The invention relates to systems and methods for protein production
TECHNICAL FIELD
The invention relates to expression systems and methods of using the same for
producing proteins of interest.
BACKGROUND
Secreted and membrane proteins undergo folding and other post-translational
modifications in the endoplasmic reticulum (ER)-Golgi system. Disruption of the
homeostasis of this system causes cellular stress that can lead to apoptosis. ER
homeostasis can be altered by changes in Ca2+ concentration or redox status, altered
glycosylation, or accumulation of unfolded or misfolded proteins in the ER lumen. To
overcome stress, the secretory system has evolved an adaptive stress response
mechanism known as the unfolded protein response (UPR). Activation of the
mammalian UPR results in at least three responses: (1) the amount of new protein
translocated to the ER lumen is reduced through reduction in translation; (2)
accumulated protein in the ER lumen is re trotrans located to the cytosol and degraded;
and (3) the ER-Golgi secretory system is remodeled so that the protein folding and
processing capacities in the system are enhanced.
The capacity enhancement of the ER-Golgi system in response to stress involves
upregulation of folding and processing enzymes. These enzymes include ER
chaperones, enzymes involved in glycosylation and disulfide bond formation, and
enzymes participating in vesicle transportation. In mammalian cells, IRE1 and ATF6
proteins are the major transducers of this branch of the UPR pathway. IRE1 protein is an
ER transmembrane glycoprotein with kinase and endonuclease activities at its C-terminal
cytosolic domain. At least two IRE1 genes have been identified in mice, IRE la and
IRElp. IRE la is essential for viability and is broadly expressed. IRE 1(3 has been
detected only in the gastrointestinal mucosa. ER stress leads to oligomerization of IRE 1
proteins and trans-autophosphorylation of their cytosolic domains. Phosphorylation of
IRE1 activates its endonuclease activity which excises an intron from the mRNA of the
transcription factor X-box binding protein 1 (XBP1). This splicing event results in the
conversion of a transcription-inactive XBP1 isoform (i.e., XBPlu) to a transcriptionactive
XBP1 isoform (i.e., XBPls or XBPlp). XBPlp then travels into the nucleus,
where it binds to its target sequences including ER stress response element (ERSE) and
UPR element (UPRE), in the regulatory regions of ER-Golgi chaperone/enzyme genes,
to induce their transcription. Many UPR target genes have one or more copies of ERSE
or UPRE sequence in their promoter regions.
ATF6 (activating transcription factor 6) is another ER transmembrane protein.
ER stress leads to the transit of ATF6 protein to the Golgi compartment where its
cytosolic domain is cleaved by Site 1 and Site 2 proteases. The cleaved cytosolic
domain travels to the nucleus and acts as a transcription factor by binding to ERSE
sequences, which in turn up-regulates a variety of chaperones and processing enzymes in
the secretory pathway.
Activation of the UPR pathway in mammalian cells also leads to a transient
inhibition of protein translation through the PERK signaling pathway. PERK is an ER
transmembrane kinase which can phosphorylate the eukaryotic translation initiation
factor eIF2a in response to ER stress. Phosphorylation of eIF2a prevents the assembly
of the 43S ribosomal pre-initiation complex and therefore results in translation
attenuation. Paradoxically, phosphorylation of eIF2a also results in rapid synthesis of
transcription factor ATF4, which in turn enhances the expression of a proapoptotic
transcription factor CHOP. CHOP potentiates cell death when the detrimental effects of
ER stress can no longer be overcome.
Overexpression of secreted recombinant proteins in mammalian cells often leads
to low production yield. A commonly used method to improve protein production is to
increase the transcriptional rates, such as using stronger promoters or increasing gene
copy numbers. However, increased transcriptional rates may exacerbate ER stress and,
therefore, often fails to significantly improve the yield. In some cases, it may even
further reduce the production yield.
SUMMARY OF THE INVENTION
The present invention provides expression systems with improved production
yields for secreted or membrane proteins. The systems employ genetically-engineered
animal or plant cells that have modified, and in many cases, enhanced protein folding or
processing capacities.
In one aspect, the present invention features genetically-engineered animal or
plant cells comprising one or more recombinant expression cassettes which encode (1) a
protein of interest and (2) a component or modulator of a UPR pathway. Suitable UPR
components or modulators include, but are not limited to, non-IREl molecules that are
functional in the UPR pathways. They can be endogenous UPR components of the host
cells, or variants or functional equivalents thereof. They can also be naturally occurring
or non-naturally occurring molecules that modulate the activity or expression of an
endogenous UPR component either directly or indirectly. In many cases, the UPR
components/modulators are selected such that their expression or activation increases the
protein folding or processing capacity of the host cells.
Exemplary UPR components/modulators include, but are not limited to,
transcription factors, such as XBP1 or ATF6 or their biologically active fragments or
variants. Other components in the XBP1- or ATF6-mediated UPR pathways can also be
used. In addition, ER-resident chaperones or processing enzymes can be used.
Proteins that can be produced according to the present invention include, but are
not limited to, erythropoietins, growth hormones, insulins, interferons, growth factors,
membrane proteins, or other therapeutic, prophylactic or diagnostic proteins. In many
embodiments, the proteins of interest are expressed by the host cells as secreted or
membrane proteins.
The genetically-engineered cells of the invention can be derived from cell lines,
primary cultures, or other isolated or cultured cells. The genetically-engineered cells can
also be hybrid cells. In many cases, the hybrid cells are generated by fusing an animal
cell and a cancer cell (such as a myeloma cell). Recombinant expression cassettes
encoding a protein of interest or a UPR component/modulator can be incorporated or
introduced into the hybrid cells before or after the fusion event. In addition, the
genetically-engineered cells of the present invention can be cells of transgenic animals or
plants. In one embodiment, the genetically-engineered cell is a mammalian cell.
A recombinant expression cassette can be incorporated into a host cell by a
variety of means. For instance, an expression cassette can be stably integrated into a
chromosome or the genome of a host cell. The integration can be either random or
targeted (e.g., by using the Cre-fot recombination system of bacteriophage PI). An
expression cassette can also be introduced into a host cell via a non-integrated expression
vector.
A recombinant expression cassette can be controlled by a constitutive or
inducible promoter. It can also be controlled by a tissue-specific or developmentallyregulated
promoter. Other types of promoters can also be used for the present invention.
In another aspect, the present invention features genetically-modified animal or
plant cells comprising one or more recombinant expression cassettes which encode (1) a
protein of interest and (2) a polypeptide capable of binding to a UPRE or ERSE of the
host cells. In one embodiment, the UPRE- or ERSE-binding polypeptide is a
transcription factor, such as XBP1 or ATF6. In another embodiment, the UPRE- or
ERSE-binding polypeptide can recruit another protein to the promoter regions of UPR
genes, and the latter protein comprises a transactivation domain capable of activating the
transcription of the UPR genes (e.g., the transcription activation domain of XBP1 or
ATF6).
A genetically-engineered cell of the invention can express a protein of interest
and a UPR component/modulator from the same or different recombinant expression
cassettes. The protein of interest and the UPR component/modulator can be controlled
by the same or different promoters.
In one embodiment, a genetically-engineered cell of the invention comprises (1) a
first recombinant expression cassette encoding a protein of interest and (2) a second
recombinant expression cassette encoding a UPR component or modulator or a UPRE or
ERSE binding protein (e.g., XBP1 or ATF6). The ratio of the total number of the first
recombinant expression cassette over the total number of the second recombinant
expression cassette in the cell can range, without limitation, from no more than 0.1:1 to
at least 10:1. In many instances, the promoter employed by the first recombinant
cassette can have the same or similar strength as the promoter employed by the second
recombinant cassette, and the ratio of the total number of the first recombinant cassette
over the total number of the second recombinant cassette ranges from 0.5:1 to 10:1 (such
as at least 1:1,2:1, 3:1,4:1, 5:1,6:1, 7:1, 8:1, or 9:1). The promoters in the first and the
second recombinant cassettes can also have different strengths. For instance, the
promoter in the first recombinant cassette can be stronger than the promoter in the
second recombinant cassette, such as by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more folds.
In still another aspect, the present invention features animals or plants which
include a genetically-engineered cell of the present invention. Methods for incorporating
a genetically-modified cell into an animal or plant are well known in the art. In many
embodiments, the animals or plants are transgenic animals or plants.
In yet another aspect, the present invention features cell cultures that are
transfected or transduced with one or more expression vectors encoding (1) a protein of
interest and (2) a component or non-IREl modulator of a UPR pathway. In many
embodiments, the expression vector(s) comprises a first recombinant expression cassette
encoding the protein of interest and a second recombinant expression cassette encoding
the UPR component or modulator (e.g., XBP1 or ATF6). The first and the second
expression cassettes can be carried by the same or different expression vectors. The
molar ratio of the first recombinant expression cassette over the second recombinant
expression cassette in the cell culture can range, for example, from no more than 0.1:1 to
at least 10:1, such as at least 1:1,2:1,3:1,4:1,5:1,6:1,7:1, 8:1,9:1, 10:1, or more. The
cell culture can be a mammalian cell culture, an insect cell culture, a plant cell culture, or
another culture that is suitable for the production of the protein of interest.
The present invention also features methods of using the genetically-engineered
cells, animals, plants, or cell cultures for the production of proteins of interest.
In addition, the present invention features an expression vector which encodes (1)
a protein of interest and (2) a component or non-IREl modulator of a UPR pathway.
Other features, objects, and advantages of the present invention are apparent in
the detailed description that follows. It should be understood, however, that the detailed
description, while indicating preferred embodiments of the invention, is given by way of
illustration only, not limitation. Various changes and modifications within the scope of
the invention will become apparent to those skilled in the art from the detailed
description.
BRIEF DESCRIPTION OF THE DRAWINGS
The drawings are provided for illustration, not limitation.
Figure 1 demonstrates that overexpression of BMP6 in PA DUKX cells causes
ER stress.
Figure 2 indicates expression of exogenous XBP1 in stably transfected cell lines
under unstressed or stressed conditions.
Figure 3 shows increased secretion of BMP6 in several XBP1 cell lines than in
parental CHO DUKX cells.
Figure 4 illustrates increased secretion of ILllRFc in several XBP1 cell lines
than in parental CHO DUKX cells.
Figure 5 demonstrates that the transfection efficiency of GFP is similar in
selected XBP1 and parental CHO DUKX cell lines.
Figure 6 shows that the transcriptional and translational efficiency of GFP is
similar in selected XBP1 and parental CHO CUKX cell lines.
Figure 7 illustrates the successful expression of inducible ATF6 protein in COS-1
cells.
Figure 8 depicts the effects of XBP1 or ATF6 in different ratios with target
protein cDNAs on the production of target proteins.
Figure 9 demonstrates the effects of XBP1 or ATF6 on the expression of different
target proteins.
DETAILED DESCRIPTION
The present invention provides systems and methods for producing proteins of
interest. The expression systems of the present invention employ genetically-engineered
animal or plant cells that have modified or improved protein folding or processing
capacities. In many embodiments, the genetically-engineered cells of the present
invention comprise one or more recombinant expression cassettes which encode a
protein of interest and a component or modulator of a UPR pathway. Co-expression of
the UPR component/modulator significantly increases the yield of the protein of interest.
UPR components/modulators suitable for the present invention include, but are not
limited to, transcription factors, such as XBP1 or ATF6, or their biologically active
fragments or variants. ER-associated chaperones or enzymes can also be used.
Various aspects of the present invention are described in further detail in the
following subsections. The use of subsections is not meant to limit the invention. Each
subsection may apply to any aspect of the invention. As used herein, the term "or"
means "and/or" unless stated otherwise.
A. Proteins of Interest
Proteins that can be produced according to the present invention include, but are
not limited to, therapeutic, prophylactic or diagnostic proteins, such as erythropoietins,
growth hormones, insulins, interleukins, growth factors, interferons, colony stimulating
factors, blood factors, vaccines, collagens, fibrinogens, human serum albumins, tissue
plasminogen activators, glucosidases, alglucerases, myelin basic proteins, hypoxanthine
guanine phosphoribosyl transferases, tyrosine hydroxylases, dopadecarboxylases, or
antibodies. Exemplary antibodies amenable to the present invention include, but are not
limited to, monoclonal antibodies, mono-specific antibodies, poly-specific antibodies,
non-specific antibodies, humanized antibodies, human antibodies, single-chain
antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid
antibodies, Fab, F(ab')2, Fv, scFv, Fd, dAb, or functional fragments thereof. Highaffinity
binders selected using in vitro display technologies or evolutionary strategies can
also be produced according to the present invention. These high-affinity binders include,
but are not limited to, peptides, antibodies and antibody mimics. See, e.g., Binz, et al.,
NAT BIOTECHNOL., 22:575-582 (2004); and Lipovsek and Pluckthun, J IMMUNOL
METHODS, 290:51-67 (2004). Other proteins of interest, such as kinases, phosphatases,
G protein coupled receptors, growth factor receptors, cytokine receptors, chemokine
receptors, cell-surface antibodies (membrane bound immunoglobulin), BMP/GDFreceptors,
neuronal receptors, ion channels, proteases, transcription factors, or
polymerases, can also be produced by the present invention.
In many embodiments, the proteins produced by the present invention are
recombinant proteins. As used herein, a recombinant protein refers to a protein that is
constructed or produced using recombinant DNA technology. A recombinant protein
can have a naturally-occurring sequence or a genetically-engineered sequence. It can be
expressed, for example, from a recombinant vector or from a gene that is endogenous to
the host cells but has a genetically-engineered regulatory sequence. For instance, a
recombinant protein can be produced from an endogenous gene but with a geneticallyengineered
viral promoter.
In many instances, the recombinant proteins are fusion proteins including a
polypeptide tag to facilitate the isolation, purification, detection, immobilization,
stabilization, folding, or targeting of the expressed products.
In many other instances, the recombinant proteins include signal peptides. A
signal peptide can be endogenous or heterologous to the protein being produced. A
signal peptide often determines whether a protein will be formed on the rough ER or on
free ribosomes. A signal peptide can interact with signal recognition particle and direct
the ribosome to the ER where co-translational insertion takes place. Many signal
peptides are highly hydrophobic with positively charged residues. A signal peptide can
be removed from the growing peptide chain by a signal peptidase, a specific protease
located on the cisternal face of the ER.
Proteins targeted to the ER by signal sequences can be released into the extracellular
space as secreted proteins. For example, vesicles containing secreted proteins can fuse
with the cell membrane and then release their contents into the extracellular space - a
process called exocytosis. Exocytosis can occur constitutively or after receipt of a
triggering signal. In the latter case, the proteins are stored in secretory vesicles (or
secretory granules) until exocytosis is triggered. Similarly, proteins residing on the cell
membrane can be secreted into the extracellular space by proteolytic cleavage of a
"linker" that holds the protein to the membrane.
A protein of interest can be isolated from an expression system by a variety of
means. Examples of initial materials for protein isolation include, but are not limited to,
culture medium or cell lysate. Suitable isolation methods include, but are not limited to,
affinity chromatography (including immunoaffinity chromatography), ionic exchange
chromatography, hydrophobic interaction chromatography, size-exclusion
chromatography, HPLC, protein precipitation (including immunoprecipitation),
differential solubilization, electrophoresis, centrifugation, crystallization, or a
combination thereof. A polypeptide tag, such as a streptavidin tag, a FLAG tag, a polyhistidine
tag, a glutathione S-transferase, or an Fc fragment, can be fused to a protein of
interest to facilitate its isolation or purification. In one example, the polypeptide tag is
cleavable from the protein of interest by a protease.
In many embodiments, a protein of interest isolated according to the present
invention is substantially free from other proteins or contaminants. For instance, an
isolated protein can be at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% pure from other
proteins. In one example, an isolated protein contains no more than an insignificant
amount of contaminants that would interfere with its intended use.
A protein of interest isolated according to the present invention can be verified by
using standard techniques such as SDS-PAGE or immunoassays. An SDS-PAGE can be
stained with coommassie blue, silver or other suitable agents to visualize the isolated
protein. Suitable immunoassays include, but are not limited, Western blots, ELISAs,
RIAs, sandwich or immunometric assays, latex or other particle agglutination, or
proteomic chips. Protein sequencing and mass spectroscopy can also be used to verify or
analyze an isolated protein.
B. UPR Components/Modulators
UPR components/modulators that are amenable to the present invention include
molecules that are functional in UPR pathways. They can be naturally occurring or nonnaturally
occurring. They can be genetically engineered, chemically synthesized, or
biologically isolated. A UPR component/modulator can be derived from the same or
different species as the host cells. Expression of the UPR component/modulator
improves the secretion or ER processing capacity of the host cells. In many cases, coexpression
of a UPR component/modulator with a protein of interest in the host cells
improves the yield of the protein of interest by at least 2, 3, 4, 5, 10, or more folds.
Examples of UPR components/modulators that are suitable for the present
invention include, but are not limited to, transcription factors, such as XBP1 or ATF6.
The functional equivalents of these transcription factors, such as fragments of XBP1 or
ATF6, can also be used. These fragments retain at least a substantial portion of the
transcription activity of the transcriptionally activated XBP1 or ATF6 protein.
Downstream effectors of XBP1 or ATF6, such as ER chaperones or enzymes involved in
protein glycosylation or vesicle translocation, can also be used.
In addition, non-IREl modulators that can activate the expression or biological
function of a component of an XBP1- or ATF6-mediated UPR pathway can be used.
Such a modulator can modulate the UPR pathway by a mechanism other than self
overexpression. For instance, such a modulator can activate the function of a UPR
component by directly binding to the component, or modulate the expression of the
component by binding to a regulatory sequence in the gene that encodes the component.
Moreover, modulators that can inhibit the expression or biological functions of
components of the PERK signaling pathway can be used. These modulators include,
without limitation, antibodies, antisense RNA, or RNAi sequences. In addition,
dominant negative mutants of the PERK pathway components can be used. An example
of such dominant negative mutants is an eIF2a S51A mutant with the replacement of
serine at position 51 (murine sequence) to alanine. This substitution eliminates the
protein's ability to be phosphorylated and therefore abolishes its inhibitory effect on the
protein translation rate during ER stress. Similarly, mutations can be introduced into the
kinase domain of PERK to eliminate or reduce its kinase activity to phosphorylate eIF2a,
thereby preventing the induction of translational attenuation or apoptosis during ER
stress.
In one embodiment, XBP1 protein or a biologically active fragment thereof is
employed to increase the yield of a protein of interest in the host cells. XBP1 protein
includes two domains commonly found in transcription factors that confer DNA binding
and dimerization capability. XBP1 is known as a transcription factor that regulates
MHC class II genes by binding to a promoter element referred to as an X box. XBP1
also binds to an enhancer in the T cell leukemia virus type 1 promoter.
Activation of the UPR pathway leads to IRE 1-dependent splicing of a small
intron from XBP1 mRNA in both Caenorhabditis elegans and mammalian model
systems. The resulting exons are joined by a tRNA ligase. This splicing event results in
a frame-shift in XBP1 mRNA, which produces a protein that has the original N-terminal
10
DNA binding domain but a new C-terminal transactivation domain. In murine cells, the
splicing event converts a 267-amino acid XBP1 isoform to a 371-amino acid XBP1
isoform (XBPls or XBPlp). See Calfon, et al, NATURE, 415: 92-96 (2002).
XBPlp protein binds to the ERSE or UPRE sequences in the promoter regions of
many ER chaperone or UPR genes, activating the transcription of these genes. In
mammals, at least two ERSE sequences have been identified, ERSE-I and ERSE-II.
ERSE-I has a conserved sequence as shown in SEQ ID NO:1
(CCAATNKNTNNNNNNCCACG). ERSE-II has a conserved sequence as depicted in
SEQ ID NO:2 (ATTGGNCCACG). In addition, at least two mammalian UPRE
sequences have been identified, one having a conserved sequence as depicted in SEQ ID
NO:3 (TGACGTGG) and the other having a conserved sequence as illustrated in SEQ ID
NO:4 (TGACGTGA).
The XBP1 protein coding sequences can be obtained from a variety of sources.
For instance, the coding sequences for human, mouse, rat, chicken, fruit fly, and
zebrafish XBP1 proteins can be obtained from the Entrez nucleotide database at National
Center for Biotechnology Information (NCBI) (Bethesda, MD). These sequences have
Entrez accession numbers NM_005080, NMJH3842, NM_001004210,
NM_001006192, NM_079983, orNM_131874, respectively.
A biologically active fragment of an XBP1 protein retains at least a substantial
portion of the transcription activation activity of the XBPlp protein. For instance, an
XBP1 fragment employed in the invention can retain at least 50%, 60%, 70%, 80%,
90%, or more of the transcription activation activity of XBPlp. Transcriptionally active
XBP1 fragments can be selected by numerous means. In one example, a
transcriptionally active XBP1 fragment is selected based on its ability to activate the
transcription of genes downstream from an ERSE or UPRE sequence.
In another embodiment, ATF6 protein or a biologically active fragment thereof is
used to improve the yield of a protein of interest in the host cells. ATF6 is a
transmembrane protein which includes a "sensing" domain located in the ER lumen and
a cytosolic transcription transactivation domain. Upon ER stress, the cytosolic domain
of ATF6 is cleaved off and transported to the nucleus where it binds to the ERSE
sequences and thereby activates the downstream UPR genes. At least two ATF6 proteins
have been identified - namely, ATF6a and ATF6p\ ATF6a and ATF6P are structurally
related and share significant similarity in their b-zip domains. Exemplary coding
sequences for human mouse, sheep, and chicken ATF6 proteins have Entrez accession
numbers NM_007348, XM_129579, AY942654, and XM_422208, respectively.
Similarly to the XBP1 fragments employed in the invention, a biologically active
fragment of an ATF6 protein retains at least a substantial portion of the transcription
activation activity of the activated ATF6 protein or its cytosolic domain. A biologically
active ATF6 fragment can be selected by monitoring its binding to an ERSE sequence
and the ability of the fragment to activate the transcription of genes downstream from the
ERSE sequence.
In still another embodiment, an ER-resident processing enzyme or chaperone is
used to increase the yield of a protein of interest in the host cells. Examples of suitable
ER-located enzymes/chaperones include, but are not limited to, GRP78, GRP94, GRP58,
the protein disulfide isomerase, calnexin, and calrecticulin. In one example, the
endogenous counterpart of an ER enzyme (or chaperone) employed in the present
invention has a promoter region including one or more ERSE-I or ERSE-II sequences.
In another example, the endogenous counterpart of an ER enzyme (or chaperone)
employed in the present invention has a promoter region including one or more UPRE
sequences.
The present invention also features the use of a UPR component that is a variant
of an endogenous protein. A variant of an endogenous UPR component can be naturallyoccurring,
such as by allelic variation or polymorphism, or deliberately engineered. The
UPR activity of a variant does not decrease substantially compared to the original protein
(e.g., an XBPlp, a transcriptionally activated ATF6 protein, or a biologically active
fragment thereof)- In many embodiments, the variants employed in the present invention
retain at least 50% of the UPR activity of the corresponding original proteins. For
instance, a variant can retain at least 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the
UPR activity of the original protein. In one embodiment, a variant employed in the
present invention exhibits an increased UPR activity as compared to the original protein.
A desirable variant of a UPR component can be selected such that the expression or
activation of the variant enhances the secretion of a co-transfected protein in the host
cells.
The amino acid sequence of a variant is substantially identical to that of the
original protein. In many instances, the amino acid sequence of a variant has at least
80%, 85%, 90%, 95%, or 99% global sequence identity or similarity to the original
protein. Sequence identity or similarity can be determined by a variety of methods. In
one embodiment, sequence identity or similarity is determined by using a sequence
alignment algorithm. Suitable algorithms for this purpose include, but are not limited to,
Basic Local Alignment Tool (BLAST) described in Altschul, et al, J. MOL. BIOL.,
215:403-410 (1990), the algorithm of Needleman, et al., J. MOL. BlOL., 48:444-453
(1970), the algorithm of Myers and Miller, COMPUTE. APPLE. BiOSCi., 4:11-17 (1988),
and dot matrix analysis. Suitable computer programs for this purpose include, but are
not limited to, the BLAST programs provided by NCBI, MegAlign provided by
DNASTAR (Madison, WI), and the Genetics Computer Group (GCG) GAP program
(Needleman-Wench algorithm). For the GAP program, default values may be used (e.g.,
the penalty for opening a gap in one of the sequences is 11 and for extending the gap is
8). Similar amino acids can be defined by the BLOSSOM substitution matrix.
Numerous methods are available for preparing a desirable variant of a UPR
component. For instance, a variant can be derived from the original protein by at least 1,
2, 3, 4, 5, 10, 20, or more amino acid substitutions, deletions, insertions, or other
modifications. The substitutions can be conservative or non-conservative. In many
instances, conservative amino acid substitutions can be introduced into a protein
sequence without significantly changing the structure or biological activity of the
protein. Conservative amino acid substitutions can be made on the basis of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity, or the amphipathic nature of
the residues. For instance, conservative amino acid substitutions can be made among
amino acids with basic side chains, such as lysine (Lys or K), arginine (Arg or R) and
histidihe (His or H); amino acids with acidic side chains, such as aspartic acid (Asp or D)
and glutamic acid (Glu or E); amino acids with uncharged polar side chains, such as
asparagine (Asn or N), glutamine (Gin or Q), serine (Ser or S), threonine (Thr or T), and
tyrosine (Tyr or Y); or amino acids with nonpolar side chains, such as alanine (Ala or A),
glycine (Gly or G), valine (Val or V), leucine (Leu or L), isoleucine (He or I), proline
(Pro or P), phenylalanine (Phe or F), methionine (Met or M), tryptophan (Trp or W) or
cysteine (Cys or C). Examples of commonly used amino acid substitutions are
illustrated in Table 1.
Table 1. Example of Amino Acid Substitutions
Original Residues
Other desirable amino acid substitutions can also be introduced into a UPR
component. For instance, amino acid substitution(s) can be introduced into a UPR
component to increase its stability. For another instance, amino acid substitution(s) can
be introduced to increase or decrease the UPR activity of a UPR component.
In addition, the present invention features the use of polypeptides that can bind to
the UPRE or ERSE sequences of the host cells. These polypeptides, either alone or in
combination with other protein(s), can function as transcription factors to activate the
transcription of the genes that have the UPRE or ERSE promoter regions.
C. Recombinant Expression Cassettes and Host Cells
A typical recombinant expression cassette employed in the present invention
comprises a protein coding sequence operatively linked to a 5' untranslated regulatory
region and a 3' untranslated regulatory region. The protein coding sequence can be a
genomic sequence, a cDNA sequence, a combination thereof, or other expressible
sequences.
In one embodiment, a recombinant expression cassette includes all of the regulatory
elements necessary to direct the expression of the encoded protein. Examples of suitable
5' untranslated regulatory elements include promoters, enhancers, or the Kozak
sequences. Examples of suitable 3' untranslated regulatory elements include
polyadenylation sequences or other transcription/translation termination sequences.
Selection of suitable promoters, enhancers, or other regulatory elements for an
expression cassette is a matter of routine design within the level of ordinary skill in the
art. Many such elements are described in the literature and are available through
commercial suppliers.
Promoters suitable for the present invention include constitutive or inducible
promoters. These promoters can be endogenous or heterologous to the host cells. In one
embodiment, a tissue-specific promoter is used. Suitable tissue-specific promoters
include, but are not limited to, liver-specific promoters, lymphoid-specific promoters, T
cell-specific promoters, neuron-specific promoters, pancreas-specific promoters, or
mammary gland-specific promoters. A developmentally-regulated promoter can also be
used. A recombinant expression cassette having a tissue-specific or developmentalregulated
promoter can be used to prepare transgenic animals or plants. Protein(s)
encoded by such a recombinant expression cassette can be produced in specific tissue(s)
or at specific developmental stage(s) of the transgenic animals or plants.
In another embodiment, an inducible expression system is used to produce a protein
of interest or a UPR component/modulator. Systems suitable for this purpose include,
but are not limited to, the Tet-on/off system, the Ecdysone system, and the Rapamycin
system. The Tet-on/off system is based on two regulatory elements derived from the
tetracycline-resistance operon of the E. coli TnlO transposon. The system includes two
components, a regulator cassette and a reporter cassette. In one format of the Tet-off
system, the regulator cassette encodes a hybrid protein comprising a Tet represser (tetR)
fused to the VP16 activation domain of herpes simplex virus (HSV). The reporter
cassette includes a tet-responsive element (TRE) operatively linked to a report gene. The
reporter gene can encode, for example, a protein of interest or a UPR
component/modulator. In the absence of inducer (e.g., tetracycline or doxycycline), the
tetR-VP16 fusion protein binds to the TRE, thereby activating the transcription of the
reporter gene. In one format of the Tet-on system, the regulator cassette encodes a
hybrid protein comprising a mutated Tet represser (rtetR) fused to the VP16 activation
domain of HSV. The rtetR is a reverse Tet represser which binds to and activates the
TRE in the presence of inducer (e.g., tetracycline or doxycycline).
The Ecdysone system is based on the molting induction system in Drosophila. In
one format, the system includes a regulator cassette which encodes a functional ecdysone
receptor, and a reporter cassette which includes an ecdysone-responsive promoter
operatively linked to a reporter gene. In the presence of an inducer (such as ponasterone
A or muristerone A), the ecdysone receptor binds to the ecdysone-responsive promoter,
activating the transcription of the reporter gene.
The Rapamycin system, also known as the CID system ("chemical inducers of
dimerization"), employs two chimeric proteins. The first chimeric protein includes
FKBP12 fused to a DNA-binding domain that binds to a DNA response element. The
second chimeric protein includes FRAP fused to a transcriptional activation domain.
The addition of rapamycin causes dimerization of the two chimeric proteins, thereby
activating the expression of genes controlled by the DNA response element.
In one example, a recombinant expression cassette employed in the present
invention comprises SEQ ID NO:5. Transcription of SEQ ID NO:5 produces a nonspliced
human XBP1 mRNA. ER stress activates IRE1, which cleaves an intron from
the non-spliced mRNA. Translation of the spliced mRNA produces a mature and
functional XBP1 protein, the amino acid sequence of which is depicted in SEQ ID NO:6.
In another example, a recombinant expression cassette employed in the present
invention comprises SEQ ID NO:7. SEQ ID NO:7 does not include any cleavable intron
sequences. Expression of SEQ ID NO:7 produces a mature and functional human XBP1
protein (SEQ ID NO:6). Other nucleic acid sequences that encode SEQ ID NO:6 or a
functional equivalent thereof can also be used to prepare a recombinant expression
cassette of the invention. These nucleic acid sequences may or may not include introns
or other removable sequences.
In still another example, a recombinant expression cassette employed in the
present invention comprises SEQ ID NO:8. Transcription and translation of SEQ ID
NO:8 produce a human ATF6 protein, the amino acid sequence of which is illustrated in
SEQ ID NO:9.
In a further example, a recombinant expression cassette employed in the present
invention comprises a nucleic acid sequence encoding amino acid residues 1-366 of SEQ
ID NO:9. An example of such a nucleic acid sequence is nucleotides 1-1098 of SEQ ID
NO:8. Amino acid residues 1-366 of SEQ ID NO:9 include the entire basic region and
the majority of the leucine zipper region of the human ATF6 protein. This ATF6
fragment has been shown to be capable of activating endogenous GRP78 genes. See
Haze, et al, MOL. BlOL. CELL, 10:3787-3799 (1999).
Recombinant cassettes encoding XBP1 or ATF6 proteins derived from non-human
species can also be used in the present invention. For instance, XBP1 or ATF6 proteins
of rodent or other animal species can be used. These XBP1 or ATF6 proteins can be
selected such that co-expression of these proteins with a protein of interest improves the
yield of the latter protein in the host cells.
A recombinant expression cassette can be incorporated into host cells by a variety of
means. In one embodiment, a recombinant expression cassette is introduced into a
eukaryotic host cell by using a transfection or transduction vector. Vectors suitable for
this purpose include, but are not limited to, insect cell expression vectors (e.g.,
baculovirus expression vectors) or mammalian expression vectors. These vectors can be
derived from a variety of sources, such as episomes, cosmids, viruses, or combinations
thereof. In many cases, these vectors include selectable markers to facilitate their
incorporation into the host cells.
In another embodiment, a recombinant expression cassette employed in the present
invention is constructed by modifying an endogenous gene in the host cells. The
endogenous gene can encode a protein of interest or a UPR component/modulator.
Many portions in the endogenous gene can be modified to achieve a desired expression
or regulation effect. For instance, the original promoter of an endogenous gene can be
replaced by a viral promoter to increase the expression level of the gene.
A recombinant expression cassette can be incorporated into a host cell in various
forms. For instance, a recombinant expression cassette can be integrated into a
chromosome or the genome of a host cell. A recombinant expression cassette can also
be carried by a non-integrated expression vector in a host cell. Methods for stably or
transiently introducing an expression vector or cassette into a host cell are known in the
art. In one example, the expression vector or cassette is incorporated into a chromosome
of the host cell by targeted integration. Methods suitable for this purpose include, but are
not limited to, the Cre-lox recombination system and those described in U.S. Patent Nos.
6,656,727, 6,537,542 and 6,541,231.
In many embodiments, a genetically-engineered cell of the present invention includes
(1) a first recombinant expression cassette encoding a protein of interest and (2) a second
recombinant expression cassette encoding a UPR component/modulator (e.g., XBP1 or
ATF6). The ratio of the total number of the first recombinant expression cassette over
the total number of the second recombinant expression cassette in the cell can range, for
example, from no more than 0.1:1 to at least 10:1. Non-limiting examples of suitable
ratios include from 0.2:1 to 5:1, from 0.5:1 to 5:1, from 1:1 to 2:1, from 1:1 to 3:1, from
1:1 to 4:1, from 1:1 to 5:1, from 2:1 to 3:1, from 2:1 to 4:1, and from 2:1 to 5:1. The first
and the second recombinant expression cassettes can be carried by the same or different
vectors and driven by the same or different promoters which have the same or different
strengths. In one example, the promoter in the first recombinant expression cassette has
the same or similar strength as that in the second recombinant expression cassette, and
the ratio of the total number of the first recombinant cassette to the total number of the
second recombinant cassette in the cell is at least 1:1, 2:1, 3:1, 4:1, 5:1, or more.
Host cells suitable for the present invention include animal or plant cells. The host
cells can be cultured cells, such as cell lines or primary cultures. They can also be cells
in transgenic animals or plants. The selection of suitable host cells and methods for
culture, transfection/transduction, amplification, screening, product production, and
purification are known in the art.
In one embodiment, the host cells employed in the present invention are
mammalian cells. Examples of suitable mammalian cells include, but are not limited to,
Chinese hamster ovary (CHO) cells, HeLa cells, COS cells, 293 cells, CV-1 cells, and
other mammalian cell lines collected by American Type Culture Collection (Manassas,
Virginia). In certain cases, it is desirable to produce therapeutic or prophylactic proteins
in human cells, which often provide desired post-translational modifications on the
expressed proteins.
The host cells employed in the present invention can also be hybrid cells created
through fusion of two or more cells. In many cases, a hybrid cell employed in the
present invention is generated by fusing an animal cell (e.g., a mammalian cell) and a
cancer/immortal cell (e.g., a myeloma or blastoma cell). The animal cell and the
cancer/immortal cell can be derived from the same species. They can also be derived
from different species. Any method known in the art may be used to produce hybrid
cells. These methods include, but are not limited to, electrofusion or chemical fusion
(e.g., polyethylene glycol fusion).
A recombinant expression cassette can be introduced or incorporated into a
hybrid cell before or after the fusion event. For instance, a recombinant expression
cassette encoding a protein of interest can be incorporated into a mammalian cell before
the cell is fused with a cancer cell expressing an exogenous UPR component or
modulator. For another instance, a mammalian cell can be first transfected or transduced
with recombinant expression vector(s) that encodes a protein of interest and a UPR
component or modulator, and then fused with a cancer cell. Other procedures can also
be used to prepare hybrid cells of the present invention.
In many embodiments, the cancer/immortal cells used for preparing hybrid cells
are sensitive to one or more selective agents. For instance, the cancer/immortal cells can
be sensitive to a culture medium containing hypoxanthine, aminopterin and thymidine,
which is known as "HAT medium." These HAT-sensitive cells are fused to cells
insensitive to HAT medium. Hybrid cells thus produced are selected against HAT,
which kills unfused cells. The fused cells are then screened for desired features.
The present invention also features animals or plants that comprise a eukaryotic
host cell of the present invention. Methods for incorporating a recombinant cell into an
animal or a plant are well known in the art. In many embodiments, the animals or plants
are transgenic animals or plants which include one or more transgenes that encode a
protein of interest and a UPR component/modulator. Transgenic animals or plants can
be prepared by using standard techniques. In one embodiment, the transgenic animals
are non-human animals.
The present invention further features animal or plant cell cultures that are
transfected or transduced with one or more expression vectors encoding (1) a protein of
interest and (2) a UPR component/modulator. The cell cultures can be mammalian cell
cultures, insect cell cultures, plant cell cultures, or other cultures suitable for the
production of proteins of interest. The expression vector(s) can be transfected or
transduced transiently or stably. In one embodiment, the expression vector(s) employed
comprises a first recombinant expression cassette encoding a protein of interest and a
second recombinant expression cassette encoding a UPR component/modulator (e.g.,
XBP1 or ATF6). The first and the second expression cassettes can be carried by the
same or different vectors. They can be driven by the same or different promoters. The
molar ratio of the first recombinant expression cassette over the second recombinant
expression cassette can range, for example, from no more than 0.1:1 to at least 10:1.
In one example, the promoter employed by the first recombinant cassette has the
same or similar strength as the promoter employed by the second recombinant cassette,
and the molar ratio of the first recombinant cassette over the second recombinant cassette
in the cell culture ranges from 0.5:1 to 10:1, such as at least 1:1, 2:1. 3:1, 4:1, 5:1, or
more.
D. Pharmaceutical Compositions
A therapeutic or prophylactic protein produced by the present invention can be
used to prepare a pharmaceutical composition for the treatment of a patient or animal in
need thereof. A pharmaceutical composition of the present invention typically includes
an effective amount of a therapeutic or prophylactic protein and a pharmaceutically
acceptable carrier. Suitable pharmaceutically acceptable carriers include solvents,
solubilizers, fillers, stabilizers, binders, absorbents, bases, buffering agents, lubricants,
controlled release vehicles, diluents, emulsifying agents, humectants, lubricants,
dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption
delaying agents, and the like, that are compatible with pharmaceutical administration.
The use of such media and agents for pharmaceutically active substances is well-known
in the art. Supplementary agents can also be incorporated into the composition.
A pharmaceutical composition of the present invention can be formulated to be
compatible with its intended route of administration. Examples of routes of
administration include parenteral, intravenous, intradermal, subcutaneous, oral,
inhalative, transdermal, rectal, transmucosal, topical, and systemic administration. In
one example, the administration is carried out by an implant.
Solutions or suspensions used for parenteral, intradermal, or subcutaneous
application can include the following components: a sterile diluent such as water for
injection, saline solution, fixed oils, polyethylene glycols, glycerine; propylene glycol or
other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and
agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be
adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose
vials made of glass or plastic.
A pharmaceutical composition of the present invention can be administered to a
patient or animal in a desired dosage. A suitable dosage may range, for example, from 5
mg to 100 mg, from 15 mg to 85 mg, from 30 mg to 70 mg, or from 40 mg to 60 mg.
Dosages below 5 mg or above 100 mg can also be used. The pharmaceutical
composition can be administered in one dose or multiple doses. The doses can be
administered at intervals such as once daily, once weekly, or once monthly.
Toxicity and therapeutic efficacy of a therapeutic protein can be determined by
standard pharmaceutical procedures in cell culture or experimental animal models. For
instance, the LD5o (the dose lethal to 50% of the population) and the EDso (the dose
therapeutically effective in 50% of the population) can be determined. The dose ratio
between toxic and therapeutic effects is the therapeutic index, and can be expressed as
the ratio LDso/EDso. In many cases, therapeutic proteins that exhibit large therapeutic
indices are selected.
The data obtained from cell culture assays and animal studies can be used to
formulate a range of dosages for use in humans. In one embodiment, the dosage lies
within a range that exhibits therapeutic effectiveness in at least 50% of the population
with little or no toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized.
The dosage regimen for the administration of a therapeutic protein produced by
the present invention can be determined by the attending physician based on various
factors such as the action of the protein, the site of pathology, the severity of disease, the
patient's age, sex and diet, the severity of any inflammation, time of administration, and
other clinical factors. In one example, systemic or injectable administration is initiated at
a dose which is minimally effective, and the dose is increased over a pre-selected time
course until a positive effect is observed. Subsequently, incremental increases in dosage
are made limiting to levels that produce a corresponding increase in effect while taking
into account any adverse affects that may appear.
Progress of a treatment can be monitored by periodic assessment of disease
progression. The progress can be monitored, for example, by X-rays, MRI or other
imaging modalities, synovial fluid analysis, or clinical examination.
A therapeutic or prophylactic protein of interest can also be introduced into a
human or animal by using a gene delivery vector. Vectors suitable for this purpose
include, but are not limited to, viral vectors such as retroviral, lentiviral, adenoviral,
adeno-associated viral (AAV), herpes viral, alpnavirus, astrovirus, coronavirus,
orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, or
togavirus vectors. Liposomally-encapsulated expression vectors can also be used for
gene delivery. In many embodiments, the gene delivery vector encodes both the protein
of interest and a UPR modulator. Co-expression of the UPR modulator enhances the
production of the protein of interest in the target cells (e.g., tumor cells or other
dysfunctional cells). The protein of interest and the UPR modulator can also be
delivered to the target cells by using different vectors. Gene delivery can be conducted
in vivo or ex vivo.
In one embodiment, cell-specific gene delivery methods are employed for
introducing a therapeutic/prophylactic protein of interest or a UPR modulator into the
target cells. Many cell-specific gene delivery methods known in the art can be used for
the present invention. For instance, a cell-specific ligand (e.g., an antibody specific to a
surface antigen of the target cell) can be incorporated or conjugated to the envelope of a
viral vector which encodes a therapeutic/prophylactic protein or a UPR modulator. This
ligand can mediate entry of the viral vector into a specific cell type. Antibodyconjugated
liposomes can also be used for delivering gene therapy vectors to specific
target cells.
It should be understood that the above-described embodiments and the following
examples are given by way of illustration, not limitation. Various changes and
modifications within the scope of the present invention will become apparent to those
skilled in the art from the present description.
E. Examples
Example 1. COS-1 and DUKXCell Lines
COS-1 cells were obtained from American Type Culture Collection (ATCC),
Manassas, VA, with ATCC number CRL-1650. CHO DUKX cells and PA DUKX cells
were both derived from CHO-K1 (ATCC number CCL-61), which is a derivative of the
Chinese hamster ovary (CHO) cells.
The CHO DUXK cells, which are also referred to as DUXK B11 or DXB11 cells,
are deficient in production of dihydrofolate reductase (dhfr), a critical enzyme in the
process of DNA replication. To select cells in which both dhfr alleles were mutated,
Urlaub and Chasin, PROC. NATL. ACAD. Sci. U.S.A., 77:4216-4220 (1980), performed
the mutagenesis and selection in two steps. The selective agent used against dhfr+ cells
was tritiated deoxyuridine. Tritiated deoxyuridine is toxic to cells due to its
incorporation into DNA and subsequent radioactive decay. Incorporation of
deoxyuridine into DNA requires its conversion to thymidilic acid, a process for which
DHFR is essential, dhfr- mutant cells are unable to incorporate deoxyuridine into their
DNA and thus are able to survive in the presence of tritiated deoxyuridine. Some dhfr+
cells as well as mutants deficient in some other enzyme necessary for the incorporation
of deoxyuridine into DNA, may survive also, dhfr- mutants may be distinguished
because they are unable to conduct de novo biosynthesis of glycine, hypoxanthine, and
thymidine; thus they require exogenous nucleosides for growth.
In the first step of selection as used by Urlaub and Chasin, supra, wild-type cells
were subjected to ethyl methane sulfonate (EMS) mutagenesis and selected with tritiated
deoxyuridine in the presence of dhfr-inhibiting methotrexate (MTX) to isolate a
presumptive heterozygote (d+/d-). By using a concentration of MTX that was sufficient
to inactivate all the DHFR in the heterozygote (d+/d-), but not in the homozygote
(d+/d+), the homozygote had residual DHFR activity and incorporated tritiated
deoxyuridine. By virtue of this incorporation, (the d+/d+) cells were selected against and
only the heterozygotes were able to survive. After three rounds of selection, pooling,
and expansion of the surviving cells, the presumptive heterozygote cell line, UKB25 was
isolated. UKB25 cells were further mutagenized with gamma-irradiation and selected in
tritiated deoxyuridine in the absence of MTX. The surviving colonies exhibited triple
auxotrophy for glycine, hypoxanthine, and thymidine, which indicated a dhfrphenotype.
Colonies exhibiting this triple auxotrophy were cloned and shown to be
deficient in dhfr activity. Analysis of one such clone by Southern Blot hybridization
revealed that the dhfr genes did not undergo any gross rearrangements. This clone was
designated DXB11.
The DXB 11 cells thus generated were examined to confirm the predicted
characteristics of the cell line. The DUKX Bll cells were found to be genotypically
similar to the CHO-K1 cell line from which they were derived. They are hypodiploid
CHO cells, with 20 chromosomes that have been extensively studied cytogenetically.
Geimsa banding of metaphase DUKX Bll chromosomes demonstrated that the DUKX
Bll cells are CHO-K1 derivatives. The DUKX Bll cells are DHFR-deficient and
therefore auxotrophic for glycine, purine nucleosides, and thymidine. This DHFR-
deficient phenotype of the DUKX Bl 1 cells is the basis of the genetic selection used for
the transfer of recombinant heterologous protein expression plasmids into the cells.
In some experiments, the medium used for culturing DUKX Bll cells did not
contain hypoxanthine or thymidine. Without dihydrofolate reductase activity, the only
means for cells to survive and replicate was by the supplementation of nucleosides in the
growth medium to compensate for the cells inability to make them. Therefore,
adenosine, deoxyadenosine, and thymidine were added to the growth medium for DUKX
Bll cells. These were each added at a concentration of 10|j.g/ml. This concentration
was in excess of what the cells required under routine small-scale growth conditions.
DUKX Bll cells are useful for introducing expression plasmids containing
cDNAs for desired proteins. Because they lack endogenous DHFR activity it can be
used as a selectable and amplifiable marker when generating cell lines to produce
heterologous proteins. By either co-transfecting another expression vector containing a
cDNA for dhfr, or by putting the dhfr cDNA in close proximity to the cDNA of interest
on the same expression vector, one can use dhfr activity as a marker for which cells have
taken up the expression plasmid containing the desired gene, and are likely to produce
the desired protein. By withholding exogenous nucleosides after transfection, only cells
that incorporate a vector containing the dhfr gene will be able to produce dhfr and
thereby survive. The survived cells can now produce the desired protein. This may be
accomplished by virtue of a bicistronic message when the desired cDNA and the dhfr are
on the same plasmid, or by individual messages when the genes are on different
plasmids, which usually co localize on the chromosome during a transfection event.
When using two separate plasmids, altering the ratio of dhfr-containing plasmid to
cDNA-containing plasmid can enhance one's ability to select cells that contain both.
The DUKX Bll cells are dhfr-deficient by virtue of a point mutation in the dhfr
gene and therefore reversion to a dhfr+ phenotype is possible. This reversion and ability
to grow without exogenous nucleosides was observed during a serum-free suspension
adaptation effort. The population of DUKX cells in culture remained dhfr- for
approximately 154 cumulative population doublings (CPD) from the initiation of a
suspension culture. However, when the population was checked again for dependence
on exogenous nucleosides at 190 CPD, a phenotypic reversion was evident. Coincident
with the dhfr+ phenotype is a significant increase in the average growth rate of these
cells. Because the dhfr- phenotype is desirable for transfection and gene amplification
strategies, a serum-free suspension of adapted DUKX cells was made after 153.8 CPD.
Because these cells were adapted to growth in serum-free suspension culture prior to an
expression vector being introduced, they are called "pre-adapted", and are referred to as
"PA DUKX."
Non-adapted, FBS-dependent DUKX monolayers can be used for transfecting
expression vectors. Once the expression of a heterologous gene and dhfr are achieved,
each new cell line can be adapted to FBS-free suspension growth. The adaptation period
after transfection using monolayer cells is often lengthy. "Pre-adapted" DUKX cells can
be also used as host cells for transfection. These PA DUKX cells often offer advantages
from a time and effort perspective as the period of re-adaptation to serum free suspension
growth post-transfection is usually shorter. See Sinacore, et al., BIOTECHNOLOGY AND
BIOENGINEERING, 52:518-528 (1996).
Example 2. Induction of ER stress by Overexpression of BMP6 in PA DUKX
Cells
pSMED2/XBPl and pSMED2/BMP6 (+) or empty pSMED2 vector (-) were
cotransfected into PA DUKX cells. pSMED2/XBPl and pSMED2/BMP6 expression
vectors encode XBP1 and BMP6, respectively. Both vectors are driven by a CMV
promoter. Transfections were carried out in 6-well plates using Fugene6 (Roche,
Indianapolis, IN). The growth medium for the cells was Alpha media (Gibco)
supplemented with nucleosides and 10% FBS (heat inactivated and dialyzed) and
Penicillin/Streptomycin/Glutamine (Gibco).
Cells were lysed in Cell Lysis Buffer (Cell Signaling Technology, Beverly, MA)
with the addition of 400mM NaCl and 1 Complete Mini (a protease inhibitor cocktail
tablet from Roche, Indianapolis, IN). Lysates were taken at 7, 24, 31, and 48h after
transfection and run on a 10% tricine gel, followed by Western blot analysis (Figure 1).
The Western blot analysis was performed by using a blocking buffer of 4% non-fat dry
milk, 1% BSA and 0.1% Tween20 in TBS, and a wash buffer of 0.1% Tween20 in TBS.
Antibodies were diluted in the blocking buffer. Titration for Western was 1:1000 anti-
XBP1 (Santa Cruz Biotechnology) followed by 1:5000 goat anti-rabbit antibody
conjugated with horseradish peroxidase (The Jackson Laboratory). Figure 1 indicates
that overexpression of BMP6 in PA DUKX cells caused ER stress, as measured by the
increase of XBPlp protein (about 54 kD).
Example 3. Cell Lines Stably Transfected with XBP1
pSMED2/XBPl vector was transfected into CHO DUKX cells to create stable
cell lines. DFHR gene on the pSMED2 vector allows for methotrexate (MIX)
resistance. Transfected cells were plated in 5, 10, or 20nM MTX concentrations. Three
5nM MTX colonies (5-2, 5-4, 5-5), one lOnM colony (10-3), and one 20nM colony (20-
10) were isolated. Cells from each colony were treated with (+) or without (-)
tunacymicin (Tu), a chemical known to cause ER stress. Lysates were run on a 10%
tricine gel, followed by Western blot analysis (Figure 2) using rabbit polyclonal anti-
XBP1 antibody (Santa Cruz Biotechnology). Figure 2 demonstrates that more mature
XBP1 was produced in XBP1 stable cell lines when the cells were stressed by Tu
treatment.
pSMED2/BMP6 was transiently transfected into XBP1 stable (5-2, 5-4, and 20-
10) and parental (CHO DUKX) cell lines . Conditioned media collected 48h after
transfection were run on a 10% tricine gel, followed by Western blot analysis. The
Western blot membrane (Figure 3) was probed with a mouse monoclonal anti-BMP5
antibody (1:2000) which strongly crossreacts with BMP6. The secondary antibody was
goat anti-mouse antibody conjugated with horseradish peroxidase (1:5000) (The Jackson
Laboratory). Each lane in Figure 3 represents a separate experiment for a respective cell
line. As demonstrated in Figure 3, more BMP6 was secreted in XBP1 stable cell lines
selected with 5nM and lOnM MTX than in parental cells.
pSMED2/ILHRFc, which encodes an ILllRFc protein, was also transiently
transfected into XBP1 stable and parental cell lines. Conditioned media collected 48h
after transfection were run on a 10% tricine gel, followed by Western blot analysis. The
Western blot membranes (Figure 4) were probed with goat anti-human Fc antibody
conjugated with horseradish peroxidase (The Jackson Laboratory). More ILllRFc was
secreted in the majority of XBP1 cell lines selected with 5nM MTX than in CHO DUKX
cells.
Example 4. Comparison of Transfection, Transcriptional and Translational
Efficiencies Among XBP1 Stable Cell Lines and Their Parental CHO Cells
5-2, 20-10 and CHO DUKX cells were counted and equal amounts were
transiently transfected with a construct encoding green fluorescent protein (GFP). Cells
were visualized at 10 x 10 magnification and transfection efficiency (% Cells Expressing
GFP) was determined by comparing GFP fluorescent cells to total cells in three visual
fields per cell line. The comparison result indicates that the transfection efficiency of
GFP is similar in all XBP1 and CHO DUKX cell lines tested (Figure 5).
In a further experiment, constructs encoding (+) or not encoding (-) GFP were
transiently transfected into XBP1 stable and parental cell lines. Cell lysate collected 48h
after transfection was run on a 10% tricine gel, followed by Western blot analysis
(Figure 6). Each lane in Figure 6 represents a separate experiment. The Western blot
membrane was probed with rabbit polyclonal anti-GFP antibody and mouse monoclonal
anti-actin antibody for loading control. As demonstrated by Figure 6, the sum of
transcriptional and translational efficiencies for GFP is similar in all cell lines
investigated.
Example 5. Cell Transiently Transfected With ATF6
Flag-tagged cDNA from the active soluble domain of ATF6 was cloned into a
Tet/off inducible expression vector ptTATOP6 and transiently transfected into COS-1
cells. The ptTATOP6 vector includes an inducible promoter which controls the
expression of the fusion protein comprising ATF6 and the Flag tag. Cells lysate
collected at 18, 48 and 60h after transfection was run on a 10% tricine gel, followed by
Western blot analysis. The Western blot membrane (Figure 7) was probed with anti-flag
antibody. "V" indicates an empty ptTATOP6 vector, and "P" represents a flag positive
control. As illustrated by Figure 7, ATF6 protein was successfully expressed in COS-1
cells in the absence of doxycycline.
Example 6. Co-Expressing Target Genes with XBP1 or ATF6 in Proper Ratios
Enhances the Secretion of the Target Genes in HEK293 Cells
HEK293-FT and HEK293-EBNA were grown and maintained in a humidified
incubator with 5% CO2 at 37°C in free-style 293 media (Invitrogen, Carlsbad, CA)
supplemented with 5% fetal bovine serum.
Transient expression was performed in 50-ml spinners or 24-well plates, or 1L
spinners. For the culture volume of 50 ml (or 1L), 25 ng (or 0.5mg for 1L) of plasmid
DNA was mixed with 400(ig (8mg for 1L) of Polyethylenimine (PEI, 25 kDa, linear,
neutralized to pH7.0 by HCI, Img/ml, Polysciences, Warrington, PA) in 2.5 ml (50ml for
1L) of serum-free 293 media. The spinners were incubated at 37°C with a rotation rate
of 170rpm on a P2005 Stirrer (Bellco) for 72 -144 hours before harvest. For a 24-well
plate format, lug of DNA was mixed with 8 ug of PEI in 0.5ml of serum-free 293
media. Then the mixtures were mixed with 0.5ml of HEK293 cells in 293 media with
10% FBS at the cell density of O.SxlO6 cells/ml. The plates were incubated at 37°C on
an Orbital shaker (BellCo) with a rotation rate of 300rpm for 72 hours before harvest.
pSMED2 and pSMEDA were used for the DNA construction. C-terminal His6-
tagged secreted frizzled-related protein 1 (sFRP-1), and C-terminal Flag-tagged
aggrecanase-2 (Agg-2) were subcloned into pSMEDA. C-terminal His6-tagged
neurotrophic tyrosine kinase, receptor, type 2 (TrkB) was subcloned into pSMED2.
These subcloned genes did not have any transmembrane or cytoplasmic domains,
thereby allowing secretion of the expressed products.
C-terminal His6-tagged Propl and Prop34-LBD were subcloned into pcDNAS.l
(Invitrogen, Carlsbad, CA). Propl and Prop34-LBD were derived from low density
lipoprotein receptor-related protein 5 (LRP5) with deletion of the transmembrane and
cytoplasmic domains. The amino acid sequences of Propl and Prop34-LBD are depicted
in SEQ ID NOs:10 and 11, respectively. SEQ ID NO: 10 includes a His6 tag at amino
acids 342-347 and a Flag tag at amino acids 348-356. SEQ ID NO:11 includes a His6
tag at amino acids 795-800 and a V5 tag at amino acids 778-794.
lug of Propl-his6-F lag in pcDNA3.1 was co-transfected with 0.3ug (1:3) or lug
(1:1) of XBPlp in pSMED2 vector into HEK293T cells. Both pcDNA3.1 and pSMED2
are driven by a CMV promoter. Conditioned media were harvested at 72 hr after DNA
transfection. Samples were separated by SDS-PAGE and immunoblotted with anti-His4
antibodies. Duplicates experiments (Set#l and Set#2) were performed. As demonstrated
in Figure 8A, co-transfection of XBP1 with Propl in the ratios of 1:1 or 1:3 drastically
improved the expression of Propl.
In another experiment, lug of Prop34-LBD-V5-his6 in pcDNA3.1 was cotransfected
with 0.3ug (1:3) or lug (1:1) of ATF6 in ptTATOP6 vector into HEK293T
cells. Like pSMED2, ptTATOP6 is also driven by a CMV promoter. Conditioned media
harvested at 72 hr after DNA transfection were analyzed by SDS-PAGE and
immunoblotting with anti-His4 antibody. Figure 8B shows that co-transfection of ATF6
with Prop34-LBD-V5-his6 in the ratio of 1:1 or 1:3 significantly enhanced the
expression of Prop34-LBD.
Figure 9 illustrates the effects of XBPlp or ATF6 on the expression of different
target proteins. Propl-his6-Flag or Prop34-LBD-V5-His6 in pcDNAS.l was cotransfected
with XBPlp in pSMED2 vector or ATF-6 in ptTATOP6 vector into
HEK293T cells. Conditioned media harvested at 72 hr after DNA transfection were
analyzed by SDS-PAGE and immunoblotting with anti-His4 antibody. Signals were
quantified by densitometry as shown in Figure 9. The results indicate that XBPlp and
ATF-6 have different effects on the expression of Propl and Prop34-LBD. Different
enhancement effects were also observed for TrkB, sFRP-1, and Agg-2 when these
proteins were co-expressed with XBPlp versus ATF-6. For instance, co-expression with
XBPlp increased the yield of TrkB by about 5-fold, while only about 2-fold increase was
observed when TrkB was co-expressed with ATF6.
The foregoing description of the present invention provides illustration and
description, but is not intended to be exhaustive or to limit the invention to the precise
one disclosed. Modifications and variations consistent with the above teachings are
possible or may be acquired from practice of the invention. Thus, it is noted that the
scope of the invention is defined by the claims and their equivalents.

We Claim:
1. An animal or plant cell comprising one or more recombinant expression
cassettes which encode:
a protein of interest, and
a component or non-IREl modulator of an unfolded protein response (UPR) pathway.
2. The cell according to claim 1, wherein said cell is an animal cell, and said
component or non-IREl modulator is an XBP1 or ATF6 protein.
3. A cell as in one of claims 1-2, wherein said one or more recombinant
expression cassettes include:
at least one recombinant expression cassette encoding said protein of interest; and
at least another recombinant expression cassette encoding said XBP1 or ATF6 protein,
and wherein the ratio of the total number of said at least one recombinant expression cassette to the total number of said at least another recombinant expression cassette in said cell is from 0.5:1 to 10:1.
4. The cell according to claim 3, wherein the ratio of the total number of said
at least one recombinant expression cassette to the total number of said at least another
recombinant expression cassette in said cell is at least 3:1.
5. A cell as in one of claims 1-4, wherein said cell is a mammalian cell.
6. A transgenic, non-human animal comprising a cell as in one of claims 1-
5.
7. A cell as in one of claims 1-4, wherein said cell is a hybrid cell produced
by fusing an animal cell and a cancer cell.
8. An animal or plant cell comprising one or more recombinant expression
cassettes which encode:
a protein of interest; and
a polypeptide capable of binding to a UPR element (UPRE) or ER-stress response element (ERSE) of said cell.
9. The cell according to claim 8, wherein said cell is an animal cell, and said
polypeptide includes a transcription activation domain.
10. The cell according to claim 9, wherein said transcription activation
domain comprises a transcription activation domain of an XBP1 or ATF6 protein.
11. An animal or plant cell culture transfected or transduced with one or more
expression vectors encoding:
a protein of interest; and
a component or non-IREl modulator of a UPR pathway.
12. The cell culture according to claim 11, wherein said component or non-
IREl modulator is an XBP1 or ATF6 protein, and said one or more expression vectors
include:
at least one recombinant expression cassette encoding said protein of interest; and
at least another recombinant expression cassette encoding said XBP1 or ATF6 protein,
and wherein the molar ratio of said at least one recombinant expression cassette over said at least another recombinant expression cassette in said cell culture is from 0.5:1 to 10:1.
13. The cell culture according to claim 12, wherein said cell culture is a
mammalian cell culture, and the molar ratio of said at least one recombinant expression
cassette over said at least another recombinant expression cassette in said cell culture is
at least 3:1.
14. A method comprising expressing a protein of interest in an animal or
plant cell, said cell including one or more recombinant expression cassettes which
encode:
said protein of interest; and
a component or non-IREl modulator of a UPR pathway.
15. The method according to claim 14, wherein said cell is an animal cell, and
said component or non-IREl modulator is an XBP1 or ATF6 protein.
16. A method comprising expressing a protein of interest in an animal or
plant cell, said cell including one or more recombinant expression cassettes which
encode:
said protein of interest; and
a polypeptide capable of binding to a UPRE or ERSE of said cell.
17. The method according to claim 16, wherein said cell is an animal cell, and
said polypeptide comprises a transcription activation domain of an XBP1 or ATF6
protein.
18. A method comprising expressing a protein of interest in an animal or
plant cell culture, said cell culture being transfected or transduced with one or more
expression vectors which include:
at least one recombinant expression cassette encoding said protein of interest; and
at least another recombinant expression cassette encoding a component or non-IREl modulator of a UPR pathway.
19. The method according to claim 18, wherein said cell culture is a
mammalian cell culture, and said component or non-IREl modulator is an XBP1 or
ATF6 protein, and wherein the molar ratio of said at least one recombinant expression
cassette over said at least another recombinant expression cassette in said cell culture is
from 0.5:1 to 10:1.
20. An expression vector encoding:a therapeutic or prophylactic protein of interest, and a component or non-IREl modulator of a UPR pathway.21. The invention substantially such as herein described.