEX2 Region in Aspergillus Niger 
The plasmid (pTrex4-VAVE-her2 light chain geneart (KR-TR) from Example 5 was digested with SpeI and AscI. The end of the DNA fragment of the AscI cutting site was blunted by T4 DNA polymerase. The fragment was isolated on a 1.2% agarose gel and ligated to A. niger expression plasmid (pSLGAMpR2-BBI as disclosed in US Patent Publication No. 2005 0153399) which was cut with NheI and BstEII with the BstEII end blunted with T4 DNA polymerase. The new plasmid, named pSLGAMpR2-VAVE-her2 LC geneart was transformed into A. niger strain dgr246:Δamy5;pyr—which is derived from the dgr246:ΔGAP:pyr—strain disclosed in US Pat. Pub. 20050153399. The difference being that the protein level of α-amylase is greatly reduced in this plasmid because of a mutation.
The dgr246ΔGAP:pyr2- is derived from strain dgr246 P2 which has the pepA gene deleted, is pyrG minus and has undergone several rounds of mutagenesis and screening or selection for improved production of a heterologous gene product (Ward, M. et al., 1993, Appl. Microbiol. Biotech. 39:738-743 and references therein). To create strain dgr246ΔGAP:pyr2- the glaA (glucoamylase) gene was deleted in strain dgr246 P2 using exactly the same deletion plasmid (pΔGAM NB-Pyr) and procedure as reported by Fowler, T. et al (1990) Curr. Genet. 18:537-545. Briefly, the deletion was achieved by transformation with a linear DNA fragment having glaA flanking sequences at either end and with part of the promoter and coding region of the glaA gene replaced by the Aspergillus nidulans pyrG gene as selectable marker. Transformants in which the linear fragment containing the glaA flanking sequences and the pyrG gene had integrated at the chromosomal glaA locus were identified by Southern blot analysis. This change had occurred in transformed strain dgr246ΔGAP. Spores from this transformant were plated onto medium containing fluoroorotic acid and spontaneous resistant mutants were obtained as described by van Hartingsveldt, W. et al. (1987) Mol. Gen. Genet. 206:71-75. One of these, dgr246ΔGAP:pyr2-, was shown to be a uridine auxotroph strain which could be complemented by transformation with plasmids bearing a wild-type pyrG gene.
More than 20 transformants were obtained and transferred to new plates. 17 transformants were grown in Promosoy medium for 5 days at 28° C. Culture broths were centrifuged and supernatants were used for protein SDS PAGE and Western analysis. Data indicated that all transformants produced antibody light chain. The transformant #A12 produced the most antibody light chain, and 60-70% of the fusion protein was cleaved (FIG. 9).
The preceding description merely illustrates principles of exemplary embodiments. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions.
Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.

## Claims

1. A fusion DNA construct encoding a fusion polypeptide comprising in operable linkage from the 5′ end of said construct,
a promoter,
a first DNA molecule, which is different from any one of the second, the third, and the fourth DNA molecules, encoding a signal sequence,
a second DNA molecule, which is different from any one of the first, the third, and the fourth DNA molecules, encoding a carrier protein,
a third DNA molecule, which is different from any one of the first, the second, and the fourth DNA molecules, encoding a KEX2 region, said region comprising a KEX2 site (B1B2) and a KEX2 site pre-sequence X4X3X2X1 immediately 5′ to the KEX2 site, wherein X4 is V, S, N, L, or K, X3 is A, V, D, W, E, or P; X2 is V, I, L or F; and X1 is E, S, T or Y, wherein the pre-sequence is not NVIS (SEQ ID NO:85; and
a fourth DNA molecule, which is different from any one of the first, the second, and the third DNA molecules, encoding a protein.

2. The fusion DNA construct of claim 1, wherein the first DNA molecule and second DNA molecule encode a Trichoderma CBH1 signal sequence and carrier protein or a Trichoderma endoglucanase signal sequence and carrier protein.

3. The fusion DNA construct of claim 1, wherein the first DNA molecule and second DNA molecule encode a glucoamylase signal sequence and carrier protein or an alpha amylase signal sequence and carrier protein.

4. The fusion DNA construct of claim 1, wherein the desired protein is an enzyme.

5. The fusion DNA construct of claim 1, wherein the desired protein is a therapeutic protein.

6. The fusion DNA construct of claim 5, wherein the therapeutic protein is an antibody.

7. The fusion DNA construct of claim 6, wherein the antibody is a light chain or heavy chain monoclonal antibody.

8. The fusion DNA construct of claim 1, wherein the first DNA molecule and second DNA molecule encode a CBH I signal sequence and carrier protein and the fourth DNA molecule encodes an antibody light chain or fragment thereof.

9. The fusion DNA construct of claim 1, wherein the first DNA molecule and second DNA molecule encode a glucoamylase signal sequence and carrier protein and the fourth DNA molecule encodes an antibody light chain or fragment thereof.

10. A host cell comprising the fusion DNA construct of claim 1.

11. The host cell of claim 10, wherein said host cell is a Trichoderma host cell.

12. The host cell of claim 11, wherein the Trichoderma cell is a T. reesei cell.

13. A vector comprising the fusion DNA construct of claim 1.

14. A host cell comprising the vector of claim 13.

15. The host cell of claim 14, wherein the host cell is a Trichoderma host cell.

16. A process for producing a desired protein in a filamentous fungal cell comprising:
a) obtaining a filamentous fungal host cell comprising a fusion DNA construct according to claim 1;
b) culturing the host cell under suitable conditions which allow for the expression and production of the desired protein; and
c) recovering the desired protein.

17. The process according to claim 16, wherein the host cell is a Trichoderma strain.

18. The process according to claim 17, wherein the Trichoderma cell is a T. reesei host cell.

19. The process according to claim 16, wherein the desired protein is an immunoglobulin.

20. The process according to claim 19, wherein the immunoglobulin is a monoclonal antibody.

21. The process according to claim 20, wherein the monoclonal antibody is a light chain or heavy chain monoclonal antibody or fragment thereof.

22. The process according to claim 20, wherein the host cell is a Trichoderma cell, the desired protein is a light chain antibody and the KEX2 region of the third DNA molecule is a sequence selected from VAVWKR (SEQ ID NO: 25), VAVGKR (SEQ ID NO: 26), VAVTKR (SEQ ID NO: 28), VAVVKR (SEQ ID NO: 29), VAVDKR (SEQ ID NO: 32), WAVEKR (SEQ ID NO: 40), PAVEKR (SEQ ID NO: 42), QAVEKR (SEQ ID NO: 47), HAVEKR (SEQ ID NO: 52), and VASEKR (SEQ ID NO: 79).

23. The method according to claim 18, wherein the KEX2 region of the third DNA molecule is a sequence selected from VAVWKR (SEQ ID NO: 25), VAVGKR (SEQ ID NO: 26), VAVTKR (SEQ ID NO: 28), VAVVKR (SEQ ID NO: 29), VAVDKR (SEQ ID NO: 32), WAVEKR (SEQ ID NO: 40), PAVEKR (SEQ ID NO: 42), QAVEKR (SEQ ID NO: 47), HAVEKR (SEQ ID NO: 52), and VASEKR (SEQ ID NO: 79).