U.S. patent application number 11/484814 was filed with the patent office on 2008-01-31 for kex2 cleavage regions of recombinant fusion proteins.
Invention is credited to Huaming Wang, Michael Ward.
Application Number | 20080026376 11/484814 |
Document ID | / |
Family ID | 38986752 |
Filed Date | 2008-01-31 |
United States Patent
Application |
20080026376 |
Kind Code |
A1 |
Wang; Huaming ; et
al. |
January 31, 2008 |
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.
Inventors: |
Wang; Huaming; (Palo Alto,
CA) ; Ward; Michael; (Palo Alto, CA) |
Correspondence
Address: |
LYNN MARCUS-WYNER;GENECOR INTERNATIONAL, INC.
925 PAGE MILL ROAD
PALO ALTO
CA
94304-1013
US
|
Family ID: |
38986752 |
Appl. No.: |
11/484814 |
Filed: |
July 11, 2006 |
Current U.S.
Class: |
435/6.15 ;
435/254.6; 435/484; 435/69.3; 530/350; 536/23.2 |
Current CPC
Class: |
C12N 15/80 20130101;
C12N 15/62 20130101; C07K 16/462 20130101; C07K 16/32 20130101;
C07K 16/46 20130101; C07K 2319/00 20130101 |
Class at
Publication: |
435/6 ; 435/69.3;
530/350; 435/484; 435/254.6; 536/23.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; C12N 15/74 20060101
C12N015/74; C07K 14/37 20060101 C07K014/37; C12N 1/16 20060101
C12N001/16 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0001] 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.
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 encoding a signal sequence, a second DNA
molecule encoding a carrier protein, a third DNA molecule encoding
a KEX2 region, said region comprising a KEX2 site (B.sub.1B.sub.2)
and a KEX2 site pre-sequence ((X)n=2 to 6) immediately 5' to the
KEX2 site; and a fourth DNA molecule encoding a desired
protein.
2. The fusion DNA construct of claim 1, wherein the KEX2 region is
X.sub.4X.sub.3X.sub.2X.sub.1KR.
3. The fusion DNA construct of claim 2, wherein X.sub.4 is V, S, N,
L, or K; X.sub.3 is A, V, D, W, E or P; X.sub.2 is V, I, L or F;
and X.sub.1 is E, S, T or Y.
4. The fusion DNA construct of claim 3, wherein X.sub.4 is V.
5. The fusion DNA construct of claim 3, wherein X.sub.3 is A.
6. The fusion DNA construct of claim 3, wherein X.sub.2 is V.
7. The fusion DNA construct of claim 3, wherein X.sub.1 is E or
Y.
8. The fusion DNA construct of claim 3, wherein the KEX2 site
pre-sequence is selected from the group consisting of VAVE (SEQ ID
NO: 84); 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).
9. 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.
10. 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.
11. The fusion DNA construct of claim 1, wherein the desired
protein is an enzyme.
12. The fusion DNA construct of claim 1, wherein the desired
protein is a therapeutic protein.
13. The fusion DNA construct of claim 12, wherein the therapeutic
protein is an antibody.
14. The fusion DNA construct of claim 13, wherein the antibody is a
light chain or heavy chain monoclonal antibody.
15. The fusion DNA construct of claim 3, wherein the first DNA
molecule and second DNA molecule encode a CBH1 signal sequence and
carrier protein and the fourth DNA molecule encodes an antibody
light chain or fragment thereof.
16. The fusion DNA construct of claim 3, 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.
17. The fusion protein encoded by the fusion DNA construct of claim
1.
18. A host cell comprising the fusion DNA construct of claim 1.
19. The host cell of claim 18, wherein said host cell is a
Trichoderma host cell.
20. The host cell of claim 19, wherein the Trichoderma cell is a T.
reesei cell.
21. A vector comprising the fusion DNA construct of claim 1.
22. A host cell comprising the vector of claim 21.
23. The host cell of claim 22, wherein the host cell is a
Trichoderma host cell.
24. 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.
25. The process according to claim 24, wherein the host cell is a
Trichoderma strain.
26. The process according to claim 25, wherein the Trichoderma cell
is a T. reesei host cell.
27. The process according to claim 24, wherein the desired protein
is an immunoglobulin.
28. The process according to claim 27, wherein the immunoglobulin
is a monoclonal antibody.
29. The process according to claim 28, wherein the monoclonal
antibody is a light chain or heavy chain monoclonal antibody or
fragment thereof.
30. The process according to claim 28, wherein the host cell is a
Trichoderma cell, the desired protein is a light chain antibody and
the KEX2 region is X.sub.4X.sub.3X.sub.2X.sub.1KR and X.sub.4 is V
and X.sub.1 is E, S, T or Y.
31. A method for cleaving a desired protein from a recombinant
fusion polypeptide comprising expressing in a filamentous fungal
cell a fusion polypeptide encoded by a fusion DNA construct
according to claim 1, wherein said KEX2 region provides a protein
cleavage site and obtaining a desired protein which is cleaved from
the expressed fusion polypeptide.
32. The method according to claim 31, wherein the desired protein
is a therapeutic protein.
33. The method according to claim 31, wherein the desired protein
is an antibody.
34. The method according to claim 31, wherein the cleavage of the
desired protein is increased compared to the cleavage of said
desired protein from an equivalent fusion polypeptide lacking the
KEX2 site pre-sequence.
35. The method according to claim 26, wherein the KEX2 region is
X.sub.4X.sub.3X.sub.2X.sub.1KR and X.sub.4 is V and X.sub.1 is E,
S, T or Y.
36. A method for increasing the production of an antibody from a
filamentous fungal cell comprising obtaining a filamentous fungal
cell comprising a fusion DNA construct of claim 3, culturing the
fungal cell under suitable conditions for expression of the fusion
polypeptide and allowing secretion of the fusion polypeptide,
wherein the secretion of the desired protein is increased compared
to the secretion of an equivalent fusion polypeptide not including
a KEX2 pre-sequence.
37. The method of claim 36, wherein the secretion of the desired
protein is increased by at least 30% compared to the secretion of
the desired protein from the equivalent fusion polypeptide.
38. A fusion polypeptide comprising from an amino terminus of said
fusion polypeptide a first amino acid sequence comprising: a) a
signal sequence functional as a secretory sequence; b) a second
amino acid sequence comprising a carrier protein; c) a third amino
acid sequence comprising a KEX2 region, said region comprising a
KEX2 site (B.sub.1B.sub.2) and a KEX2 site pre-sequence ((X)n=2 to
6) immediately 5' to the KEX2 site; and d) a fourth amino acid
sequence comprising a desired protein. wherein B.sub.1 or B.sub.2
is K or R and X is any amino acid residue.
39. 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
(B.sub.1B.sub.2) and a KEX2 site pre-sequence ((X)n=4 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
recombinant fusion polypeptides that are identical to said parental
fusion polypeptide except for said KEX2 site pre-sequence; b)
evaluating secretion and/or cleavage of said test fusion
polypeptides and said parental fusion polypeptide by a filamentous
fungal cell; and c) identifying a test fusion polypeptide that has
enhanced secretion and/or cleavage as compared to said parental
fusion polypeptide.
40. The method according to claim 39 further comprising identifying
an optimized KEX2 site pre-sequence which comprises, testing a
plurality of different test fusion polypeptides, 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
recombinant fusion polypeptide that has the greatest secretion
and/or protein cleavage.
Description
FIELD OF THE INVENTION
[0002] 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
[0003] 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 .alpha.-factor pheromone and
killer toxin precursors during their secretion.
[0004] 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.
[0005] 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.
[0006] 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
[0007] 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.
[0008] 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.
[0009] 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.
[0010] In further embodiments, the invention relates to a KEX2
region (X.sub.4X.sub.3X.sub.2X.sub.1B.sub.1B.sub.2) comprising a
KEX2 site (B.sub.1B.sub.2) and a KEX2 site pre-sequence
(X.sub.4X.sub.3X.sub.2X.sub.1) immediately N-terminal to said KEX2
site.
[0011] 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.
[0012] 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.
[0013] 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
[0014] 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:
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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).
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] 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 A10, A11 and A12.
Definitions
[0024] 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.
[0025] 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.
[0026] 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).
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] 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.
[0032] A "promoter" is a regulatory sequence that is involved in
binding RNA polymerase to initiate transcription of a gene.
[0033] 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.
[0034] "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.
[0035] "Under translational control" is a term well understood in
the art that indicates a regulatory process which occurs after mRNA
has been formed.
[0036] 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.
[0037] 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.
[0038] The terms "protein" and "polypeptide" are used
interchangeably herein. The conventional one-letter or three-letter
code for amino acid residues is used herein.
[0039] The term "carrier protein" refers to a polypeptide sequence
or functional portion thereof from a naturally secreted fungal
polypeptide.
[0040] 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').sub.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.
[0041] 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 .gamma. 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).
[0042] 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.
[0043] The term "derived" encompasses the terms "originated from",
"obtained" or "obtainable from", and "isolated from".
[0044] "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.
[0045] 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.
[0046] The term "culturing" refers to growing a population of
microbial cells under suitable conditions in a liquid or solid
medium.
[0047] 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.
[0048] The term "homologous" with reference to a polynucleotide or
protein refers to a polynucleotide or protein that occurs naturally
in the host cell.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] The term "glycosylated" protein means a protein that has
oligosaccharide molecules added to particular amino acid residue on
the protein.
[0053] The term "non-glycosylated" protein is a protein that does
not have oligosaccharide molecules attached to the protein.
[0054] 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).
[0055] 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.
[0056] 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.
[0057] 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).
[0058] The term "KEX2 site pre-sequence" refers to the two to six
contiguous amino acids [(X).sub.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.
[0059] The term "variant" refers to a region of a protein that
contains one or more different amino acids as compared to a
reference protein.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] Other definitions of terms may appear throughout the
specification
DETAILED DESCRIPTION
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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--
[0069] 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.
[0070] 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--
[0071] 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.
[0072] 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, .alpha.-amylase, aspartyl
proteases, glucoamylase, phytase, mannanase, .alpha. and .beta.
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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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--
[0080] The KEX2 region comprises a KEX2 site (BIB.sub.2) and a KEX2
site pre-sequence ((X).sub.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.
[0081] 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.
[0082] The KEX2 site contains amino acid sequence "B.sub.1B.sub.2"
wherein B.sub.1 and B.sub.2, are independently, basic amino acids.
Preferably the KEX2 site includes any one of KK, KR, RK or RR and
more preferably is KR.
[0083] The KEX2 site pre-sequence comprises amino acid sequence
(X).sub.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).sub.n=2-6 amino acid residues on the C-terminus of
the carrier protein. However, the contiguous (X).sub.n=2-6 amino
acid residues may be found in other parts of the carrier protein
and may be linked with a KEX2 site (B.sub.1B.sub.2) but the KEX2
region will not be attached to the N-terminus of the desired
protein.
[0084] In some embodiments, when the KEX2 site pre-sequence is
defined as X.sub.4X.sub.3X.sub.2X.sub.1B.sub.1B.sub.2,
[0085] a) X.sub.1, X.sub.2 and X.sub.3 are not G;
[0086] b) X.sub.1 is not S, if X.sub.2 and X.sub.3 are G, X.sub.4
is A, or X.sub.3 is S;
[0087] c) X.sub.4 is not T, if X.sub.3 is A and X.sub.2 is S;
or
[0088] d) X.sub.1 is not D.
[0089] In some preferred embodiments, the KEX2 region is
X.sub.4X.sub.3X.sub.2X.sub.1B.sub.1B.sub.2 wherein B.sub.1B.sub.2
is KR and
[0090] a) X.sub.1, X.sub.2 and X.sub.3 are not G;
[0091] b) X.sub.1 is not S, if X.sub.2 and X.sub.3 are G, X.sub.4
is A, or X.sub.3 is S;
[0092] c) X.sub.4 is not T, if X.sub.3 is A and X.sub.2 is S;
or
[0093] d) d) X.sub.1 is not D.
[0094] In other embodiments, the KEX2 site pre-sequence is defined
as X.sub.4X.sub.3X.sub.2X.sub.1 wherein,
[0095] a) X.sub.4 is V, S, N, L, or K;
[0096] b) X.sub.3 is A, V, D, W, E or P;
[0097] c) X.sub.2 is V, I, L or F; and
[0098] d) X.sub.1 is E, S, T or Y.
[0099] In yet other embodiments the KEX2 site pre-sequence is
defined as X.sub.4X.sub.3X.sub.2X.sub.1 wherein,
[0100] a) X.sub.4 is V, N, or L;
[0101] b) X.sub.3 is A, V, D, W, E or P;
[0102] c) X.sub.2 is V, I, L or F; and
[0103] d) X.sub.1 is E or Y.
[0104] In yet further embodiments the
X.sub.4X.sub.3X.sub.2X.sub.1KR KEX2 region may be selected from the
group of X.sub.4 is V; X.sub.3 is A; X.sub.2 is V; X.sub.1 is E or
Y and combinations thereof.
[0105] 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); VAE (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).
[0106] 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).
[0107] While the preferred KEX2 region is defined as
X.sub.4X.sub.3X.sub.2X.sub.1B.sub.1B.sub.2 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
(X.sub.3X.sub.2X.sub.1B.sub.1B.sub.2 or
X.sub.2X.sub.1B.sub.1B.sub.2). In this embodiment,
[0108] a) X.sub.1, X.sub.2 and X.sub.3 are not G (e.g.
GGGB.sub.1B.sub.2 or GGB.sub.1B.sub.2),
[0109] b) X.sub.1 is not S, if X.sub.2 and X.sub.3 are G or X.sub.3
is S (e.g. SX.sub.2S or SGS); and
[0110] c) X.sub.1 is not D.
[0111] 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.
[0112] 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.
[0113] 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--
[0114] 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.
[0115] In one embodiment, the desired protein may be an enzyme such
as a carbohydrase, such as a starch hydrolyzing .alpha.-amylase, an
alkaline .alpha.-amylase, a .beta.-amylase, a cellulase; a
dextranase, an .alpha.-glucosidase, an .alpha.-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 .beta.-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 .alpha.-galactosidase, a
.beta.-galactosidase, a .beta.-glucosidase, a laccase, a
mannosidase, a mutanase, a pectinolytic enzyme, a
polyphenoloxidase, ribonuclease or transglutaminase.
[0116] 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-.alpha.,
interferon-.beta., interferon-.gamma., 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 .alpha.-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,
.alpha.-1-antitrypsin, .alpha.-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.
[0117] 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').sub.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).
[0118] 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--
[0119] 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--
[0120] 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 II.
Vectors--
[0121] 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.
[0122] 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.).
[0123] 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.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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); theA. oryzae or A. niger alpha amylase gene and the
Rhizomucor miehei carboxylprotease gene. Any fungal polyadenylation
sequence is likely to be functional in the present invention.
[0128] 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 Kinghom
et al (1992) Applied Molecular Genetics of Filamentous Fungi,
Blackie Academic and Professional, Chapman and Hall, London.
Host cells--
[0129] 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.
[0130] 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).
[0131] 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.
[0132] 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
[0133] 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.
[0134] 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.
[0135] 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.
[0136] 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.
[0137] 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.degree. C. in shake flasks on a rotary
shaker) until desired levels of immunoglobulin expression is
achieved.
Protein Production Methods
[0138] 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.
[0139] 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.
[0140] 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 CO.sub.2. Batch and fed-batch
fermentations are common and known in the art.
[0141] 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.
[0142] 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--
[0143] 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.
[0144] 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.
[0145] 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.
[0146] 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.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] In some embodiments the increase in secretion and/or
cleavage may be measured against a standard KEX2 region defined as
GGGB.sub.1B.sub.2, wherein BIB.sub.2 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.times.,
at least 3.times., at least 5.times., and at least 10.times.
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--
[0151] 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).
[0152] 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.
[0153] 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.
[0154] 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.
[0155] 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.
[0156] 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.
[0157] 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
[0158] 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 (.degree.
C.), pressure is at or near atmospheric and the following
abbreviations apply, M (Molar); .mu.M (micromolar); N (Normal); mol
(moles); mmol (millimoles); .mu.mol (micromoles); nmol (nanomoles);
g (grams); mg (milligrams) kg (kilograms); .mu.g (micrograms); L
(liters); ml (milliliters); h (hours); min (minutes); PAGE
(polyacrylamide gel electrophoresis); kDa (kilodaltons); and bp
(base pairs). The following assays and methods are used in the
examples provided below:
A. Construction of the pTrex4 Vector:
[0159] Synthetic DNA was cloned into a Trichoderma expression
vector (pTrex4) to generate appropriate expression plasmids for use
in the examples described below.
[0160] 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.
[0161] 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)
TABLE-US-00001 ATGTATCGGAAGTTGGCCGTCATCTCGGCCTTCTTGGCCACAGCTCGTGC
TCAGTCGGCCTGCACTCTCCAATCGGAGACTCACCCGCCTCTGACATGGC
AGAAATGCTCGTCTGGTGGCACTTGCACTCAACAGACAGGCTCCGTGGTC
ATCGACGCCAACTGGCGCTCGGCTGGACTCACGCTACGAACAGCAGCACG
AACTGCTACGATGGCAACACTTGGAGCTCGACCCTATGTCCTGACAACGA
GACCTGCGCGAAGAACTGCTGTCTGGACGGTGCCGCCTACGCGTCCACGT
ACGGAGTTACCACGAGCGGTAACAGCCTCTCCATTGGCTTTGTCACCCAG
TCTGCGCAGAAGAACGTTGGCGCTCGCCTTTACCTTATGGCGAGCGACAC
GACCTACCAGGAATTCACCCTGCTTGGCAACGAGTTCTCTTTCGATGTTG
ATGTTTCGCAGCTGCCGTAAGTGACTTACCATGAACCCCTGACGTATCTT
CTTGTGGGCTCCCAGCTGACTGGCCAATTTAAGGTGCGGCTTGAACGGAG
CTCTCTACTTCGTGTCCATGGACGCGGATGGTGGCGTGAGCAAGTATCCC
ACCAACACCGCTGGCGCCAAGTACGGCACGGGGTACTGTGACAGCCAGTG
TCCCCGCGATCTGAAGTTCATCAATGGCCAGGCCAACGTTGAGGGCTGGG
AGCCGTCATCCAACAACGCAAACACGGGCATTGGAGGACACGGAAGCTGC
TGCTCTGAGATGGATATCTGGGAGGCCAACTCCATCTCCGAGGCTCTTAC
CCCCCACCCTTGCACGACTGTCGGCCAGGAGATCTGCGAGGGTGATGGGT
GCGGCGGAACTTACTCCGATAACAGATATGGCGGCACTTGCGATCCCGAT
GGCTGCGACTGGAACCCATACCGCCTGGGCAACACCAGCTTCTACGGCCC
TGGCTCAAGCTTTACCCTCGATACCACCAAGAAATTGACCGTTGTCACCC
AGTTCGAGACGTCGGGTGCCATCAACCGATACTATGTCCAGAATGGCGTC
ACTTTCCAGCAGCCCAACGCCGAGCTTGGTAGTTACTCTGGCAACGAGCT
CAACGATGATTACTGCACAGCTGAGGAGGCAGAATTCGGCGGATCCTCTT
TCTCAGACAAGGGCGGCCTGACTCAGTTCAAGAAGGCTACCTCTGGCGGC
ATGGTTCTGGTCATGAGTCTGTGGGATGATGTGAGTTTGATGGACAAACA
TGCGCGTTGACAAAGAGTCAAGCAGCTGACTGAGATGTTACAGTACTACG
CCAACATGCTGTGGCTGGACTCCACCTACCCGACAAACGAGACCTCCTCC
ACACCCGGTGCCGTGCGCGGAAGCTGCTCCACCAGCTCCGGTGTCCCTGC
TCAGGTCGAATCTCAGTCTCCCAACGCCAAGGTCACCTTCTCCAACATCA
AGTTCGGACCCATTGGCAGCACCGGCAACCCTAGCGGCGGCAACCCTCC
CGGCGGAAACCCGCCTGGCACCACCACCACCCGCCGCCCAGCCACTACCA
CTGGAAGCTCTCCCGGACCTACTAGT
[0162] The amino acid sequence of the T. reesei cbh1 signal
sequence; catalytic domain; linker (480 amino acids) is represented
below (SEQ ID NO: 105)
TABLE-US-00002 MYRKLAVISAFLATARAQSACTLQSETHPPLTWQKCSSGGTCTQQTGSVV
IDANWRWTHATNSSTNCYDGNTWSSTLCPDNETCAKNCCLDGAAYASTYG
VTTSGNSLSIGFVTQSAQKNVGARLYLMASDTTYQEFTLLGNEFSFDVDV
SQLPCGLNGALYFVSMDADGGVSKYPTNTAGAKYGTGYCDSQCPRDLKFI
NGQANVEGWEPSSNNANTGIGGHGSCCSEMDIWEANSISEALTPHPCTTV
GQEICEGDGCGGTYSDNRYGGTCDPDGCDWNPYRLGNTSFYGPGSSFTLD
TTKKLTVVTQFETSGAINRYYVQNGVTFQQPNAELGSYSGNELNDDYCTA
EEAEFGGSSFSDKGGLTQFKKATSGGMVLVMSLWDDYYANMLWLDSTYPT
NETSSTPGAVRGSCSTSSGVPAQVESQSPNAKVTFSNIKFGPIGSTGNPS
GGNPPGGNPPGTTTTTRRPATTTGSSPGPTS
[0163] 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:
[0164] In all examples below transformation was performed on a
derivative of the quad deleted (.DELTA.chba1, .DELTA.cbh2,
.DELTA.egl1, and .DELTA.egl2) 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.
[0165] A suspension of spores (approximately 5.times.10.sup.8
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 KH.sub.2PO.sub.4; 0.6 g/L CaCl.sub.2.2H.sub.2O; 1 ml/L
1000.times. trace elements solution; 20 g/L Noble agar; pH 5.5.
1000.times. trace elements solution contained 5.0 g/l
FeSO.sub.4.7H.sub.2O, 1.6 g/l MnSO.sub.4.H.sub.2O, 1.4 g/l
ZnSO.sub.4.7H.sub.2O and 1.0 g/l CoCl.sub.2.6H.sub.2O. The spore
suspension was allowed to dry on the surface of the MM acetamide
medium.
[0166] Transformation of the Trichoderma strain by the biolistic
transformation method was accomplished using a Biolistic.RTM.
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--
[0167] 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 USPat. 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:
[0168] 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 .alpha.-lactose; 6.5 g/L
(NH4).sub.2SO.sub.4; 2 g/L KH.sub.2PO.sub.4; 0.3 g/L
MgSO.sub.4.7H.sub.2O; 0.2 g/L CaCl.sub.2; 1 ml/L 1000.times. 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
CaCO.sub.3. F. Defined Media contains: 5 g/L (NH4).sub.2SO.sub.4;
33 g/L PIPPS buffer; 9 g/L casamino acids; 4.5 g/L
KH.sub.2PO.sub.4; 1 g/L CaCl.sub.2; 1 g/L MgSO.sub.4.7H.sub.2O; 5
ml/L Mazu DF60-P antifoam (Mazur Chemicals, Gurnee, Ill.); 1 mL
1000.times. trace elements solution. After autoclaving 40 ml of 40%
lactose was added. G. 1000.times. trace elements solution contains:
5.0 gA FeSO.sub.4.7H.sub.2O, 1.6 g/l MnSO.sub.4.H.sub.2O, 1.4 g/l
ZnSO.sub.4.7H.sub.2O and 1.0 g/l CoCl.sub.2.6H.sub.2O
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
[0169] 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).
TABLE-US-00003 (SEQ ID NO: 1)
ACTAGTAAACGCGGTGGCGGTGATATTCAAATGACACAATCTCCTTCTTC
TCTGTCAGCCTCAGTGGGCGACCGTGTGACGATTACTTGCCGCGCCTCTC
AGGACGTTAACACTGCCGTCGCATGGTACCAGCAGAAGCCAGGCAAGGCG
CCCAAGCTTCTGATTTACAGCGCTTCGTTCCTGTACTCTGGCGTGCCATC
CCGCTTCTCTGGCAGCCGAAGCGGCACGGATTTCACCCTGACCATTTCGT
CCCTGCAGCCCGAGGATTTCGCCACGTATTACTGCCAGCAGCACTACACC
ACTCCACCCACCTTTGGCCAAGGAACGAGAGTCGAAATCACTCGCACGGT
CGCTGCCCCTTCAGTCTTCATCTTCCCCCCCAGCGACGAACAGCTGAAGT
CTGGTACGGCCAGCGTCGTTTGCTTGCTTAATAACTTCTATCCGCGAGAG
GCGAAGGTCCAATGGAAGGTTGATAACGTTCTGCAGTCCGGCAATTCGCA
GGAGAGCGTGACCGAGCAGGATTCAAAGGATAGCACCTACTCACTCAGCA
GCACCCTGACGTTGTCCAAGGCCGATTACGAGAAGCATAAGTTGTATGCA
TGCGAGGTCACCCACCAGGGACTGTCAAGCCCAGTTACCAAGTCGTTCAA
TCGAGGCGAGTGCTAAGGCGCGCC.
[0170] 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.degree. 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.degree. 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
(transformant1010-18, 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
[0171] 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.
[0172] 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.degree. 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.degree. 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 CBH1
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
[0173] 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.degree. 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.degree. 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
[0174] 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
polypetide 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.
[0175] 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.degree. 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.degree. 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
[0176] DNA (SEQ ID NO: 10) encoding the trastuzumab antibody light
chain was synthesized by Geneart (Josef-Engert-Strasse 11, 93053
Regesburg, Germany).
TABLE-US-00004 (SEQ ID NO: 10)
ACTAGTAAGCGCGGCGGCGGCGAGGTCCAGCTCGTCGAGAGCGGCGGCG
GCCTCGTCCAGCCCGGCGGCAGCCTCCGCCTCAGCTGCGCCGCCAGCGGC
TTCAACATCAAGGACACCTACATCCACTGGGTCCGCCAGGCCCCCGGCAA
GGGCCTCGAGTGGGTCGCCCGCATCTACCCCACCAACGGCTACACCCGCT
ACGCCGACAGCGTCAAGGGCCGCTTCACCATCAGCGCCGACACCAGCAAG
AACACCGCCTACCTCCAGATGAACAGCCTCCGCGCCGAGGACACCGCCGT
CTACTACTGCAGCCGCTGGGGCGGCGACGGCTTCTACGCCATGGACTACT
GGGGCCAGGGCACCCTCGTCACGGTCTCCAGCGCCAGCACCAAGGGCCCA
AGCGTCTTTCCCCTCGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACCGC
CGCCCTCGGCTGCCTCGTCAAGGACTACTTCCCCGAGCCCGTCACTGTCA
GCTGGAACAGCGGCGCTCTCACCAGCGGCGTCCACACCTTCCCCGCCGTC
CTCCAGAGCAGCGGCCTCTACAGCCTCAGCAGCGTCGTCACCGTCCCCAG
CAGCAGCCTCGGCACCCAGACCTACATCTGCAACGTCAACCACAAGCCCA
GCAACACCAAGGTCGACAAGCGCGTCGAGCCCAAGAGCTGCGACAAGACC
CACACCTGCCCCCCCTGCCCCGCCCCCGAGCTGCTCGGCGGCCCCTCCGT
CTTCTCTTCCCCCCCAAGCCCAAGGACACCCTCATGATCAGCCGCACCCC
CGAGGTCACCTGCGTCGTCGTCGATGTCAGCCACGAGGACCCCGAGGTCA
AGTTCAACTGGTACGTCGACGGCGTCGAGGTCCACAACGCCAAGACCAAG
CCCCGCGAGGAGCAGTACAACAGCACCTACCGCGTCGTCAGCGTCCTGAC
CGTCCTCCACCAGGACTGGCTCAACGGCAAGGAGTACAAGTGCAAGGTCT
CCAACAAGGCCCTCCCCGCCCCCATCGAAAAGACCATCAGCAAGGCCAAG
GGCCAGCCCCGCGAGCCCCAGGTCTACACCCTCCCCCCCAGCCGCGAGGA
GATGACCAAGAACCAGGTCTCCCTCACCTGCCTGGTCAAGGGCTTCTACC
CCAGCGACATCGCCGTCGAGTGGGAGAGCAACGGCCAGCCCGAGAACAAC
TACAAGACCACCCCCCCCGTCCTCGACAGCGACGGCAGCTTCTTCCTCTA
CAGCAAGCTCACCGTCGACAAGAGCCGCTGGCAGCAGGGCAACGTCTTTA
GCTGCAGCGTCATGCACGAGGCCCTCCACAACCACTACACCCAGAAGAGC
CTCAGCCTCAGCCCCGGCAAGTAAGGCGCG
[0177] 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)
[0178] 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.degree. 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.degree. 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).
[0179] 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.degree.
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.degree. C. Culture broths were centrifuged and supernatants were
used for protein analysis.
[0180] 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.degree. 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.degree. C. Culture broths were
centrifuged and supernatants were used for protein analysis.
[0181] 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).
[0182] 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.
[0183] 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.degree. 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.degree. 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.
[0184] 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.degree. 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.degree. 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.
[0185] 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.degree. 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.degree. 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-US-00005 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
[0186] A NVISKR KEX2 region is found naturally in the prosequence
of the A. niger glucoainylase (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.degree. 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.degree. 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
[0187] 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.degree. 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.degree. 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
[0188] 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:.DELTA.amy5;pyr- which is derived from the
dgr246:.DELTA.GAP:pyr- strain disclosed in US Pat. Pub.
20050153399. The difference being that the protein level of
.alpha.-amylase is greatly reduced in this plasmid because of a
mutation.
[0189] The dgr246.DELTA.GAP: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 dgr246.DELTA.GAP:pyr2- the glaA
(glucoamylase) gene was deleted in strain dgr246 P2 using exactly
the same deletion plasmid (p.DELTA.GAM 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 dgr246.DELTA.GAP.
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, dgr246.DELTA.GAP:pyr2-, was shown
to be a uridine auxotroph strain which could be complemented by
transformation with plasmids bearing a wild-type pyrG gene.
[0190] More than 20 transformants were obtained and transferred to
new plates. 17 transformants were grown in Promosoy medium for 5
days at 28.degree. 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).
[0191] 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.
[0192] 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.
* * * * *