U.S. patent application number 11/548013 was filed with the patent office on 2007-02-22 for hybrid vector having a cytomegalovirus enhancer and myeloproliferative sarcoma virus promoter.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Margaret Dow Moore.
Application Number | 20070042464 11/548013 |
Document ID | / |
Family ID | 29736660 |
Filed Date | 2007-02-22 |
United States Patent
Application |
20070042464 |
Kind Code |
A1 |
Moore; Margaret Dow |
February 22, 2007 |
HYBRID VECTOR HAVING A CYTOMEGALOVIRUS ENHANCER AND
MYELOPROLIFERATIVE SARCOMA VIRUS PROMOTER
Abstract
An expression vector capable of expressing high levels of
heterologous proteins having a cytomegalovirus (CMV) enhancer 5'
upstream from a myeloproliferative sarcoma virus (MPSV)
promoter.
Inventors: |
Moore; Margaret Dow;
(Seattle, WA) |
Correspondence
Address: |
ZYMOGENETICS, INC.;INTELLECTUAL PROPERTY DEPARTMENT
1201 EASTLAKE AVENUE EAST
SEATTLE
WA
98102-3702
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
29736660 |
Appl. No.: |
11/548013 |
Filed: |
October 10, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10465156 |
Jun 18, 2003 |
|
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|
11548013 |
Oct 10, 2006 |
|
|
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60389612 |
Jun 18, 2002 |
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Current U.S.
Class: |
435/69.1 ;
435/358; 435/456; 530/350; 536/23.5 |
Current CPC
Class: |
C12N 2840/203 20130101;
C12N 9/6429 20130101; C12N 2830/00 20130101; C12N 15/86 20130101;
C12Y 304/21005 20130101; C12N 15/85 20130101; C12N 2830/60
20130101; C12N 2830/15 20130101; C12N 2740/13043 20130101 |
Class at
Publication: |
435/069.1 ;
435/456; 435/358; 530/350; 536/023.5 |
International
Class: |
C12P 21/06 20060101
C12P021/06; C07H 21/04 20060101 C07H021/04; C12N 15/86 20060101
C12N015/86; C12N 5/06 20060101 C12N005/06; C07K 14/47 20070101
C07K014/47 |
Claims
1. A non-retroviral expression vector comprising a cytomegalovirus
(CMV) enhancer and a myeloproliferative sarcoma virus (MPSV)
promoter.
2. The vector of claim 1 wherein the CMV enhancer is located
upstream from the 5' end of the MPSV promoter.
3. The vector of claim 2 wherein the CMV enhancer and MPSV promoter
comprises the polynucleotide sequence of SEQ ID NO:1.
4. The vector of claim 1 that further comprises at least one
additional element selected from the group consisting of a
consensus Ig intron, a tPA pre-proleader sequence, a polio IRES, a
.DELTA. CD8 selection marker, and a human growth hormone polyA
signal sequence.
5. The vector of claim 1 that further comprises a consensus Ig
intron, a tPA pre-porleader sequence, and a polio IRES.
6. The vector of claim 2 that further comprises a consensus Ig
intron, a tPA pre-proleader sequence, and a polio IRES.
7. The vector of claim 3 that further comprises a consensus Ig
intron, a tPA pre-proleader sequence, and a polio IRES.
8. The vector of claim 7 further comprising a structural gene such
that the gene is operably linked to the CMV enhancer and MPSV
promoter.
9. The vector pZMP21 as deposited with the ATCC, having the
reference number ATCC PTA-5266.
10. A mammalian cell transfected with the vector of claim 1.
11. The mammalian cell of claim 10 wherein the CMV enhancer and the
MPSV promoter comprises the polynucleotide sequence of SEQ ID NO:
1.
12. The mammalian cell of claim 11 wherein the cell is a CHO
cell.
13. The mammalian cell of claim 12 wherein the CHO cell is of
strain DXB11.
14. A method of producing a recombinant protein comprising a.
transfecting a mammalian host cell with the vector of claim 1; b.
growing the cells under conditions that selectively propagates
those cells that have integrated the vector of claim 1 into its
genome; c. growing the cells of step b) under conditions that cause
the recombinant protein to be secreted into the cell medium; d.
isolating the recombinant protein from the cell medium.
15. The method of claim 14 wherein the transfection occurs by
electroporation.
16. The method of claim 14 wherein the conditions that selectively
propagates cells that have integrated the vector of claim 1 into
its genome comprises growing the cells in the presence of
methotrexate.
17. A method of producing a recombinant protein comprising a.
randomly integrating the vector of claim 8 into the genome of CHO
cells; b. growing the cells in the presence of increasing
concentrations of methotrexate; c. isolating cells from step b) and
growing under conditions such that the CHO cells produce the
recombinant protein into the culture medium; d. isolating the
recombinant protein from the culture medium.
18. The method of claim 17 wherein the CHO cells are of the strain
DXB11.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This is a continuation of U.S. patent application Ser. No.
10/465,156, filed Jun. 18, 2003, which claims the benefit under 35
U.S.C. 119(e) of provisional application No. 60/389,612, filed Jun.
18, 2002, both of which are herein incorporated by reference.
INTRODUCTION
[0002] The present invention is related to the construction and
utilization of a DNA plasmid vector, in particular, those hybrid
non-retroviral vectors that comprise the cytomegalovirus (CMV)
enhancer and the myeloproliferative sarcoma virus (MPSV) promoter
minus its negative control region. This hybrid sequence promotes
the high expression of cloned genes under its transcriptional
control when the vector is transfected into mammalian cell lines.
Preferably, the vector also comprises other functional sequences to
increase expression of the cloned sequence such as the Ig intron
sequence, a viral internal ribosome entry site (IRES), a leader
sequence to allow for secreted protein expression, and
polyadenylation signals. The vector can also comprise selectable
markers and other features that facilitate the replication of the
vector in mammalian, yeast, and prokaryotic host cells, thus
increasing the stability of the vector in whatever expression
system is being used.
BACKGROUND OF THE INVENTION
[0003] The expression of foreign proteins by bacteria, yeast or
mammalian cell lines has become routine. One type of commonly used
means involves the construction of virion-plasmid hybrid vectors
that possess the capacity to express cloned inserts in mammalian
cells. The expression of the cloned gene with such hybrid vectors
can occur in a transient, extrachromosomal manner, but higher
production is usually obtained through random insertion of the
vector into the host cell genome. The typical mammalian expression
vector will contain regulatory elements, usually in the form of
viral promoter or enhancer sequences and characterized by a broad
host and tissue range, a polylinker sequence facilitating the
insertion of a DNA fragment within the plasmid vector, and the
sequences responsible for intron splicing and polyadenylation of
mRNA transcripts. This contiguous region of
promoter-polylinker-polyadenylation site is commonly referred to as
the transcription unit. Viral promoter and enhancer regions have
long been utilized as regulatory elements for use in mammalian host
cells. For example, the strength of the CMV enhancer caused it to
be a suggested component in eukaryotic expression vectors upon its
discovery (Boshart et al., Cell, 41 (2):521-30 (1985)) and it has
been utilized as a universal cell control element in transgenic
mice (Schmidt et al. Mol. Cell. Biol. 10: 4406-4411 (1990)). The
MPSV promoter coveys a wide host cell specificity to the virus
including fibroblasts and hematopoietic stem cells (Stocking et al.
Proc. Natl. Acad. Sci. USA, 82: 5746-5750 (1985)). Accordingly,
this promoter has been used to express heterologous genes in a
number of cell types, including skin fibroblasts (Pamer et al.,
Blood, 73: 438-445 (1989), primary hepatocytes (Ponder et al., Hum.
Gene Ther. 2:41-52 (1991), and rodent cells lines and human
fibroblast cell lines (van den Wollenberg, Gene 144: 237-241
(1994)).
[0004] Generally, there are two types of expression vectors
suitable for use in eukaryotic cells, retrovirally-based systems
and virion-plasmid hybrids described above. van den Wollenberg et
al. describe a retroviral vector that comprises the CMV enhancer
genetically engineered within the U3 region of the MPSV promoter.
However, retroviral vectors have significant drawbacks for use in
industrial level protein production. First, the level of protein
production is severely hampered by the retroviral packaging
sequence, a necessary component of such vectors, as it interferes
with translational initiation. Second, protein production is
reduced because the transport of retroviral messenger RNA is less
efficient than a standard mRNA and there is competition between
retroviral packaging and translation. Third, it is impossible to
reach the gene copy numbers routinely achieved by standard vectors
with an amplifying selection marker, due to the fact that a
retroviral vector implants two promoters for each random
integration, thus randomly activating downstream sequences with
deleterious effects to the cell. Fourth, there are serious safety
concerns with large-scale production of retroviral cultures due to
random recombination to replication competency. Finally,
retrovirally-established cell lines are harder to document and less
efficient to develop since a viral production cell line must first
be used to make a master cell bank, then the actual production cell
line is produced, requiring a second round of analysis and banking.
Accordingly, industrial production of protein is not routinely
performed with retroviral vectors.
[0005] Thus, the expression of foreign proteins in commercially
acceptable quantities remains a challenge. This is especially true
in mammalian cell lines. Very often expression of a mammalian
protein in a mammalian cell line is required in order to mimic the
native form of the protein in all respects: structure, catalytic
activity, immunological reactivity, and biological function. Often
glycosylation or other post-translational modifications are the key
to the production of the desired form of the protein, and bacteria
or yeast systems are unable to accomplish these modifications.
Thus, there remains a need for improved plasmids that promote the
production of mammalian proteins in commercially viable quantities
within mammalian host systems.
BRIEF SUMMARY OF THE INVENTION
[0006] One aspect of the present invention is a non-retroviral
expression vector comprising a cytomegalovirus (CMV) enhancer and a
myeloproliferative sarcoma virus (MPSV) promoter. Preferably, the
CMV enhancer is located upstream from the 5' end of the MPSV
promoter. Most preferably, the CMV enhancer and MPSV promoter
construct comprises the polynucleotide sequence of SEQ ID NO:1.
[0007] The vector of the present invention can further comprise at
least one additional element selected from the group consisting of
a consensus Ig intron, a tPA pre-proleader sequence, a polio IRES,
a .DELTA. CD8 selection marker, and a human growth hormone polyA
signal sequence. Preferably, the vector comprises a consensus Ig
intron, a tPA pre proleader sequence, and a polio IRES. The vector
can also comprise a structural gene, such as prethrombin.
[0008] A further aspect of the present invention is a mammalian
cell transfected with the vector. The mammalian cell of the present
invention is preferably a CHO cell, and most preferably a CHO of
the strain DXB11. The present invention also encompasses a method
of producing a recombinant protein comprising transfecting a
mammalian host cell with the vector of the present invention,
growing the cells under conditions that selectively propagates
those cells that have integrated the vector into its genome, and
growing the cells with the integrated vector under conditions that
cause the recombinant protein to be secreted into the cell medium,
and isolating the recombinant protein from the cell medium.
BRIEF DESCRIPTION OF THE FIGURES
[0009] FIG. 1 shows a plasmid map containing the MPSV/CMV
promoter/enhancer of the present invention. Clockwise the plasmid
contains the CMV enhancer, the MPSV LTR promoter minus the negative
signal sequence, a consensus Ig intron, a tPA pre-pro leader, polio
IRES, a .DELTA.CD8 selection marker, a human growth hormone (hGH)
polyA sequence, a dihydrofolate reductase (DHFR) selection cassette
with the SV40 promoter/enhancer and SV40 polyA, pUC ori, .beta.
lactamase selection, yeast CEN/ARS and URA3 selection. This vector
has been named pZMP21.
[0010] FIG. 2 compares the picograms per cell per day (pg/cell-day)
of prethrombin production for Chinese Hamster Ovary (CHO) cells
transfected with pZMP20 (CMV promoter/enhancer) or pZMP21 (MPSV
promoter/CMV enhancer).
DESCRIPTION OF THE INVENTION
[0011] The present invention fills this need by providing for a
novel non-retroviral expression vector, which is able to transfect
mammalian cell lines such as Chinese Hamster Ovary Cells (CHO
cells) and promote the production of foreign proteins in
unexpectedly high quantities. The plasmid of the present invention
is comprised of a cytomegalovirus enhancer upstream from the 5' end
of a myeloproliferative sarcoma virus (MPSV) promoter. Preferably
the MPSV promoter is fused to a cytomegalovirus (CMV) enhancer.
1. Overview
[0012] SEQ ID NO: 1 shows a CMV enhancer/MPSV LTR promoter
construct of the present invention. The CMV enhancer extends from
nucleotide 1 to and including nucleotide 374 of SEQ ID NO: 1. The
MPSV LTR promoter extends from nucleotide 375 to and including
nucleotide 851.
2. Definitions
[0013] In the description that follows, a number of terms are used
extensively. The following definitions are provided to facilitate
understanding of the invention.
[0014] As used herein, "nucleic acid" or "nucleic acid molecule"
refers to polynucleotides, such as deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA), oligonucleotides, fragments generated by
the polymerase chain reaction (PCR), and fragments generated by any
of ligation, scission, endonuclease action, and exonuclease action.
Nucleic acid molecules can be composed of monomers that are
naturally occurring nucleotides (such as DNA and RNA), or analogs
of naturally occurring nucleotides (e.g., .alpha.-enantiomeric
forms of naturally-occurring nucleotides), or a combination of
both. Modified nucleotides can have alterations in sugar moieties
and/or in pyrimidine or purine base moieties. Sugar modifications
include, for example, replacement of one or more hydroxyl groups
with halogens, alkyl groups, amines, and azido groups, or sugars
can be functionalized as ethers or esters. Moreover, the entire
sugar moiety can be replaced with sterically and electronically
similar structures, such as aza-sugars and carbocyclic sugar
analogs. Examples of modifications in a base moiety include
alkylated purines and pyrimidines, acylated purines or pyrimidines,
or other well-known heterocyclic substitutes. Nucleic acid monomers
can be linked by phosphodiester bonds or analogs of such linkages.
Analogs of phosphodiester linkages include phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the
like. The term "nucleic acid molecule" also includes so-called
"peptide nucleic acids," which comprise naturally occurring or
modified nucleic acid bases attached to a polyamide backbone.
Nucleic acids can be either single stranded or double stranded.
[0015] The term "complement of a nucleic acid molecule" refers to a
nucleic acid molecule having a complementary nucleotide sequence
and reverse orientation as compared to a reference nucleotide
sequence.
[0016] The term "contig" denotes a nucleic acid molecule that has a
contiguous stretch of identical or complementary sequence to
another nucleic acid molecule. Contiguous sequences are said to
"overlap" a given stretch of a nucleic acid molecule either in
their entirety or along a partial stretch of the nucleic acid
molecule.
[0017] The term "structural gene" refers to a nucleic acid molecule
that is transcribed into messenger RNA (mRNA), which is then
translated into a sequence of amino acids characteristic of a
specific polypeptide.
[0018] An "isolated nucleic acid molecule" is a nucleic acid
molecule that is not integrated in the genomic DNA of an organism.
For example, a DNA molecule that encodes a growth factor that has
been separated from the genomic DNA of a cell is an isolated DNA
molecule. Another example of an isolated nucleic acid molecule is a
chemically-synthesized nucleic acid molecule that is not integrated
in the genome of an organism. A nucleic acid molecule that has been
isolated from a particular species is smaller than the complete DNA
molecule of a chromosome from that species.
[0019] A "nucleic acid molecule construct" is a nucleic acid
molecule, either single- or double-stranded, that has been modified
through human intervention to contain segments of nucleic acid
combined and juxtaposed in an arrangement not existing in
nature.
[0020] "Linear DNA" denotes non-circular DNA molecules having free
5' and 3' ends. Linear DNA can be prepared from closed circular DNA
molecules, such as plasmids, by enzymatic digestion or physical
disruption.
[0021] "Complementary DNA (cDNA)" is a single-stranded DNA molecule
that is formed from an mRNA template by the enzyme reverse
transcriptase. Typically, a primer complementary to portions of
mRNA is employed for the initiation of reverse transcription. Those
skilled in the art also use the term "cDNA" to refer to a
double-stranded DNA molecule consisting of such a single-stranded
DNA molecule and its complementary DNA strand. The term "cDNA" also
refers to a clone of a cDNA molecule synthesized from an RNA
template.
[0022] A "promoter" is a nucleotide sequence that directs the
transcription of a structural gene. Typically, a promoter is
located in the 5' non-coding region of a gene, proximal to the
transcriptional start site of a structural gene. Sequence elements
within promoters that function in the initiation of transcription
are often characterized by consensus nucleotide sequences. These
promoter elements include RNA polymerase binding sites, TATA
sequences, CAAT sequences, differentiation-specific elements [DSEs;
McGehee et al., Mol. Endocrinol. 7:551 (1993)], cyclic AMP response
elements (CREs), serum response elements [SREs; Treisman, Seminars
in Cancer Biol. 1:47 (1990)], glucocorticoid response elements
(GREs), and binding sites for other transcription factors, such as
CRE/ATF [O'Reilly et al., J. Biol. Chem. 267:19938 (1992)], AP2 [Ye
et al., J. Biol. Chem. 269:25728 (1994)], SP1, cAMP response
element binding protein [CREB; Loeken, Gene Expr. 3:253 (1993)] and
octamer factors [see, in general, Watson et al., eds., Molecular
Biology of the Gene, 4th ed. (The Benjamin/Cummings Publishing
Company, Inc. 1987), and Lemaigre and Rousseau, Biochem. J. 303:1
(1994)]. If a promoter is an inducible promoter, then the rate of
transcription increases in response to an inducing agent. In
contrast, the rate of transcription is not regulated by an inducing
agent if the promoter is a constitutive promoter. Repressible
promoters are also known.
[0023] A "core promoter" contains essential nucleotide sequences
for promoter function, including the TATA box and start of
transcription. By this definition, a core promoter may or may not
have detectable activity in the absence of specific sequences that
may enhance the activity or confer tissue specific activity.
[0024] A "regulatory element" is a nucleotide sequence that
modulates the activity of a core promoter or increases the
translation of the mRNA product that results from transcription
driven by the core promoter. For example, a regulatory element may
contain a nucleotide sequence that binds with cellular factors that
increases transcription over basal levels or imparts transcription
exclusively or preferentially in particular cells, tissues, or
organelles. Other regulatory elements increase translation of the
mRNA message that results because of sequences that are now
included in the message, such as an IRES (due to increased ribosome
entry) or a poly-A tail (due to increased mRNA stability).
[0025] An "enhancer" is a type of regulatory element that can
increase the efficiency of transcription, regardless of the
distance or orientation of the enhancer relative to the start site
of transcription.
[0026] "Heterologous DNA" refers to a DNA molecule, or a population
of DNA molecules, that does not exist naturally within a given host
cell. DNA molecules heterologous to a particular host cell may
contain DNA derived from the host cell species (i.e., endogenous
DNA) so long as that host DNA is combined with non-host DNA (i.e.,
exogenous DNA). For example, a DNA molecule containing a non-host
DNA segment encoding a polypeptide operably linked to a host DNA
segment comprising a transcription promoter is considered to be a
heterologous DNA molecule. Conversely, a heterologous DNA molecule
can comprise an endogenous gene operably linked with an exogenous
promoter. As another illustration, a DNA molecule comprising a gene
derived from a wild-type cell is considered to be heterologous DNA
if that DNA molecule is introduced into a mutant cell that lacks
the wild-type gene.
[0027] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 10 amino acid residues are commonly
referred to as "peptides."
[0028] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
non-peptidic substituents may be added to a protein by the cell in
which the protein is produced, and will vary with the type of cell.
Proteins are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are generally
not specified, but may be present nonetheless.
[0029] A peptide or polypeptide encoded by a non-host DNA molecule
is a "heterologous" peptide or polypeptide.
[0030] An "integrated genetic element" is a segment of DNA that has
been incorporated into a chromosome of a host cell after that
element is introduced into the cell through human manipulation.
Within the present invention, integrated genetic elements are most
commonly derived from linearized plasmids that are introduced into
the cells by electroporation or other techniques. Integrated
genetic elements are passed from the original host cell to its
progeny.
[0031] A "cloning vector" is a nucleic acid molecule, such as a
plasmid, cosmid, or bacteriophage that has the capability of
replicating autonomously in a host cell. Cloning vectors typically
contain one or a small number of restriction endonuclease
recognition sites that allow insertion of a nucleic acid molecule
in a determinable fashion without loss of an essential biological
function of the vector, as well as nucleotide sequences encoding a
marker gene that is suitable for use in the identification and
selection of cells transformed with the cloning vector. Marker
genes typically include genes that provide tetracycline resistance
or ampicillin resistance.
[0032] An "expression vector" is a nucleic acid molecule encoding a
gene that is expressed in a host cell. Typically, an expression
vector comprises a transcription promoter, a gene, and a
transcription terminator. Gene expression is usually placed under
the control of a promoter, and such a gene is said to be "operably
linked to" the promoter. Similarly, a regulatory element and a core
promoter are operably linked if the regulatory element modulates
the activity of the core promoter.
[0033] A "non-retroviral vector expression vector" is an expression
vector that does not contain a polynucleotide sequence encoding a
retroviral packaging element.
[0034] A "recombinant host" is a cell that contains a heterologous
nucleic acid molecule, such as a cloning vector or expression
vector. "Integrative transformants" are recombinant host cells, in
which heterologous DNA has become integrated into the genomic DNA
of the cells.
[0035] The term "secretory signal sequence" denotes a DNA sequence
that encodes a peptide (a "secretory peptide") that, as a component
of a larger polypeptide, directs the larger polypeptide through a
secretory pathway of a cell in which it is synthesized. The larger
polypeptide is commonly cleaved to remove the secretory peptide
during transit through the secretory pathway.
[0036] An "isolated polypeptide" is a polypeptide that is
essentially free from contaminating cellular components, such as
carbohydrate, lipid, or other proteinaceous impurities associated
with the polypeptide in nature. Typically, a preparation of
isolated polypeptide contains the polypeptide in a highly purified
form, i.e., at least about 80% pure, at least about 90% pure, at
least about 95% pure, greater than 95% pure, or greater than 99%
pure. One way to show that a particular protein preparation
contains an isolated polypeptide is by the appearance of a single
band following sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis of the protein preparation and Coomassie Brilliant
Blue staining of the gel. However, the term "isolated" does not
exclude the presence of the same polypeptide in alternative
physical forms, such as dimers or alternatively glycosylated or
derivatized forms.
[0037] The terms "amino-terminal or N-terminal" and
"carboxyl-terminal or C-terminal" are used herein to denote
positions within polypeptides. Where the context allows, these
terms are used with reference to a particular sequence or portion
of a polypeptide to denote proximity or relative position. For
example, a certain sequence positioned carboxyl-terminal to a
reference sequence within a polypeptide is located proximal to the
carboxyl terminus of the reference sequence, but is not necessarily
at the carboxyl terminus of the complete polypeptide.
[0038] The term "expression" refers to the biosynthesis of a gene
product. For example, in the case of a structural gene, expression
involves transcription of the structural gene into mRNA and the
translation of mRNA into one or more polypeptides.
[0039] The term "complement/anti-complement pair" denotes
non-identical moieties that form a non-covalently associated,
stable pair under appropriate conditions. For instance, biotin and
avidin (or streptavidin) are prototypical members of a
complement/anti-complement pair. Other exemplary
complement/anti-complement pairs include receptor/ligand pairs,
antibody/antigen (or hapten or epitope) pairs, sense/antisense
polynucleotide pairs, and the like. Where subsequent dissociation
of the complement/anti-complement pair is desirable, the
complement/anti-complement pair preferably has a binding affinity
of less than 10.sup.9 M.sup.-1.
[0040] "Upstream" and "downstream" are terms used to describe the
relative orientation between two elements present in a nucleotide
sequence. An element that is "upstream" of another is located in a
position closer to the 5' end of the sequence (i.e., closer to the
end of the molecule that has a phosphate group attached to the 5'
carbon of the ribose or deoxyribose backbone if the molecule is
linear) than the other element. An element is said to be
"downstream" when it is located in a position closer to the 3' end
of the sequence (i.e., the end of the molecule that has an hydroxyl
group attached to the 3' carbon of the ribose or deoxyribose
backbone in the linear molecule) when compared to the other
element.
[0041] In eukaryotes, RNA polymerase II catalyzes the transcription
of a structural gene to produce mRNA. A nucleic acid molecule can
be designed to contain an RNA polymerase II template in which the
RNA transcript has a sequence that is complementary to that of a
specific mRNA. The RNA transcript is termed an "anti-sense RNA" and
a nucleic acid molecule that encodes the anti-sense RNA is termed
an "anti-sense gene." Anti-sense RNA molecules are capable of
binding to mRNA molecules, resulting in an inhibition of mRNA
translation.
[0042] Due to the imprecision of standard analytical methods,
molecular weights and lengths of polymers are understood to be
approximate values. When such a value is expressed as "about" X or
"approximately" X, the stated value of X will be understood to be
accurate to .+-.10%.
[0043] Polynucleotides, generally a cDNA sequence, of the present
invention encode the described polypeptides herein. A cDNA sequence
which encodes a polypeptide of the present invention is comprised
of a series of codons, each amino acid residue of the polypeptide
being encoded by a codon and each codon being comprised of three
nucleotides. The amino acid residues are encoded by their
respective codons as follows.
[0044] Alanine (Ala) is encoded by GCA, GCC, GCG or GCT;
[0045] Cysteine (Cys) is encoded by TGC or TGT;
[0046] Aspartic acid (Asp) is encoded by GAC or GAT;
[0047] Glutamic acid (Glu) is encoded by GAA or GAG;
[0048] Phenylalanine (Phe) is encoded by TTC or TTT;
[0049] Glycine (Gly) is encoded by GGA, GGC, GGG or GGT;
[0050] Histidine (His) is encoded by CAC or CAT;
[0051] Isoleucine (Ile) is encoded by ATA, ATC or ATT;
[0052] Lysine (Lys) is encoded by AAA, or AAG;
[0053] Leucine (Leu) is encoded by TTA, TTG, CTA, CTC, CTG or
CTT;
[0054] Methionine (Met) is encoded by ATG;
[0055] Asparagine (Asn) is encoded by AAC or AAT;
[0056] Proline (Pro) is encoded by CCA, CCC, CCG or CCT;
[0057] Glutamine (Gln) is encoded by CAA or CAG;
[0058] Arginine (Arg) is encoded by AGA, AGG, CGA, CGC, CGG or
CGT;
[0059] Serine (Ser) is encoded by AGC, AGT, TCA, TCC, TCG or
TCT;
[0060] Threonine (Thr) is encoded by ACA, ACC, ACG or ACT;
[0061] Valine (Val) is encoded by GTA, GTC, GTG or GTT;
[0062] Tryptophan (Trp) is encoded by TGG; and
[0063] Tyrosine (Tyr) is encoded by TAC or TAT.
[0064] It is to be recognized that according to the present
invention, when a polynucleotide is claimed as described herein, it
is understood that what is claimed are both the sense strand, the
anti-sense strand, and the DNA as double-stranded having both the
sense and anti-sense strand annealed together by their respective
hydrogen bonds. Also claimed is the messenger RNA (mRNA) that
encodes the polypeptides of the president invention, and which mRNA
is encoded by the cDNA described herein. Messenger RNA (mRNA) will
encode a polypeptide using the same codons as those defined herein,
with the exception that each thymine nucleotide (T) is replaced by
a uracil nucleotide (U).
3. Detailed Description
[0065] The vector of the present invention can be used to produce
polypeptides having value in industry, therapeutics, diagnostics,
or research. Illustrative proteins include antibodies and antibody
fragments, receptors, immunomodulators, hormones, and the like. For
example, the expression vector can include a nucleic acid molecule
that encodes a pharmaceutically active molecule, such as
prethrombin, Factor VIIa, proinsulin, insulin, follicle stimulating
hormone, tissue type plasminogen activator, tumor necrosis factor,
interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,
IL-16, IL-17, IL-18, and IL-19), colony stimulating factors (e.g.,
granulocyte-colony stimulating factor, and granulocyte
macrophage-colony stimulating factor), interferons (e.g.,
interferons-.alpha., -.beta., -.gamma., -.omega., -.delta., -.tau.,
and -.epsilon.), a stem cell growth factor, erythropoietin, and
thrombopoietin. Additional examples of a protein of interest
include an antibody, an antibody fragment, an anti-idiotype
antibody (or, fragment thereof), a chimeric antibody, a humanized
antibody, an antibody fusion protein, and the like. An example of
such an antibody fusion protein would be a fusion of the
extracellular portion of the transmembrane activator and
CAML-interactor (TACI) protein, such as amino acids 30-110, fused
to the Fc portion of human IgG1. The Fc portion can be the native
sequence, or one that has been mutated to remove the immunoglobulin
effector functions. Examples of these mutations include changes at
amino acids 234, 235, 237, 330 and 331 of the IgG1 Fc sequence.
[0066] The vectors of the present invention have been found to
produce these proteins of interest at higher than expected levels.
Without being bound by theory, it is anticipated that the greater
than average protein expression displayed by the vectors of the
present invention is due, at least in part, to the greater than
average stability of expression exhibited by this vector when
integrated into the genome of a mammalian host cell.
[0067] The gene of interest can be isolated from genomic or cDNA
sequences using methods well known to one of ordinary skill or
chemically synthesized. If chemically synthesized and double
stranded DNA is required, then each complementary strand is made
separately. The production of short genes (60 to 80 base pairs) is
technically straightforward and can be accomplished by synthesizing
the complementary strands and then annealing them. For the
production of longer genes (>300 base pairs), however, special
strategies may be required, because the coupling efficiency of each
cycle during chemical DNA synthesis is seldom 100%. To overcome
this problem, synthetic genes (double-stranded) are assembled in
modular form from single-stranded fragments that are from 20 to 100
nucleotides in length.
[0068] One method for building a synthetic gene requires the
initial production of a set of overlapping, complementary
oligonucleotides, each of which is between 20 to 60 nucleotides
long. The sequences of the strands are planned so that, after
annealing, the two end segments of the gene are aligned to give
blunt ends. Each internal section of the gene has complementary 3'
and 5' terminal extensions that are designed to base pair precisely
with an adjacent section. Thus, after the gene is assembled, the
only remaining requirement to complete the process is to seal the
nicks along the backbones of the two strands with T4 DNA ligase. In
addition to the protein coding sequence, synthetic genes can be
designed with terminal sequences that facilitate insertion into a
restriction endonuclease sites of a cloning vector and other
sequences should also be added that contain signals for the proper
initiation and termination of transcription and translation.
[0069] An alternative way to prepare a full-size gene is to
synthesize a specified set of overlapping oligonucleotides (40 to
100 nucleotides). After the 3' and 5' extensions (6 to 10
nucleotides) are annealed, large gaps still remain, but the
base-paired regions are both long enough and stable enough to hold
the structure together. The duplex is completed and the gaps filled
by enzymatic DNA synthesis with E. coli DNA polymerase I. This
enzyme uses the 3'-hydroxyl groups as replication initiation points
and the single-stranded regions as templates. After the enzymatic
synthesis is completed, the nicks are sealed with T4 DNA ligase.
For larger genes, the complete gene sequence is usually assembled
from double-stranded fragments that are each put together by
joining four to six overlapping oligonucleotides (20 to 60 base
pairs each). If there is a sufficient amount of the double-stranded
fragments after each synthesis and annealing step, they are simply
joined to one another. Otherwise, each fragment is cloned into a
vector to amplify the amount of DNA available. In both cases, the
double-stranded constructs are sequentially linked to one another
to form the entire gene sequence. Each double-stranded fragment and
the complete sequence should be characterized by DNA sequence
analysis to verify that the chemically synthesized gene has the
correct nucleotide sequence. For reviews on polynucleotide
synthesis, see, for example, Glick and Pasternak, Molecular
Biotechnology, Principles and Applications of Recombinant DNA (ASM
Press 1994), Itakura et al., Annu. Rev. Biochem. 53:323 (1984), and
Climie et al., Proc. Nat'l Acad. Sci. USA 87:633 (1990).
[0070] Expression vectors that are suitable for production of an
amino acid sequence of interest in eukaryotic cells typically
contain (1) eukaryotic or viral DNA elements that control
initiation and level of transcription, such as a promoter and an
enhancer; (2) DNA elements that control the processing of
transcripts, such as a transcription termination/polyadenylation
sequence; and (3) one or more selectable marker gene(s) and other
sequences useful for stable gene expression for all anticipated
host cells. Expression vectors can also include nucleotide
sequences encoding a secretory sequence that directs the
heterologous polypeptide into the secretory pathway of a host
cell.
[0071] To express a gene of interest or a selectable marker gene, a
nucleic acid molecule encoding the amino acid sequence must be
operably linked to regulatory sequences that control
transcriptional expression and then, introduced into a host cell.
The vector of the present invention comprises the MPSV promoter
with the CMV enhancer in a 5' position to the promoter. MPSV is a
member of the Moloney murine sarcoma virus family (Mo-MuSV) and can
transform fibroblasts in vitro and cause sarcoma in vivo.
Additionally, MPSV causes an acute myeloprolerative disease in
adult mice. The mos oncogene, which is a component of the virus
genome, is necessary for the virus' transforming function, but it
is sequences specific to its long terminal repeat (LTR) that
account for expanded cell target specificity when compared to
Mo-MuSV. These additional cell targets makes the MPSV LTR an
attractive promoter for mammalian cell line expression. The MPSV
LTR is generally defined as nucleotides between -416 to +31 in
relation to the transcription initiation site located within the
LTR sequences, although other sequences of the MPSV LTR that
function as a promoter can also be used. Preferably, the MPSV
promoter has the sequence of nucleotides 375 to 851 of SEQ ID NO:
1. The MPSV LTR includes sequences that have been identified as
negatively controlling transcription. Although deletion of these
sequences proved to have marginal effect on protein production, so
they remain in pZMP21, they can optionally be deleted in the vector
of the present invention.
[0072] The second regulatory element of the present invention is
the CMV enhancer and can be generally defined as the nucleotides
between -118 and -524 5' of the transcription initiation site of
the major immediate-early gene of CMV. Preferably, the CMV enhancer
has the sequence of nucletodes 1 to 374 of SEQ ID NO: 1. The
enhancer function of this fragment of the viral genome was
discovered based on its ability to produce recombinant viruses when
cotransfected with enhancerless SV40 viral genome (Boshart et al.,
Cell, 41(2):521-30 (1985)). For the vectors of the present
invention, this sequence, or functionally fragments thereof, is
placed within the vector such that an increase in transcription
results when compared to the transcription without the presence of
the CMV enhancer. Preferably, this location is 5' of the MPSV
promoter sequence.
[0073] The vector of the present invention can comprise other
regulatory elements that can increase the expression of the
recombinant protein of interest within mammalian host cells. Among
the other regulatory elements that can be included is the
transcription enhancer located within the intron of an
immunoglobulin gene. Particularly preferred is a consensus Ig
intron sequence that comprises sequences that have been optimized
for use in mammalian host cells such as CHO DXB11. A second
additional regulatory element is an internal ribosome entry site
(IRES), a sequence derived from viral genomes that allows for the
translation of a dicistronic message. Particularly preferred is the
IRES derived from the polio virus. A third regulatory element is a
poly-A signal sequence that results in the addition of adenosine
residues on the end of the mRNA message, which increases the
message stability. Particularly preferred is the poly-A signal
sequence derived from the human growth hormone (hGH) gene
sequence.
[0074] Recombinant host cells can be produced that secrete the
amino acid sequence of interest into surrounding medium.
Accordingly, the present invention contemplates expression vectors
comprising a nucleotide sequence that encodes a secretory signal
sequence, which is also known as a "signal peptide," a "leader
sequence," a "prepro sequence," or a "pre sequence." The secretory
signal sequence is operably linked to a gene of interest such that
the two sequences are joined in the correct reading frame and
positioned to direct the newly synthesized polypeptide of interest
into the secretory pathway of the host cell. Secretory signal
sequences are commonly positioned 5' to the nucleotide sequence
encoding the amino acid sequence of interest, although certain
secretory signal sequences may be positioned elsewhere in the
nucleotide sequence of interest (see, e.g., Welch et al., U.S. Pat.
No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830). The
present invention can utilize a tissue plasminogen activator (tPA)
pre-proleader derived from the sequence described in U.S. Pat. No.
5,641,655. Mutations have been introduced into the pre-proleader so
that it is optimized for use within mammalian expression
systems.
[0075] Expression vectors can also comprise nucleotide sequences
that encode a peptide tag to aid the purification of the
polypeptide of interest. Peptide tags that are useful for isolating
recombinant polypeptides include polyHistidine tags (which have an
affinity for nickel-chelating resin), c-myc tags, calmodulin
binding protein (isolated with calmodulin affinity chromatography),
substance P, the RYIRS tag (which binds with anti-RYIRS
antibodies), the Glu-Glu tag, and the FLAG tag (which binds with
anti-FLAG antibodies). See, for example, Luo et al., Arch. Biochem.
Biophys. 329:215 (1996), Morganti et al., Biotechnol. Appl.
Biochem. 23:67 (1996), and Zheng et al., Gene 186:55 (1997).
Nucleic acid molecules encoding such peptide tags are available,
for example, from Sigma-Aldrich Corporation (St. Louis, Mo.).
[0076] A wide variety of selectable marker genes for use in
mammalian expression vectors are available (see, for example,
Kaufman, Meth. Enzymol. 185:487 (1990); Kaufman, Meth. Enzymol.
185:537 (1990)). Selectable marker genes generally confer growth
resistance to a chemical or drug, that allow selection of initial
positive transformants in bacterial, yeast, or mammalian host
cells. Selectable markers fall into two functional categories:
recessive and dominant. The recessive markers are usually genes
that encode products that are not produced in the host cells, i.e.,
host cells that lack the "marker" product or function. Marker genes
for thymidine kinase (TK), dihydrofolate reductase (DHFR), adenine
phosphoribosyl transferase (APRT), and hypoxanthine-guanine
phosphoribosyl transferase (HGPRT) are in this category. (see, for
example, Srivastava and Schlessinger, Gene 103:53 (1991); Romanos
et al., "Expression of Cloned Genes in Yeast," in DNA Cloning 2:
Expression Systems, 2.sup.nd Edition, pages 123-167 (IRL Press
1995); Markie, Methods Mol. Biol. 54:359 (1996); Pfeifer et al.,
Gene 188:183 (1997); Tucker and Burke, Gene 199:25 (1997);
Hashida-Okado et al., FEBS Letters 425:117 (1998)).
[0077] Dominant markers include genes that encode products that
confer resistance to growth-suppressing compounds (such as
antibiotics or other drugs) and/or permit growth of the host cells
in metabolically restrictive environments. Commonly used markers
within this category include a mutant DHFR gene that confers
resistance to methotrexate; the gpt gene for xanthine-guanine
phosphoribosyl transferase, which permits host cell growth in
mycophenolic acid/xanthine containing media; and the neo gene for
aminoglycoside 3'-phosphotransferase, which can confer resistance
to G418, gentamycin, kanamycin, and neomycin. More newly developed
markers include resistance to zeocin, bleomycin, blastocidin, and
hygromycin (see, e.g., Gatignol et al., Mol. Gen. Genet. 207:342
(1987); Drocourt et al., Nucl. Acids Res. 18:4009 (1990)).
[0078] The use of selectable markers has been extended beyond
isolation of cells that have incorporated the vector sequences to
selection for cells that are expressing the recombinant protein at
a high level. An example of this selection process is co-expression
of green fluorescent protein with the recombinant protein. The use
of autofluorescent proteins provides a visual mechanism to assess
if host cells are overexpressing recombinant protein. Similar
selection can be performed with a cell surface protein that can be
detected with an antibody (e.g. CD4, CD8, Class I major
histocompatibility complex (MHC) protein, etc.). Preferably, the
cytoplasmic domain of the cell surface protein has been deleted, in
order to reduce the cytological effect on the host cell of
over-expression of the protein. The expression products of such
selectable marker genes can be used to sort transfected cells from
untransfected cells by such standard means as FACS sorting or
magnetic bead separation technology. Selectable marker genes can be
cloned or synthesized using published nucleotide sequences, or
marker genes can be obtained commercially.
[0079] The present vector preferably utilizes as selectable makers
a DHFR cassette with the SV40 promoter/enhancer for use in
mammalian host cells, a CD8 .DELTA. construct (.DELTA. indicating
that the sequence encoding the cytoplasmic domain of the protein
has been deleted) to determine recombinant gene expression at the
cell surface of mammalian cells, .beta. lactamase for use in
bacterial host cells, and URA3 for use in yeast host cells.
[0080] A final common component of expression vectors are sequences
that facilitate the replication of the vector in mammalian, yeast,
and bacterial hosts such as centromeres, origins of replication,
chromatin stability sequences, and the like, that increase the
stability of the vector in the host system. For example, the vector
of present invention can comprise the pUC origin of replication for
use in bacterial host cells and the S. cerevisiae CEN/ARS origin of
replication for use in yeast host cells. Chromatin elements that
may modulate protein expression levels and/or stability are: locus
control regions (LCR), matrix or scaffold attachment regions (MAR
or SAR) or insulators.
[0081] Both during and after construction of the expression vector
comprising the amino acid-encoding sequences of interest, the
vector is typically propagated in a host cell. Vector propagation
can be carried out in a prokaryotic host cell, such as E. coli.
Suitable strains of E. coli include BL21(DE3), BL21(DE3)pLysS,
BL21(DE3)pLysE, DH1, DH41, DH5, DH51, DH51F', DH51MCR, DH10B,
DH10B/p3, DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1,
Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for example, Brown
(ed.), Molecular Biology Labfax (Academic Press 1991)). Standard
techniques for propagating vectors in prokaryotic hosts are
well-known to those of skill in the art (see, for example, Ausubel
et al. (eds.), Short Protocols in Molecular Biology, 3.sup.rd
Edition (John Wiley & Sons 1995) ["Ausubel 1995"]; Wu et al.,
Methods in Gene Biotechnology (CRC Press, Inc. 1997)).
[0082] Alternatively, vector propagation both during or after
vector construction can be carried out in eukaryotic cells, such as
yeast. Yeast species of particular interest in this regard include
Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.
Methods for transforming S. cerevisiae cells with exogenous DNA and
producing recombinant polypeptides therefrom are disclosed by, for
example, Kawasaki, U.S. Pat. No. 4,599,311, Kawasaki et al., U.S.
Pat. No. 4,931,373, Brake, U.S. Pat. No. 4,870,008, Welch et al.,
U.S. Pat. No. 5,037,743, and Murray et al., U.S. Pat. No.
4,845,075. Transformed cells are selected by phenotype determined
by the selectable marker, commonly drug resistance or the ability
to grow in the absence of a particular nutrient (e.g., leucine).
Transformation systems for other yeasts, including Hansenula
polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis,
Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia
methanolica, Pichia guillermondii and Candida maltosa are known in
the art. See, for example, Gleeson et al., J. Gen. Microbiol.
132:3459 (1986), and Cregg, U.S. Pat. No. 4,882,279.
[0083] Ultimately, the amino acid sequence of interest may be
expressed in any prokaryotic or eukaryotic host cell as described
above. Preferably, using the vector of the present invention, the
amino acid sequence of interest is produced by a eukaryotic cell,
such as a mammalian cell. Examples of suitable mammalian host cells
include African green monkey kidney cells (Vero; ATCC CRL 1587),
human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamster
kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314),
canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary
cells (CHO--K1; ATCC CCL61; CHO DG44; CHO DXB11 (Hyclone, Logan,
Utah); see also, e.g., Chasin et al., Som. Cell. Molec. Genet.
12:555, 1986)), rat pituitary cells (GH1; ATCC CCL82), HeLa S3
cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCC CRL 1548)
SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650) and
murine embryonic cells (NIH-3T3; ATCC CRL 1658). The CHO strain
DXB11 is the preferred host cell for protein production utilizing
the vector of the present invention.
[0084] An expression vector can be introduced into host cells using
a variety of standard techniques including calcium phosphate
transfection, liposome-mediated transfection,
microprojectile-mediated delivery, electroporation, and the like.
Transfected cells can be selected and propagated to provide
recombinant host cells that comprise the gene of interest stably
integrated in the host cell genome. Standard methods for
introducing nucleic acid molecules into bacterial, yeast, insect,
mammalian, and plant cells are provided, for example, by Ausubel
(1995). General methods for expressing and recovering foreign
protein produced by a mammalian cell system are provided by, for
example, Etcheverry, "Expression of Engineered Proteins in
Mammalian Cell Culture," in Protein Engineering: Principles and
Practice, Cleland et al. (eds.), pages 163 (Wiley-Liss, Inc.
1996).
[0085] The present invention, thus generally described, will be
understood more readily by reference to the following examples,
which are provided by way of illustration and are not intended to
be limiting of the present invention.
EXAMPLE 1
[0086] Construction of MPSV Promoter and pZMP21
[0087] The MPSV LTR promoter was constructed synthetically by
assembling oligonucleotides in sets of four using PCR.
[0088] First the oligos were assembled in pairs by PCR:SEQ ID NOs:
4+5, 6+7, 8+9, 10+11, 12+13, 14+15. Then the pairs were assembled
into three sets of four oligos SEQ ID NOS: 4+5 and 6+7, with oligos
SEQ ID NOs: 4 and 7 as primers, 8+9 and 10+11 with oligos 8 and 11
as primers, and 12+13 and 14+15 with oligos 12 and 15 as primers in
PCR reactions. When the three PCR fragments were assembled a
smaller than expected product was observed. A new primer, 16, was
made to get around the internal repeat that lead to this deletion.
The product of 4+7 was extended with primers 4 and 16 to make a
better overlap with the product of 8+15.4+16 and 8+15 were
assembled with primers 4 and 15 by PCR to make a full length
product.
[0089] The PCR reactions were run as follows: to a 100 .mu.l final
volume was added, 10 .mu.l 10.times.Taq polymerase Reaction Buffer
(Perkin Elmer), 8 .mu.l of 2.5 mM dNTPs, 78 .mu.l dH.sub.2O, 2
.mu.l each of a 20 mM stock solution of the two primers described
above, and taq polymerase (2.5 units, Life Technology). An equal
volume of mineral oil was added and the reaction was heated to
94.degree. C. for 2 minutes, followed by 25 cycles at 94.degree. C.
for 30 seconds, 45.degree. C. for 30 seconds, 72.degree. C. for 30
seconds followed by a 5 minute extension at 72.degree. C. In the
case of the first stage of assembly the primers were also the
templates of the reaction. For the later steps, 10 .mu.l of PCR
product was used as template for the each level of assembly.
[0090] Ten .mu.l of the 100 .mu.l PCR reaction is run on a 1.0%
agarose gel with 1.times.TBE buffer for analysis. The remaining 90
.mu.l of PCR reaction is precipitated with the addition of 5 .mu.l
1 M NaCl and 250 .mu.l of absolute ethanol. The plasmid pZMP20
which has been cut with NheI is used for recombination with the PCR
fragment. Plasmid pZMP20 was constructed from pZP9 (deposited at
the American Type Culture Collection, 10801 University Boulevard,
Manassas, Va. 20110-2209, and is designated No. 98668) with the
yeast genetic elements taken from pRS316 (deposited at the American
Type Culture Collection, 10801 University Boulevard, Manassas, Va.
20110-2209, and designated No. 77145), an IRES element from
poliovirus, and the extracellular domain of CD8, truncated at the
carboxyl terminal end of the transmembrane domain. pZMP20 is a
mammalian expression vector containing an expression cassette
having the cytomegalovirus immediate early promoter, immunoglobulin
signal peptide intron, multiple restriction sites for insertion of
coding sequences, a stop codon and a human growth hormone
terminator. The plasmid also has an E. coli origin of replication,
a mammalian selectable marker expression unit having an SV40
promoter, enhancer and origin of replication, a DHFR gene, the SV40
terminator, as well as the URA3 and CEN-ARS sequences required for
selection and replication in S. cerevisiae.
[0091] One hundred microliters of competent yeast cells (S.
cerevisiae) are independently combined with 10 .mu.l of the various
DNA mixtures from above and transferred to a 0.2 cm electroporation
cuvette. The yeast/DNA mixtures are electropulsed at 0.75 kV (5
kV/cm), .infin. ohms, 25 .mu.F. To each cuvette is added 600 .mu.l
of 1.2 M sorbitol and the yeast is plated in two 300 .mu.l aliquots
onto two URA-D plates and incubated at 30.degree. C. After about 48
hours, the Ura+ yeast transformants from a single plate are
resuspended in 1 ml H.sub.2O and spun briefly to pellet the yeast
cells. The cell pellet is resuspended in 1 ml of lysis buffer (2%
Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA).
Five hundred microliters of the lysis mixture is added to an
Eppendorf tube containing 300 .mu.l acid washed glass beads and 200
.mu.l phenol-chloroform, vortexed for 1 minute intervals two or
three times, followed by a 5 minute spin in a Eppendorf centrifuge
at maximum speed. Three hundred microliters of the aqueous phase is
transferred to a fresh tube, and the DNA precipitated with 600
.mu.l ethanol (EtOH), followed by centrifugation for 10 minutes at
4.degree. C. The DNA pellet is resuspended in 10 .mu.l
H.sub.2O.
[0092] Transformation of electrocompetent E. coli cells (DH10B,
GibcoBRL) is done with 0.5-2 ml yeast DNA prep and 40 ul of DH10B
cells. The cells are electropulsed at 1.7 kV, 25 .mu.F and 400
ohms. Following electroporation, 1 ml SOC (2% Bacto' Tryptone
(Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl,
2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose) is plated in
250 .mu.l aliquots on four LB AMP plates (LB broth (Lennox), 1.8%
Bacto Agar (Difco), 100 mg/L Ampicillin).
[0093] Individual clones harboring the correct construct are
identified by restriction digest to verify the presence of the MPSV
promoter and to confirm that the various DNA sequences have been
joined correctly to one another. The insert of positive clones are
subjected to sequence analysis. Larger scale plasmid DNA is
isolated using the Qiagen Maxi kit (Qiagen) according to
manufacturer's instruction. pZMP21 was deposited on Jun. 17, 2003
at the American Type Culture Collection (ATCC) 10801 University
Boulevard, Manassas, Va. 20110-2209, designated as ATCC #
PTA-5266.
EXAMPLE 2
[0094] Construction of Prethrombin Expression Vectors
[0095] An expression plasmid containing all or part of a
polynucleotide encoding prethrombin is constructed via homologous
recombination. A fragment of prethrombin cDNA is isolated using PCR
that includes the polynucleotide sequence from nucleotide 1 to
nucleotide 1380 of SEQ ID NO: 15 with flanking regions at the 5'
and 3' ends corresponding to the vectors sequences flanking the
prethrombin insertion point. The primers for PCR each include from
5' to 3' end: 40 bp of flanking sequence from the vector and 17 bp
corresponding to the amino and carboxyl termini from the open
reading frame of prethrombin.
[0096] Ten .mu.l of the 100 .mu.l PCR reaction is run on a 0.8% LMP
agarose gel (Seaplaque GTG) with 1.times.TBE buffer for analysis.
The remaining 90 .mu.l of PCR reaction is precipitated with the
addition of 5 .mu.l 1 M NaCl and 250 .mu.l of absolute ethanol. The
plasmids pZMP20 and pZMP21, described in the previous example,
which were cut with BglII were used for recombination with the PCR
fragment.
[0097] One hundred microliters of competent yeast cells (S.
cerevisiae) are independently combined with 10 .mu.l of the various
DNA mixtures from above and transferred to a 0.2 cm electroporation
cuvette. The yeast/DNA mixtures are electropulsed at 0.75 kV (5
kV/cm), .infin. ohms, 25 .mu.F. To each cuvette is added 600 .mu.l
of 1.2 M sorbitol and the yeast is plated in two 300 .mu.l aliquots
onto two URA-D plates and incubated at 30.degree. C. After about 48
hours, the Ura+ yeast transformants from a single plate are
resuspended in 1 ml H.sub.2O and spun briefly to pellet the yeast
cells. The cell pellet is resuspended in 1 ml of lysis buffer (2%
Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA).
Five hundred microliters of the lysis mixture is added to an
Eppendorf tube containing 300 .mu.l acid washed glass beads and 200
.mu.l phenol-chloroform, vortexed for 1 minute intervals two or
three times, followed by a 5 minute spin in a Eppendorf centrifuge
at maximum speed. Three hundred microliters of the aqueous phase is
transferred to a fresh tube, and the DNA precipitated with 600
.mu.l ethanol (EtOH), followed by centrifugation for 10 minutes at
4.degree. C. The DNA pellet is resuspended in 10 .mu.l
H.sub.2O.
[0098] Transformation of electrocompetent E. coli cells (DH10B,
Invitrogen) is done with 0.5-2 ml yeast DNA prep and 40 ul of DH10B
cells. The cells are electropulsed at 1.7 kV, 25 .mu.F and 400
ohms. Following electroporation, 1 ml SOC (2% Bacto' Tryptone
(Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl,
2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose) is plated in
250 .mu.l aliquots on four LB AMP plates (LB broth (Lennox), 1.8%
Bacto Agar (Difco), 100 mg/L Ampicillin).
[0099] Individual clones harboring the correct expression construct
for prethrombin are identified by restriction digest to verify the
presence of the prethrombin insert and to confirm that the various
DNA sequences have been joined correctly to one another. The insert
of positive clones are subjected to sequence analysis. Larger scale
plasmid DNA is isolated using the Qiagen Maxi kit (Qiagen)
according to manufacturer's instruction.
EXAMPLE 3
[0100] Expression of Prethrombin in Protein-Free,
Suspension-Adapted CHO Cells
[0101] Serum-free, suspension-adapted CHO DG44 cells were
electroporated with two of the plasmids described above:
pZMP21-prethrombin and the control plasmid, pZMP20-prethrombin, by
the following method. The plasmids were linearized by digestion
with PvuI, precipitated with sodium acetate and ethanol then rinsed
with 70% ethanol and dried. The pellets were resuspended at a
concentration of 200 .mu.g/100 .mu.l per electroporation in PFCHO
medium supplemented with 4 mM L-Glut, 1% Hypoxanthine/Thymidine, 1%
vitamins, and 1% Na pyruvate (Invitrogen). Cells, growing at log
phase, were pelleted and resuspended at 5E6/800 .mu.l per
electroporation reaction. The electroporation was performed in a
BioRad GenePulser II with Capacitance extender (BioRad, Hercules,
Calif.), at 300 v and 950 .mu.Fd in 4 mm cuvettes. The cells were
suspended in 25 ml of the medium described above in 125 mL shake
flasks and put on shakers in cell culture incubators at 37.degree.
C., at 80 rpm for 24 h to recover. The cells were then pelleted and
resuspended at 2.5E5 in selective medium, consisting of PFCHO
supplemented with 4 mM L-Glut, 1% vitamins, 1% Na Pyruvate. Cell
lines were further cultured in increasing concentrations of
methotrexate up to 1 .mu.M once the cultures were capable of
growing in the absence of hypoxanthine/thymidine supplementation.
Once the cultures were growing actively in selection media and the
viability had increased to over 95%, cultures were established for
harvest and analysis of protein. Cultures were seeded at 5E5/mL at
25 mL in shake flasks, and allowed to grow for 48 h then harvested.
The supernatants were filtered through 0.22 .mu.m filters and
analyzed by ELISA assay.
[0102] The ELISA assay was performed using two polyclonal
antibodies: capture antibody, sheep anti-humain prethrombin
fragment 2 (Accurate Chemical #20112AP) and detection antibody,
sheep anti-human prethrombin-HRP conjugate (Accurate Chemical
#20110HP). The coating antibody was diluted in 0.1 M Na carbonate
pH9.6 at 1 .mu.g/mL, dispensed into 96 wells and incubated at
4.degree. C. overnight. The plates were rinsed five times in wash
buffer (PBS plus 0.05% Tween) and blocked by incubating twices with
SuperBlock (Pierce, Rockford, Ill., #37515) 200 .mu.l/well 5
minutes at room temperature. The samples and standards were applied
to the plate in binding buffer (PBS, 0.05% Tween, 1 mg/mL BSA) and
incubated 1 hour at 37.degree. C. The plates were washed five times
in wash buffer and detection antibody diluted to 2 ng/mL in binding
buffer. The detection antibody was applied to the wells and
incubated 1 h at 37.degree. C. The plates were rinsed five times
with wash buffer and the detection reagent, OPD, was applied. OPD
was prepared by adding hydrogen peroxide immediately before use
according to the manufacturer's instructions (Pierce, Rockford,
Ill., #34006), 100 .mu.l added to each well, allowed to develop 10
minutes at RT and stopped with 100 .mu.l per well of 1 N H2SO4.
Plates were read at 492 nm. The results were calculated via
SoftMaxPro. Production rates of prethrombin by CHO cell pools was
calculated by dividing the prethrombin titer by the average number
of cells and the number of days in culture. These comparative
results are shown in a bar graph in FIG. 2 and indicate that
pZMP21-prethrombin produces approximately 3.6 times the amount of
recombinant protein as the pZMP20-prethrombin control.
EXAMPLE 4
[0103] Construction of zsig37 Expression Vectors
[0104] An expression plasmid containing all or part of a
polynucleotide encoding zsig37 is constructed via homologous
recombination. A fragment of zsig37 cDNA is isolated using PCR that
includes the polynucleotide sequence from nucleotide 1 to
nucleotide 873 of SEQ ID NO: 16 with flanking regions at the 5' and
3' ends corresponding to the vectors sequences flanking the zsig37
insertion point. The primers for PCR each include from 5' to 3'
end: 40 bp of flanking sequence from the vector and 17 bp
corresponding to the amino and carboxyl termini from the open
reading frame of zsig37.
[0105] Ten .mu.l of the 100 .mu.l PCR reaction is run on a 0.8% LMP
agarose gel (Seaplaque GTG) with 1.times.TBE buffer for analysis.
The remaining 90 .mu.l of PCR reaction is precipitated with the
addition of 5 .mu.l 1 M NaCl and 250 .mu.l of absolute ethanol. The
plasmids pZMP20 and pZMP21, described in the previous example,
which were cut with BglII were used for recombination with the PCR
fragment.
[0106] One hundred microliters of competent yeast cells (S.
cerevisiae) are independently combined with 10 .mu.l of the various
DNA mixtures from above and transferred to a 0.2 cm electroporation
cuvette. The yeast/DNA mixtures are electropulsed at 0.75 kV (5
kV/cm), .infin. ohms, 25 .mu.F. To each cuvette is added 600 .mu.l
of 1.2 M sorbitol and the yeast is plated in two 300 .mu.l aliquots
onto two URA-D plates and incubated at 30.degree. C. After about 48
hours, the Ura+ yeast transformants from a single plate are
resuspended in 1 ml H.sub.2O and spun briefly to pellet the yeast
cells. The cell pellet is resuspended in 1 ml of lysis buffer (2%
Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA).
Five hundred microliters of the lysis mixture is added to an
Eppendorf tube containing 300 .mu.l acid washed glass beads and 200
.mu.l phenol-chloroform, vortexed for 1 minute intervals two or
three times, followed by a 5 minute spin in a Eppendorf centrifuge
at maximum speed. Three hundred microliters of the aqueous phase is
transferred to a fresh tube, and the DNA precipitated with 600
.mu.l ethanol (EtOH), followed by centrifugation for 10 minutes at
4.degree. C. The DNA pellet is resuspended in 10 .mu.l
H.sub.2O.
[0107] Transformation of electrocompetent E. coli cells (DH10B,
Invitrogen) is done with 0.5-2 ml yeast DNA prep and 40 ul of DH10B
cells. The cells are electropulsed at 1.7 kV, 25 .mu.F and 400
ohms. Following electroporation, 1 ml SOC (2% Bacto' Tryptone
(Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl,
2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose) is plated in
250 .mu.l aliquots on four LB AMP plates (LB broth (Lennox), 1.8%
Bacto Agar (Difco), 100 mg/L Ampicillin).
[0108] Individual clones harboring the correct expression construct
for zsig37 are identified by restriction digest to verify the
presence of the zsig37 insert and to confirm that the various DNA
sequences have been joined correctly to one another. The insert of
positive clones are subjected to sequence analysis. Larger scale
plasmid DNA is isolated using the Qiagen Maxi kit (Qiagen)
according to manufacturer's instruction.
EXAMPLE 5
Analysis of the Stability of Production of zsig37 by Cells
Transfected with MPSV vs. CMV Expression Vectors
[0109] Serum-free, suspension-adapted CHO DG44 cells are
electroporated with the plasmids described above, by the following
method. The plasmids are linearized by digestion with PvuI,
precipitated with sodium acetate and ethanol then rinsed with 70%
ethanol and dried. The pellets are resuspended at a concentration
of 200 .mu.g/100 .mu.l per electroporation in PFCHO medium
supplemented with 4 mM L-Glut, 1% Hypoxanthine/Thymidine, 1%
vitamins, and 1% Na pyruvate (Invitrogen). Cells, growing at log
phase, are pelleted and resuspended at 5E6/800 .mu.l per
electroporation reaction. The electroporation is performed in, at
300 v and 950 .mu.Fd in 4 mm cuvettes. The cells are suspended in
25 ml of the medium described above in 125 mL shake flasks and put
on shakers in cell culture incubators at 37.degree. C., at 80 rpm
for 24 h to recover. The cells are then pelleted and resuspended at
2.5E5 in selective medium, consisting of PFCHO supplemented with 4
mM L-Glut, 1% vitamins, 1% Na Pyruvate. Cell lines are further
cultured in increasing concentrations of methotrexate up to 1 .mu.M
once the cultures are capable of growing in the absence of
hypoxanthine/thymidine supplementation. Once the cultures are
growing actively in selection media and the viability has increased
to over 95%, cultures are established for harvest and analysis of
protein. Cultures are passaged over a period of three months and
samples are removed weekly for analysis by ELISA. The supernatants
were filtered through 0.22 .mu.m filters and analyzed by ELISA
assay.
[0110] The ELISA assay is performed using two polyclonal
antibodies: capture antibody, sheep anti-human zsig37 and detection
antibody, sheep anti-human zsig37-HRP conjugate. The coating
antibody is diluted in 0.1 M Na carbonate pH9.6 at 1 .mu.g/mL,
dispensed into 96 wells and incubated at 4.degree. C. overnight.
The plates are rinsed five times in wash buffer (PBS plus 0.05%
Tween) and blocked by incubating twices with SuperBlock (Pierce,
Rockford, Ill., #37515) 200 .mu.l/well 5 minutes at room
temperature. The samples and standards are applied to the plate in
binding buffer (PBS, 0.05% Tween, 1 mg/mL BSA) and incubated 1 hour
at 37.degree. C. The plates are washed five times in wash buffer
and detection antibody diluted to 2 ng/mL in binding buffer. The
detection antibody is applied to the wells and incubated 1 h at
37.degree. C. The plates are rinsed five times with wash buffer and
the detection reagent, OPD, was applied. OPD is prepared by adding
hydrogen peroxide immediately before use according to the
manufacturer's instructions (Pierce, Rockford, Ill., #34006), 100
.mu.l added to each well, allowed to develop 10 minutes at RT and
stopped with 100 .mu.l per well of 1 N H2SO4. Plates are read at
492 nm. The results are calculated via SoftMaxPro. Production rates
of zsig37 by CHO cell pools is calculated by dividing the zsig37
titer by the average number of cells and the number of days in
culture. The levels of productivity as a function of time are
calculated for the two cultures for comparison.
[0111] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
16 1 851 DNA Artificial Sequence hybrid cytomegalovirus and
myeloproliferative sarcoma virus regulatory sequence 1 ggctgaccgc
ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 60
acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
120 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
caatgacggt 180 aaatggcccg cctggcatta tgcccagtac atgaccttat
gggactttcc tacttggcag 240 tacatctacg tattagtcat cgctattacc
atggtgatgc ggttttggca gtacatcaat 300 gggcgtggat agcggtttga
ctcacgggga tttccaagtc tccaccccat tgacgtcaat 360 gggagtttgt
tttgaatgaa agaccccacc tgtaggtttg gcaagctagc ttaagtaacg 420
ccatttgcaa ggcatggaaa aatacataac tgagaataga gaagttcaga tcaaggtcag
480 gaacagagaa acaggagaat atgggccaaa caggatatct gtggtaagca
gttcctgccc 540 cgctcagggc caagaacagt tggaacagga gaatatgggc
caaacaggat atctgtggta 600 agcagttcct gccccgctca gggccaagaa
cagatggtcc ccagatcggt cccgccctca 660 gcagtttcta gagaaccatc
agatgtttcc agggtgcccc aaggacctga aatgaccctg 720 tgccttattt
gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc 780
ccgagctcaa taaaagagcc cacaacccct cactcggcgc gccagtcctc cgatagactg
840 cgtcgcccgg g 851 2 60 DNA cytomegalovirus 2 caagtctcca
ccccattgac gtcaatggga gtttgttttg caatgaaaga ccccacctgt 60 3 65 DNA
cytomegalovirus 3 ttccatgcct tgcaaatggc gttacttaag ctagcttgcc
aaacctacag gtggggtctt 60 tcatt 65 4 63 DNA cytomegalovirus 4
ccatttgcaa ggcatggaaa aatacataac tgagaataga gaagttcaga tcaaggtcag
60 gaa 63 5 63 DNA cytomegalovirus 5 cttaccacag atatcctgtt
tggcccatat tctcctgttt ctctgttcct gaccttgatc 60 tga 63 6 64 DNA
cytomegalovirus 6 acaggatatc tgtggtaagc agttcctgcc ccgctcaggg
ccaagaacag ttggaacagg 60 agaa 64 7 64 DNA cytomegalovirus 7
gagcggggca ggaactgctt accacagata tcctgtttgg cccatattct cctgttccaa
60 ctgt 64 8 65 DNA cytomegalovirus 8 agcagttcct gccccgctca
gggccaagaa cagatggtcc ccagatcggt cccgccctca 60 gcagt 65 9 66 DNA
cytomegalovirus 9 atttcaggtc cttggggcac cctggaaaca tctgatggtt
ctctagaaac tgctgagggc 60 gggacc 66 10 65 DNA cytomegalovirus 10
gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc
60 gcttc 65 11 67 DNA cytomegalovirus 11 ggggttgtgg gctcttttat
tgagctcggg gagcagaagc gcgcgaacag aagcgagaag 60 cgaactg 67 12 63 DNA
cytomegalovirus 12 taaaagagcc cacaacccct cactcggcgc gccagtcctc
cgatagactg cgtcgcccgg 60 ggc 63 13 64 DNA cytomegalovirus 13
ctactgtgag ccccttacct gtagctgaga tccacgagcc gctagccccg ggcgacgcag
60 tcta 64 14 64 DNA cytomegalovirus 14 ttctcctgtt ccaactgttc
ttggccctga gcggggcagg aactgcttac cacagatatc 60 ctgt 64 15 1380 DNA
Homo sapien CDS (1)...(1380) 15 atg gat gca atg aag aga ggg ctc tgc
tgt gtg ctg ctg ctg tgt ggc 48 Met Asp Ala Met Lys Arg Gly Leu Cys
Cys Val Leu Leu Leu Cys Gly 1 5 10 15 gcc gtc ttc gtt tcg ctc agc
cag gaa atc cat gcc gag ttg aga cgc 96 Ala Val Phe Val Ser Leu Ser
Gln Glu Ile His Ala Glu Leu Arg Arg 20 25 30 ttc cgt aga tct gaa
ggc tcc agt gtg aat ctg tca cct cca ctc gag 144 Phe Arg Arg Ser Glu
Gly Ser Ser Val Asn Leu Ser Pro Pro Leu Glu 35 40 45 cag tgt gtc
cct gat cgg ggg cag cag tac cag ggg cgc ctg gcg gtg 192 Gln Cys Val
Pro Asp Arg Gly Gln Gln Tyr Gln Gly Arg Leu Ala Val 50 55 60 acc
aca cat ggg ctc ccc tgc ctg gcc tgg gcc agc gca cag gcc aag 240 Thr
Thr His Gly Leu Pro Cys Leu Ala Trp Ala Ser Ala Gln Ala Lys 65 70
75 80 gcc ctg agc aag cac cag gac ttc aac tca gct gtg cag ctg gtg
gag 288 Ala Leu Ser Lys His Gln Asp Phe Asn Ser Ala Val Gln Leu Val
Glu 85 90 95 aac ttc tgc cgc aac cca gac ggg gat gag gag ggc gtg
tgg tgc tat 336 Asn Phe Cys Arg Asn Pro Asp Gly Asp Glu Glu Gly Val
Trp Cys Tyr 100 105 110 gtg gcc ggg aag cct ggc gac ttt ggg tac tgc
gac ctc aac tat tgt 384 Val Ala Gly Lys Pro Gly Asp Phe Gly Tyr Cys
Asp Leu Asn Tyr Cys 115 120 125 gag gag gcc gtg gag gag gag aca gga
gat ggg ctg gat gag gac tca 432 Glu Glu Ala Val Glu Glu Glu Thr Gly
Asp Gly Leu Asp Glu Asp Ser 130 135 140 gac agg gcc atc gaa ggg cgt
acc gcc aca agt gag tac cag act ttc 480 Asp Arg Ala Ile Glu Gly Arg
Thr Ala Thr Ser Glu Tyr Gln Thr Phe 145 150 155 160 ttc aat ccg agg
acc ttt ggc tcg gga gag gca gac tgt ggg ctg cga 528 Phe Asn Pro Arg
Thr Phe Gly Ser Gly Glu Ala Asp Cys Gly Leu Arg 165 170 175 cct ctg
ttc gag aag aag tcg ctg gag gac aaa acc gaa aga gag ctc 576 Pro Leu
Phe Glu Lys Lys Ser Leu Glu Asp Lys Thr Glu Arg Glu Leu 180 185 190
ctg gaa tcc tac atc gac ggg cgc att gtg gag ggc tcg gat gca gag 624
Leu Glu Ser Tyr Ile Asp Gly Arg Ile Val Glu Gly Ser Asp Ala Glu 195
200 205 atc ggc atg tca cct tgg cag gtg atg ctt ttc cgg aag agt ccc
cag 672 Ile Gly Met Ser Pro Trp Gln Val Met Leu Phe Arg Lys Ser Pro
Gln 210 215 220 gag ctg ctg tgt ggg gcc agc ctc atc agt gac cgc tgg
gtc ctc acc 720 Glu Leu Leu Cys Gly Ala Ser Leu Ile Ser Asp Arg Trp
Val Leu Thr 225 230 235 240 gcc gcc cac tgc ctc ctg tac ccg ccc tgg
gac aag aac ttc acc gag 768 Ala Ala His Cys Leu Leu Tyr Pro Pro Trp
Asp Lys Asn Phe Thr Glu 245 250 255 aat gac ctt ctg gtg cgc att ggc
aag cac tcc cgc acc agg tac gag 816 Asn Asp Leu Leu Val Arg Ile Gly
Lys His Ser Arg Thr Arg Tyr Glu 260 265 270 cga aac att gaa aag ata
tcc atg ttg gaa aag atc tac atc cac ccc 864 Arg Asn Ile Glu Lys Ile
Ser Met Leu Glu Lys Ile Tyr Ile His Pro 275 280 285 agg tac aac tgg
cgg gag aac ctg gac cgg gac att gcc ctg atg aag 912 Arg Tyr Asn Trp
Arg Glu Asn Leu Asp Arg Asp Ile Ala Leu Met Lys 290 295 300 ctg aag
aag cct gtt gcc ttc agt gac tac att cac cct gtg tgt ctg 960 Leu Lys
Lys Pro Val Ala Phe Ser Asp Tyr Ile His Pro Val Cys Leu 305 310 315
320 ccc gac agg gag acg gca gcc agc ttg ctc cag gct gga tac aag ggg
1008 Pro Asp Arg Glu Thr Ala Ala Ser Leu Leu Gln Ala Gly Tyr Lys
Gly 325 330 335 cgg gtg aca ggc tgg ggc aac ctg aag gag acg tgg aca
gcc aac gtt 1056 Arg Val Thr Gly Trp Gly Asn Leu Lys Glu Thr Trp
Thr Ala Asn Val 340 345 350 ggt aag ggg cag ccc agt gtc ctg cag gtg
gtg aac ctg ccc att gtg 1104 Gly Lys Gly Gln Pro Ser Val Leu Gln
Val Val Asn Leu Pro Ile Val 355 360 365 gag cgg ccg gtc tgc aag gac
tcc acc cgg atc cgc atc act gac aac 1152 Glu Arg Pro Val Cys Lys
Asp Ser Thr Arg Ile Arg Ile Thr Asp Asn 370 375 380 atg ttc tgt gct
ggt tac aag cct gat gaa ggg aaa cga ggg gat gcc 1200 Met Phe Cys
Ala Gly Tyr Lys Pro Asp Glu Gly Lys Arg Gly Asp Ala 385 390 395 400
tgt gaa ggt gac agt ggg gga ccc ttt gtc atg aag agc ccc ttt aac
1248 Cys Glu Gly Asp Ser Gly Gly Pro Phe Val Met Lys Ser Pro Phe
Asn 405 410 415 aac cgc tgg tat caa atg ggc atc gtc tca tgg ggt gaa
ggc tgt gac 1296 Asn Arg Trp Tyr Gln Met Gly Ile Val Ser Trp Gly
Glu Gly Cys Asp 420 425 430 cgg gat ggg aaa tat ggc ttc tac aca cat
gtg ttc cgc ctg aag aag 1344 Arg Asp Gly Lys Tyr Gly Phe Tyr Thr
His Val Phe Arg Leu Lys Lys 435 440 445 tgg ata cag aag gtc att gat
cag ttt gga gag taa 1380 Trp Ile Gln Lys Val Ile Asp Gln Phe Gly
Glu * 450 455 16 2769 DNA Homo sapien CDS (171)...(1016) 16
gaattcgaat tcctttgttt ccactgggac ggaatcggag ctctggaggc tgggctggcc
60 aagcgccccg aaggcccgat gcctgacggc tcatgcggcc tccttgtttg
cagggcctgg 120 gcaaaaattt acactgagtc ccactcttcg ctccagggcc
cggcaggaag atg ggc 176 Met Gly 1 tcc cgt gga cag gga ctc ttg ctg
gcg tac tgc ctg ctc ctt gcc ttt 224 Ser Arg Gly Gln Gly Leu Leu Leu
Ala Tyr Cys Leu Leu Leu Ala Phe 5 10 15 gcc tct ggc ctg gtc ctg agt
cgc gtg ccc cat gtc cag ggg gaa cag 272 Ala Ser Gly Leu Val Leu Ser
Arg Val Pro His Val Gln Gly Glu Gln 20 25 30 cag gag tgg gag ggg
act gag gag ctg ccg tcc cct ccg gac cat gcc 320 Gln Glu Trp Glu Gly
Thr Glu Glu Leu Pro Ser Pro Pro Asp His Ala 35 40 45 50 gag agg gct
gaa gaa caa cat gaa aaa tac agg ccc agt cag gac cag 368 Glu Arg Ala
Glu Glu Gln His Glu Lys Tyr Arg Pro Ser Gln Asp Gln 55 60 65 ggg
ctc cct gct tcc cgg tgc ttg cgc tgc tgt gac cct ggt acc tcc 416 Gly
Leu Pro Ala Ser Arg Cys Leu Arg Cys Cys Asp Pro Gly Thr Ser 70 75
80 atg tac ccg gcg acc gcc gtg ccc cag atc aac atc act atc ttg aaa
464 Met Tyr Pro Ala Thr Ala Val Pro Gln Ile Asn Ile Thr Ile Leu Lys
85 90 95 ggg gag aag ggt gac cgc gga gat cga ggc ctc caa ggg aaa
tat ggc 512 Gly Glu Lys Gly Asp Arg Gly Asp Arg Gly Leu Gln Gly Lys
Tyr Gly 100 105 110 aaa aca ggc tca gca ggg gcc agg ggc cac act gga
ccc aaa ggg cag 560 Lys Thr Gly Ser Ala Gly Ala Arg Gly His Thr Gly
Pro Lys Gly Gln 115 120 125 130 aag ggc tcc atg ggg gcc cct ggg gag
cgg tgc aag agc cac tac gcc 608 Lys Gly Ser Met Gly Ala Pro Gly Glu
Arg Cys Lys Ser His Tyr Ala 135 140 145 gcc ttt tcg gtg ggc cgg aag
aag ccc atg cac agc aac cac tac tac 656 Ala Phe Ser Val Gly Arg Lys
Lys Pro Met His Ser Asn His Tyr Tyr 150 155 160 cag acg gtg atc ttc
gac acg gag ttc gtg aac ctc tac gac cac ttc 704 Gln Thr Val Ile Phe
Asp Thr Glu Phe Val Asn Leu Tyr Asp His Phe 165 170 175 aac atg ttc
acc ggc aag ttc tac tgc tac gtg ccc ggc ctc tac ttc 752 Asn Met Phe
Thr Gly Lys Phe Tyr Cys Tyr Val Pro Gly Leu Tyr Phe 180 185 190 ttc
agc ctc aac gtg cac acc tgg aac cag aag gag acc tac ctg cac 800 Phe
Ser Leu Asn Val His Thr Trp Asn Gln Lys Glu Thr Tyr Leu His 195 200
205 210 atc atg aag aac gag gag gag gtg gtg atc ttg ttc gcg cag gtg
ggc 848 Ile Met Lys Asn Glu Glu Glu Val Val Ile Leu Phe Ala Gln Val
Gly 215 220 225 gac cgc agc atc atg caa agc cag agc ctg atg ctg gag
ctg cga gag 896 Asp Arg Ser Ile Met Gln Ser Gln Ser Leu Met Leu Glu
Leu Arg Glu 230 235 240 cag gac cag gtg tgg gta cgc ctc tac aag ggc
gaa cgt gag aac gcc 944 Gln Asp Gln Val Trp Val Arg Leu Tyr Lys Gly
Glu Arg Glu Asn Ala 245 250 255 atc ttc agc gag gag ctg gac acc tac
atc acc ttc agt ggc tac ctg 992 Ile Phe Ser Glu Glu Leu Asp Thr Tyr
Ile Thr Phe Ser Gly Tyr Leu 260 265 270 gtc aag cac gcc acc gag ccc
tag ctggccggcc acctcctttc ctctcgccac 1046 Val Lys His Ala Thr Glu
Pro * 275 280 cttccacccc tgcgctgtgc tgaccccagg gctcagcacc
aggctgaccc caccgcctct 1106 tccccgatcc ctggactccg actccctggc
tttggcattc agtgagacgc cctgcacaca 1166 cagaaagcca aagcgatcgg
tgctcccaga tcccgcagcc tctggagaga gctgacggca 1226 gatgaaatca
ccagggcggg gcacccgcga gaaccctctg ggaccttccg cggccctctc 1286
tgcacacatc ctcaagtgac cccgcacggc gagacgcggg tggcggcagg gcgtcccagg
1346 gtgcggcacc gcggctccag tccttggaaa taattaggca aattctaaag
gtctcaaaag 1406 gagcaaagta aaccgtggag gacaaagaaa agggttgtta
tttttgtctt tccagccagc 1466 ctgctggctc ccaagagaga ggccttttca
gttgagactc tgcttaagag aagatccaaa 1526 gttaaagctc tggggtcagg
ggaggggccg ggggcaggaa actacctctg gcttaattct 1586 tttaagccac
gtaggaactt tcttgaggga taggtggacc ctgacatccc tgtggccttg 1646
cccaagggct ctgctggtct ttctgagtca cagctgcgag gtgatggggg ctggggcccc
1706 aggcgtcagc ctcccagagg gacagctgag ccccctgcct tggctccagg
ttggtagaag 1766 cagccgaagg gctcctgaca gtggccaggg acccctgggt
cccccaggcc tgcagatgtt 1826 tctatgaggg gcagagctcc tggtacatcc
atgtgtggct ctgctccacc cctgtgccac 1886 cccagagccc tggggggtgg
tctccatgcc tgccaccctg gcatcggctt tctgtgccgc 1946 ctcccacaca
aatcagcccc agaaggcccc ggggctttgg cttctgtttt ttataaaaca 2006
cctcaagcag cactgcagtc tcccatctcc tcgtgggcta agcatcaccg cttccacgtg
2066 tgttgtgttg gttggcagca aggctgatcc agaccccttc tgcccccact
gccctcatcc 2126 aggcctctga ccagtagcct gagaggggct ttttctaggc
ttcagagcag gggagagctg 2186 gaaggggcta gaaagctccc gcttgtctgt
ttctcaggct cctgtgagcc tcagtcctga 2246 gaccagagtc aagaggaagt
acacatccca atcacccgtg tcaggattca ctctcaggag 2306 ctgggtggca
ggagaggcaa tagcccctgt ggcaattgca ggaccagctg gagcagggtt 2366
gcggtgtctc cgcggtgctc tcgccctgcc catggccacc ccagactctg atctccagga
2426 accccatagc ccctctccac ctcaccccat gttgatgccc agggtcactc
ttgctacccg 2486 ctgggccccc aaacccccgc tgcctctctt ccttcccccc
atcccccacc tggttttgac 2546 taatcctgct tccctctctg ggcctggctg
ccgggatctg gggtccctaa gtccctctct 2606 ttaaagaact tctgcgggtc
agactctgaa gccgagttgc tgtgggcgtg cccggaagca 2666 gagcgccaca
ctcgctgctt aagctccccc agctctttcc agaaaacatt aaactcagaa 2726
ttgtgttttc agcaaaaaaa aaaaaaaaaa aaagggcggc cgc 2769
* * * * *