U.S. patent application number 15/399432 was filed with the patent office on 2017-07-27 for expression vector comprising a polynucleotide encoding a modified glutamine synthetase and a method for preparing a target protein employing the same.
The applicant listed for this patent is ARES TRADING S.A.. Invention is credited to Yong Ho Ahn, Hyun Sook Jang, Sun Kyu Kim, Dong Heon Lee, Sang Kyung Park.
Application Number | 20170211057 15/399432 |
Document ID | / |
Family ID | 49161429 |
Filed Date | 2017-07-27 |
United States Patent
Application |
20170211057 |
Kind Code |
A1 |
Jang; Hyun Sook ; et
al. |
July 27, 2017 |
Expression Vector Comprising a Polynucleotide Encoding a Modified
Glutamine Synthetase and a Method for Preparing a Target Protein
Employing the Same
Abstract
The present invention relates to a vector comprising a
polynucleotide encoding a modified glutamine synthetase (GS), and a
method for preparing a target protein employing the same. More
particulary, the present invention relates to a modified GS having
an increased sensitivity to a glutamine synthetase (GS) inhibitor,
a polynucleotide encoding the modified GS, a vector comprising the
polynucleotide, a transformat comprising the vector, and a method
for preparing a target protein using the transformat.
Inventors: |
Jang; Hyun Sook; (Daejeon,
KR) ; Lee; Dong Heon; (Daejeon, KR) ; Kim; Sun
Kyu; (Daejeon, KR) ; Ahn; Yong Ho; (Daejeon,
KR) ; Park; Sang Kyung; (Daejeon, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ARES TRADING S.A. |
Aubonne |
|
CH |
|
|
Family ID: |
49161429 |
Appl. No.: |
15/399432 |
Filed: |
January 5, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14384594 |
Sep 11, 2014 |
9567577 |
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PCT/KR2013/001779 |
Mar 5, 2013 |
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15399432 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/85 20130101;
C07K 14/71 20130101; C12N 2840/203 20130101; C12Y 603/01002
20130101; A61K 35/12 20130101; C12N 9/93 20130101; C07K 2319/30
20130101 |
International
Class: |
C12N 9/00 20060101
C12N009/00; C07K 14/71 20060101 C07K014/71; A61K 35/12 20060101
A61K035/12; C12N 15/85 20060101 C12N015/85 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 12, 2012 |
KR |
10-2012-0025197 |
Claims
1. A modified glutamine synthetase (GS), wherein glycine (Gly, G)
at position 299 of a glutamine synthetase having an amino acid
sequence shown in SEQ ID NO. 4 is substituted with arginine (Arg,
R).
2. The modified glutamine synthetase according to claim 1, having
an increased sensitivity to a glutamine synthetase inhibitor,
compared to the glutamine synthetase having an amino acid sequence
shown in SEQ ID NO.4.
3. The modified glutamine synthetase according to claim 2, wherein
the glutamine synthetase inhibitor is selected from the group
consisting of glycine, alanine, tryptophan, histidine,
glucosamine-6-phosphate, cytidine triphosphate, methionine
sulphoximine (MSX), and combinations thereof.
4. (canceled)
5. (canceled)
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. (canceled)
11. (canceled)
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. A target protein prepared by a method comprising culturing a
transformant comprising a vector for expression of a target
protein, comprising the polynucleotide encoding a modified
glutamine synthetase wherein glycine (Gly, G) at position 299 of a
glutamine synthetase having an amino acid sequence shown in SEQ ID
NO. 4 is substituted with arginine (Arg, R).
Description
TECHNICAL FIELD
[0001] The present invention relates to a vector comprising a
polynucleotide encoding a modified glutamine synthetase (GS), and a
method for preparing a target protein employing the same. More
particularly, the present invention relates to a modified GS having
an increased sensitivity to a glutamine synthetase (GS) inhibitor,
a polynucleotide encoding the modified GS, a vector comprising the
polynucleotide, a transformant comprising the vector, and a method
for preparing a target protein using the transformant.
BACKGROUND ART
[0002] Recombinant proteins can be expressed in different types of
host cells including prokaryotic and eukaryotic cells. However,
glycoproteins, such as antibodies and Fc fusion proteins, consist
of a polypeptide linked to a carbohydrate moiety which influences
the safety and efficacy thereof, and thus they are usually
expressed in animal cells that are capable of glycosylation during
post-translational modification. In the production of recombinant
protein drugs, the difference in their sugar chains with those of
human (native) glycoproteins is associated with immunogenicity of
the protein drugs. Thus, it is important to produce glycoproteins
having sugar chains identical or similar to those of human
glycoproteins.
[0003] Until recently, animal cells such as hybridoma, mouse
myeloma, and CHO cells have been commonly used in the expression
and production of recombinant protein drugs. The protein expression
in these animal cells is appropriate for producing proteins similar
to human proteins, but there are disadvantages of a significantly
low expression yield and difficulty in scale-up of the production.
In particular, therapeutic antibodies need to be produced in
kilogram quantities, and thus animal cell culturing is not suitable
for a large-scale production of the therapeutic antibodies.
Therefore, for a high level expression of a target gene in the host
cells, the target gene needs to be integrated into a
transcriptionally active region of the genome when the target DNA
is randomly introduced to the animal cell. The introduced foreign
gene replicates along with the genome of the host cell. However a
homologous recombination technique for integration of the gene into
transcriptionally active regions is not generalized for common use
yet.
[0004] Another method for increasing the expression rate of
randomly integrated DNA is by amplifying the integrated gene, and
this method needs the step of cloning the gene into the vector
engineered with gene amplification system. A DHFR system (Takeshi
omasa, gene amplification and its application in cell and tissue
engineering, J. Bios. and Bioe (2002), Vol. 94, No. 6, 600-605) is
a common gene amplification system, and this system increases the
protein expression level by co-amplification of a target gene and
DHFR using methotrexate (MTX) which is an inhibitor of
dihydrofolate reductase (DHFR). DHFR is an enzyme involved in
nucleotide biosynthesis, and thus inhibition of DHFR activity can
effectively interrupt DNA synthesis which is essential for cell
maintenance, thereby leading to cell death. Therefore, only those
clones having exogenous DHFR gene inserted in their genome can
survive under this condition. Furthermore, when the concentration
of MTX being added is increased and a strong promoter is used, DHFR
gene can be amplified to hundreds to thousands of copies. That is,
the more MTX, a DHFR inhibitor, is added to the cell culture, in
order for them to survive they increase the expression of DHFR
along with the introduced target gene. Consequently, several copies
of DNA will be incorporated, leading to the generation of various
molecular variants.
[0005] A DHFR system using CHO cell line has been reported and
commercialized as an expression system for various protein drugs,
verifying its safety and efficacy in use. However, the DHFR system
has a disadvantage in that it requires several months to isolate a
single cell line that shows the expression level higher than the
normal level. In addition, when the cell becomes resistant to MTX,
even with an increase in MTX concentration, the target gene cannot
be amplified anymore. Furthermore, in the CHO DUKX cells used for
DHFR system, revertants may appear easily. As a result, there has
been a high demand for the development of a high-level gene
expression system for protein production other than the DHFR
system.
[0006] GS system is a high-level gene expression system that was
first developed by Celltech (U.S. Pat. No. 5,122,464), and it
overcame the limitations of the DHFR-based gene expression system,
that is, low time-efficiency for isolating the single cell line of
interest and low productivity of target protein. The GS system
utilizes glutamine synthetase (GS) which is an enzyme involved in
the sole synthetic pathway for producing glutamine from glutamate
and ammonia, based on the fact that animal cells cannot grow
properly in the glutamine-deficient condition. The GS system has
advantage in that it requires less number of DNA copies per cell
compared to the DHFR system and allows for the selection of single
cell line having high expression rate at the early stage of
screening. Consequently, an increasing number of organizations
adopt the GS system as a protein drug expression system. NS0 cell
line and CHO cell line are the most commonly used cell lines for GS
system. Between two, NS0 cell line which is a mouse myeloma cell
line cannot express sufficient amount of GS, and thus in the
glutamine-deficient condition, those cells where the target gene is
inserted into their genome can be easily selected. Unlike the NSO
cell line, a CHO cell line can express sufficient amount of GS that
they can survive even in the glutamine-deficient medium. However,
if the CHO cells are treated with a high concentration of
GS-specific inhibitor such as methionine sulphoximine (MSX), the
cells cannot survive only with the endogenous GS activity, and thus
only those cells introduced with the vector comprising the GS gene
and the gene for a target protein can survive. Through the above
mechanism, the cells inserted with the gene for target protein can
be isolated, and the target protein can be produced at high yield.
In other words, as more GS-specific inhibitor is added to the
cells, in order for them to survive, they will amplify exogenous GS
gene as well as the target gene which is introduced together with
the GS gene, thereby increasing the amount of target protein in the
cell. However, even this GS system has a limitation in production
amount when the cells transfected with the vector comprising the
genes for target protein and GS are treated with the GS-specific
inhibitor for producing the target protein. Also when the cells
were cultured for a long time, the production amount of the target
protein was reduced. Due to these limitations, there has been a
high demand for the development of a modified GS protein that
responses more sensitively to the GS inhibitor and thus can amplify
the target gene introduced with GS to the greater level.
DISCLOSURE OF INVENTION
Technical Problem
[0007] In an effort to develop a modified GS protein that has a
significantly higher sensitivity to GS inhibitor compared to the
wildtype GS protein, the present inventors have developed a
modified GS with a significantly higher sensitivity to GS inhibitor
having one amino acid substituted compared to the wildtype GS
protein. Then, the present inventors confirmed the production of
the target protein in the presence of GS inhibitor after
transfecting the animal cells with the vector comprising the gene
encoding the modified GS and target protein, and further confirmed
that the modified GS demonstrates high sensitivity towards GS
inhibitor, showing the remarkably higher level of target protein
production compared to the vector system comprising the wildtype GS
protein and also no reduction in the production level of target
protein even after long period of culturing, thereby completing the
present invention.
Solution to Problem
[0008] An object of the present invention is to provide a modified
glutamine synthetase (GS), wherein glycine (Gly, G) at position 299
of a glutamine synthetase having an amino acid sequence shown in
SEQ ID NO. 4 is substituted with arginine (Arg, R).
[0009] Another object of the present invention is to provide a
polynucleotide encoding the modified glutamine synthetase.
[0010] Still another object of the present invention is to provide
a vector for the expression of a target protein, comprising the
polynucleotide and the gene encoding the target protein.
[0011] Still another object of the present invention is to provide
a transformant comprising the vector.
[0012] Still another object of the present invention is to provide
a method for the preparation of a target protein comprising
culturing of the transformant and to provide the target protein
prepared by said method.
Advantageous Effects of Invention
[0013] The expression vector comprising a modified GS gene of the
present invention allows for the selection of the host cells
introduced with the expression vector comprising the modified GS
gene even under the condition where CHO cells are used as a host
cell and the cells are treated with a GS inhibitor. Therefore, the
present expression vector can be widely used for the efficient
production of a target protein.
BRIEF DESCRIPTION OF DRAWINGS
[0014] FIG. 1 is a schematic diagram showing the cloning method for
preparing pcDNA3.1-Kozak-TNFR-Fc-IRES-GS or
pcDNA3.1-kozak-TNFR-Fc-IRES-GS PM of the present invention;
[0015] FIG. 2 is a cleavage map of the recombinant expression
vector, pcDNA3.1-kozak-TNFR-Fc-IRES-GS or
pcDNA3.1-kozak-TNFR-Fc-IRES-GS PM, comprising a gene encoding a
TNFR-Fc fusion protein which is a representative target protein of
the present invention;
[0016] FIG. 3 is a schematic diagram showing the cloning method for
preparing the recombinant expression vector,
pcDNA3.1-kozak-TNFR-Fc-SV40-GS or pcDNA3.1-kozak-TNFR-Fc-SV40-GS
PM, comprising a gene encoding a TNFR-Fc fusion protein which is a
representative target protein of the present invention;
[0017] FIG. 4 is a cleavage map of the recombinant expression
vector, pcDNA3.1-kozak-TNFR-Fc-SV40-GS or
pcDNA3.1-kozak-TNFR-Fc-SV40-GS PM, comprising a gene encoding a
TNFR-Fc fusion protein which is a representative target protein of
the present invention;
[0018] FIG. 5 is a graph showing the expression level of TNFR-Fc in
CHO K-1 cells transfected with one of the three types of TNFR-Fc
expression vectors (IRES-GS PM, IRES-GS, or SV40-GS);
[0019] FIG. 6 is a graph showing the changes in expression level of
TNFR-Fc protein with MSX treatment over time after transfection of
CHO K-1 cells with one of the three types of TNFR-Fc expression
vectors (IRES-GS PM, IRES-GS, or SV40-GS);
[0020] FIG. 7 is a graph demonstrating the changes in TNFR-Fc
expression level confirmed by the secondary transient transfection,
showing a high expression level in the SV40-GS vector-transfected
group during the early phase of culturing, but after longer period
of culturing with the MSX treatment the highest expression level
was observed in IRES-GS PM vector-transfected group; and
[0021] FIG. 8 is a graph showing the TNFR-Fc expression level in
two groups of stable CHO-S cell line each transfected with either
IRES-GS PM vector or SV40-GS PM vector.
BEST MODE FOR CARRYING OUT THE INVENTION
[0022] As one aspect, the present invention provides a modified
glutamine synthetase (GS), wherein glycine (Gly, G) at position 299
of a glutamine synthetase having an amino acid sequence shown in
SEQ ID NO. 4 is substituted with arginine (Arg, R).
[0023] As used herein, the term `glutamine synthetase (GS)` refers
to an enzyme that is found in the mammalian organs and
microorganisms and that catalyzes the synthesis of glutamine from
glutamate and ammonia in the presence of ATP. For activation of
this enzyme, divalent metal ions are required, and its enzymatic
activity is inhibited by the presence of glycine, alanine,
tryptophan, histidine, glucosamine-6-phosphate, cytidine
triphosphate, etc. For the purpose of the present invention, the
glutamine synthetase refers to an enzyme that can be used to select
the cells transfected with the vector comprising the gene encoding
a target protein or to enhance the expression of the target protein
by treatment with GS inhibitor, but is not limited thereto.
[0024] The information on the glutamine synthetase can be obtained
from the common database such as NCBI GenBank. For instance, the
information on glutamine synthetase derived from hamster can be
found from GenBank with the accession number X03495.1, but is not
limited thereto. The nucleotide sequence of the representative wild
type glutamine synthetase is shown as SEQ ID NO. 3 and its amino
acid sequence is shown as SEQ ID NO. 4.
[0025] As used herein, the term `modified glutamine synthetase`
refers to an enzyme wherein glycine (Gly, G) at position 299 of a
glutamine synthetase modified glutamine synthetase (GS), wherein
glycine (Gly, G) at position 299 of a glutamine synthetase having
an amino acid sequence shown in SEQ ID NO. 4 is substituted with
arginine (Arg, R).
[0026] The modified glutamine synthetase may be used as a selection
marker by transfection of the cells that can or cannot produce GS
endogenously with the target protein expression vector comprising
the gene encoding the modified glutamine synthetase, or preferably
refers to the protein that can enhance the target protein
expression by treatment with GS inhibitor through polycistronic
translation with the gene encoding the target protein in the form
of `polynucleotide encoding the promoter-target protein encoding
gene-IRES-modified glutamine synthetase` or `polynucleotide
encoding the promoter-modified glutamine synthetase-IRES-target
protein encoding gene`, but is not limited thereto.
[0027] The inventor of the present invention has identified for the
first time that if guanosine (G) in the codon of glycine at
position 299 of the glutamine synthetase with SEQ ID NO. 4 is
substituted with cytidine (C), the sensitivity of glutamine
synthetase towards GS inhibitor is remarkably increased, as
compared to the wild type GS. As the modified GS of the present
invention has a significantly higher sensitivity towards GS
inhibitor, it can be effectively used in the target protein
expression system.
[0028] Furthermore, the modified glutamine synthetase comprises the
amino acid sequence wherein glycine (Gly, G) at position 299 of a
glutamine synthetase consisting of an amino acid sequence of SEQ ID
NO. 4 is substituted with arginine (Arg, R), and as long as the
enzyme demonstrates increased sensitivity towards GS inhibitor
compared to the wildtype GS, the modified glutamine synthetase of
the present invention may comprise the amino acid sequence having a
sequence homology of 70% or higher, preferably 80% or higher, more
preferably 90% or higher, even more preferably 95% or higher, even
much more preferably 98% or higher, and most preferably 99% or
higher to the above-described amino acid sequence of modified
GS.
[0029] As used herein, the term `GS inhibitor` refers to an
external factor that is capable of inhibiting the GS activity.
Examples of such inhibitor include glycine, alanine, tryptophan,
histidine, glucosamine-6-phosphate, cytidine triphosphate, and
methionine sulphoximine (MSX), but is not limited thereto. With
respect to the objects of the present invention, the GS inhibitor
is preferably MSX, but is not limited thereto.
[0030] As used herein, the term `sensitivity` generally refers to
the feature of responding to external stimuli. With respect to
enzyme, sensitivity refers to the feature of enhancing or reducing
enzymatic activity in response to external factors that regulate
enzymatic activity. For the purpose of the present invention, the
sensitivity refers to the suppression of the GS activity in
response to the GS inhibitor, but is not limited thereto.
[0031] In one Example of the present invention, the gene encoding
the modified glutamine synthetase was to be obtained through
cloning the glutamine synthetase from RNA of hamster cells via PCR.
The results confirmed that the modified glutamine synthetase was
obtained in which glycine (Gly, G) at position 299 of wildtype
glutamine synthetase of SEQ ID NO. 4 is substituted with arginine
(Arg, R), and it was named GS PM (Example 3). Subsequently, IRES-GS
PM was prepared by linking the polynucleotide encoding the modified
glutamine synthetase with IRES, which was further connected to the
gene encoding the target protein in order to prepare the vector
comprising the expression cassette wherein the target protein
encoding gene-IRES-GS PM are operably connected together (Example
4). Furthermore, when the target protein was expressed using the
vector comprising the modified glutamine synthetase of the present
invention, the production level of the target protein was
significantly higher than when the wildtype glutamine synthetase
was used (Examples 6 and 7).
[0032] As another aspect, the present invention provides a
polynucleotide encoding the modified glutamine synthetase.
[0033] The modified glutamine synthetase of the present invention
is characterized by having a substitution of the amino acid at
position 299 of wild type GS from glycine to arginine. Thus, DNA
codon encoding the amino acid at position 299 of the modified
glutamine synthetase may be selected from the group consisting of
CGT, CGC, CGA, CGG, AGA and AGG, but is not limited thereto.
[0034] Furthermore, the polynucleotide encoding the modified
glutamine synthetase of the present invention may be preferably for
enhancing the expression of target protein.
[0035] The polynucleotide encoding the modified glutamine
synthetase of the present invention may be present in the vector to
be translated by polycistronic translation with the gene encoding
target protein. After transfection of the host cells with the above
vector, when the cells are treated with GS inhibitor, the activity
of the modified glutamine synthetase expressed from the introduced
vector is inhibited, thereby reducing the synthesis of glutamine.
However, in order for the cells to survive, glutamine is essential,
and thus under the suppression by GS inhibitor those cells tend to
synthesize more of the glutamine synthetase. Here, the target
protein-encoding gene introduced along with the polynucleotide
encoding the modified GS gets amplified as well, and through this
mechanism the expression of the target protein can be
increased.
[0036] As another aspect, the present invention provides a vector
for expression of a target protein, comprising the polynucleotide
encoding the modified GS and a gene encoding the target
protein.
[0037] The polynucleotide encoding the modified GS is the same as
described above.
[0038] As used herein, the term `target protein` refers to the
protein of interest to be produced in the host cells. For the
purpose of the present invention, it refers to the protein whose
expression is enhanced by the modified glutamine synthetase, but is
not limited thereto. The type of target protein is not specifically
limited as long as it can be expressed by the vector of the present
invention. In one Example of the present invention, tumor necrosis
factor receptor (TNFR)-Fc fusion protein was used as a
representative target protein that can be expressed by the modified
glutamine synthetase of the present invention.
[0039] As used herein, the term `tumor necrosis factor receptor
(TNFR)-Fc fusion protein` refers to the product prepared by
connecting the entire or a part of TNFR protein with immunoglobulin
Fc region by enzymatic action, or the product prepared by
expressing two polypeptides into a single polypeptide by genomic
manipulation. In the TNFR-Fc fusion protein, the TNFR protein and
the immunoglobulin Fc region may be directly linked with each
other, or linked via a peptide linker, but is not limited thereto.
The polynucleotide encoding the TNFR-Fc fusion protein may be a
polynucleotide of SEQ ID NO. 5, but is not limited thereto.
[0040] As used herein, the term `expression vector` refers to a DNA
construct comprising an essential control component which is
operably linked to an insert gene so that the insert gene is only
expressed when introduced into the host cell. The expression vector
may be prepared and purified by a standard recombinant DNA
technology. The type of the expression vector is not particularly
limited, as long as it expresses and produces a target gene in a
variety of host cells of prokaryotic and eukaryotic cells.
Preferably, the expression vector is a vector capable of producing
a large amount of a foreign protein in a similar form to the native
protein while it retains a strong promoter activity and a strong
expression ability. The expression vector is preferably a vector
comprising at least a promoter, a start codon, a gene encoding a
target protein, a stop codon, and a terminator. In addition, it may
comprise a DNA encoding a signal peptide, an enhancer sequence,
untranslated regions at the 5' and 3' ends of a target gene, a
selectable marker region or a replicable unit, etc., if desired.
Moreover, the type of the expression vector may be a mono-cistronic
vector including a polynucleotide encoding one recombinant protein,
a bi-cistronic vector including a polynucleotide encoding two
recombinant proteins, a poly-cistronic vector including a
polynucleotide encoding three recombinant proteins or more. With
respect to the objects of the present invention, the expression
vector is preferably a mono-cistronic vector including a SV40
promoter or a bi-cistronic vector including an IRES sequence, more
preferably, an expression vector including a promoter, a gene
encoding a target protein, IRES, and a modified GS gene in this
order or an expression vector including a promoter, a modified GS
gene, IRES, and a gene encoding a target protein, but is not
limited thereto.
[0041] According to one embodiment of the present invention, a
polynucleotide encoding GS PM was acquired from CHO DG44, and a
pGEMT-GS PM vector was obtained by connecting each of the acquired
polynucleotides to a pGEMT vector. Subsequently, the pGEMT-GS PM
vector was inserted into a cleaved TOPO-IRES-DHFR vector to obtain
a pCR2.1-TOPO-IRES-GS PM vector, and an IRES-GS PM fragment was
obtained from the vector. The obtained IRES-GS PM fragment was
inserted into a cleaved pcDNA-Kozak-TNFR-Fc-IRES-DHFR vector so as
to construct a kozak-TNFR-Fc-IRES-GS PM vector ('IRES-GS PM
vector') (FIG. 1).
[0042] According to another embodiment of the present invention,
the polynucleotide encoding GS PM was inserted into the cleaved
pcDNA3.1-TNFR-Fc-SV40-DHFR vector so as to obtain a
pcDNA3.1-TNFR-Fc-SV40-GS PM vector. Thereafter, the
pcDNA3.1-TNFR-Fc-SV40-GS PM vector was cleaved, and a TNFR-Fc
fragment from the pcDNA3.1-Kozak-TNFR-Fc-IRES-DHFR vector was
inserted into the cleaved region so as to construct a
pcDNA-Kozak-TNFR-Fc-SV40-GS PM vector ('SV40-GS PM vector') (FIG.
3).
[0043] As another aspect, the present invention provides a
transformant comprising the vector.
[0044] As used herein, the term `transformant` refers to the cell
transformed with the expression vector so as to express the
polynucleotide encoding the recombinant protein included in the
expression vector. It may be recombinant mammalian cells, rodent
cells, preferably animal cells or animal-derived cells, and most
preferably NS0 or CHO cells, but is not limited thereto. With
respect to the objects of the present invention, the transformant
is preferably a transformant prepared by introducing the expression
vector into a NS0 or CHO cell line, but is not limited thereto.
[0045] As another aspect, the present invention provides a method
for preparing a target protein, comprising culturing the
transformant.
[0046] The transformant and target protein are the same as
described above.
[0047] To be specific, the above method comprises (a) culturing the
transformant; and (b) adding a GS inhibitor to a culture medium. In
addition, the method further comprises (c) isolating the target
protein from the culture medium.
[0048] Preferably, when the GS inhibitor is added to the medium,
the expression system using the modified GS protein of the present
invention, which has an enhanced sensitivity to GS inhibitor can
lead to the increased expression of a target protein as compared to
the expression system using the wildtype GS.
[0049] As another aspect, the present invention provides a target
protein prepared by the above described method.
[0050] The method and the target protein are the same as described
above.
MODE FOR THE INVENTION
[0051] Hereinafter, the present invention will be described in more
detail with reference to Examples. However, these Examples are for
illustrative purposes only, and the invention is not intended to be
limited by these Examples.
EXAMPLE 1
Synthesis of TNFR-Fc Fusion Protein-Encoding Gene
[0052] In order to examine the expression level of a recombinant
protein produced using a recombinant protein expression vector
system, of the present invention, a TNFR-Fc fusion protein was used
as a representative target protein.
[0053] The fusion protein-encoding gene (SEQ ID NO. 5) was
synthesized by GeneArt Inc., so as to meet the following criteria:
(1) it must include a TNFR signal sequence (2) it must express the
TNFR amino acids at position 1 to 235 (3) it must be
codon-optimized for CHO cells in order to be transfected into CHO
cells (4) it must have a NheI restriction site at 5'-end and a NotI
restriction site at 3'-end, considering insertion into a pcDNA3.1
vector of Invitrogen.
[0054] The nucleotide sequence of the synthesized fusion
protein-encoding gene was finally analyzed using the VectorNTl
program.
EXAMPLE 2
Construction of Expression Vector Comprising TNFR-Fc Fusion
Protein-Encoding Gene
[0055] In the present invention, a DHFR system that is a common
recombinant protein expression system was utilized as a control for
the recombinant protein expression system using the modified GS
protein. For this, a hamster dihydrofolate reductase (DHFR) gene
was cloned as detailed below:
[0056] In order to obtain the hamster DHFR gene, a pSVA3 vector
(ATCC 77273) having a mutant type of hamster DHFR gene was
purchased, and then a wild type of DHFR gene was obtained by
performing point mutation using the DHFR gene as a template. In
addition, an IRES sequence was obtained by PCR from a Clontech
vector (Cat. #6029-1, PT3267-5) having the corresponding DNA
sequence.
[0057] The obtained DHFR gene and internal ribosome entry site
(IRES) sequence were cloned into a pCR2.1 vector so as to construct
a pCR2.1-IRES-DHFR expression vector.
[0058] Each of the TNFR-Fc-inserted pcDNA3.1-TNFR-Fc vector
obtained in Example 1 and the obtained pCR2.1-IRES-DHFR vector was
digested with restriction enzymes, SalI and XbaI, and ligated so as
to obtain a TNFR-Fc-inserted pcDNA3.1-TNFR-Fc-IRES-DHFR expression
vector.
[0059] In order to clone it into a vector having a
kanamycin-resistant gene, the kanamycin-resistant gene was obtained
from a pAC-GFP vector (#632483) of Clontech, so as to introduce a
Kan/Neo gene. The vector is a vector having a Kozak sequence at a
transcription initiation sequence of the TNFR-Fc gene and the
Kan/Neo gene as an antibiotic selection marker, and it was used as
a basic frame for cloning 4 different expression vector systems in
order to compare the expression levels of the recombinant protein
using CHO cells.
EXAMPLE 3
Preparation of a Modified GS Gene
[0060] In order to acquire a modified GS gene which has an
increased sensitivity to GS inhibitor as compared to wildtype GS
and thus can be applied to the target protein expression system,
the following procedures were performed.
[0061] To acquire the modified GS gene, a hamster cell line, CHO
DG44 (Invitrogen, 12609-012) was cultured, and then total RNA was
isolated using a TRIZOL reagent (Invitrogen). After that, RT-PCR
was performed using the obtained total RNA so as to obtain cDNA.
PCR (25 cycles of denaturation at 94.degree. C. for 5 minutes;
denaturation at 94.degree. C. for 30 seconds, annealing at
50.degree. C. for 30 seconds, elongation at 72.degree. C. for 90
seconds; and elongation at 72.degree. C. for 7 minutes) was
performed using the obtained cDNA as a template and a pair of
primers (GS SalI-F primer and GS XbaI-R primer) for acquisition of
the following GS PM gene, so as to obtain a PCR product.
TABLE-US-00001 GS SalI-F (Forward primer): (SEQ ID NO. 6)
5'-gtcgacatggccacctcagcaagttccc-3' GS XbaI-R (Reverse primer): (SEQ
ID NO. 7) 5'-tctagattagtttttgtattggaaaggg-3'
[0062] The obtained PCR product was electrophoresed on a 0.8%
agarose gel, and then the corresponding band was cut, followed by
clean-up using a Quiagen Cleaning kit (#28204). Then, the resultant
was inserted into a gene cloning vector, pGEMT vector (Promega,
USA). The PCR product-inserted pGEMT vector was introduced into a
TOP10 cell so as to obtain a total of 10 colonies. After that, a
nucleotide sequence and an amino acid sequence encoded by the
nucleotide sequence of each colony were analyzed.
[0063] By performing the above procedures numerous times, a
modified GS gene showing a difference in one amino acid at position
299 as compared to the amino acid sequence of the wild type hamster
GS (NCBI GenBank: X03495.1), was acquired. As a result of sequence
analysis, this difference is attributed to the alteration of the
895.sup.th nucleotide of the wildtype GS gene (SEQ ID NO. 3) from G
(Guanosine) to C (Cytidine). That is, the modified GS has the
characteristic of altered amino acids at position 299 from glycine
(Gly, G) to arginine (Arg, R). In the present invention, the
acquired modified GS was named `GS PM`.
[0064] Further, to acquire a polynucleotide encoding the wild type
hamster GS protein, the 895.sup.th C in the nucleotide sequence of
GS-PM was substituted with G. Specifically, cloning was performed
by point mutation for replacement of one amino acid. More
specifically, in order to obtain the wild type GS having one amino
acid different from those of GS PM of the present invention, PCR
(30 cycles of denaturation at 94.degree. C. for 5 minutes;
denaturation at 94.degree. C. for 30 seconds, annealing at
54.degree. C. for 30 seconds, elongation at 72.degree. C. for 30
seconds; and elongation at 72.degree. C. for 7 minutes) was
performed using the GS PM DNA as a template and a pair of primers
(KpnI F-primer and XbaI R-primer) that was synthesized to contain a
point mutation region (CGT.fwdarw.GGT). As a result, a GS PCR
fragment having an alteration from CGT to GGT was obtained.
TABLE-US-00002 KpnI F-primer (Forward primer): (SEQ ID NO. 8)
5'-caccggtaccacattcgagcctacgatcccaaggggggcctggacaa tgcccgtggtctg-3'
XbaI R-primer (Reverse primer): (SEQ ID NO. 9)
5'-tctagattagtttttgtattggaaggg-3'
[0065] In addition, the nucleotide sequence of the PCR product was
analyzed. As a result, a point mutation from CGT to GGT was
observed. The corresponding PCR product was digested with KpnI and
XbaI, and then ligated with a pGEMT-GS PM vector treated with KpnI
and XbaI, so as to obtain a pGEMT-GS vector.
EXAMPLE 4
Cloning of a Mammalian Cellular Protein-Expressing GS Vector
[0066] The GS PM gene included in the pGEMT-GS PM vector obtained
in Example 3 was cloned to have SalI and XbaI restriction sites at
its N- and C-terminals, respectively. Therefore, in order to obtain
IRES-GS PM, a fragment obtained by treating pGEMT-GS PM with SalI
and XbaI restriction enzymes was inserted into a TOPO-IRES-DHFR
vector that was previously digested with SalI and XbaI restriction
enzymes, so as to obtain a pCR2.1-TOPO-IRES-GS PM gene.
[0067] Next, in order to connect the TNFR-Fc gene and the IRES-GS
PM gene, the TNFR-Fc-IRES-DHFR gene and the IRES-GS PM fragment
digested with XhoI and XbaI were ligated so as to construct a
kozak-TNFR-Fc-IRES-GS PM vector ("IRES-GS PM vector") (FIG. 1).
FIG. 1 is a schematic diagram showing the cloning method of
pcDNA3.1-kozak-TNFR-Fc-IRES-GS PM of the present invention.
[0068] Further, the kozak-TNFR-Fc-IRES-GS PM vector and the TOPO-GS
vector were used to construct a Kozak-TNFR-Fc-IRES-GS vector
("IRES-GS vector") (FIG. 2).
[0069] FIG. 2 is a cleavage map showing
pcDNA3.1-kozak-TNFR-Fc-IRES-GS or pcDNA3.1-kozak-TNFR-Fc-IRES-GS
PM, which is a recombinant expression vector including the
TNFR-Fc-encoding gene of the present invention.
[0070] Meanwhile, the cloned pcDNA3.1-TNFR-Fc-SV40-DHFR vector was
used in order to prepare a SV40 promoter-GS system identical to
Lonza's GS system among GS systems. Since the
pcDNA3.1-TNFR-Fc-SV40-DHFR has no restriction sites suitable for GS
gene insertion, new restriction sites were first inserted into both
ends of the GS gene.
[0071] A pair of primers (GS-BsaBI-F primer and GS-BstBI-R primer)
containing BsaBI at the N-terminal of GS gene and BstBI at
C-terminal of GS gene were synthesized to perform PCR. Thus, the
BsaBI and BstBI sites were inserted into both ends of the GS gene,
and DHFR was removed from pcDNA3.1-TNFR-FC-SV40-DHFR by treatment
with BsaBI and BstBI. Subsequently, the GS gene digested with
BsaBI/BstBI was inserted thereto so as to construct a
pcDNA3.1-TNFR-Fc-SV40-GS PM vector.
TABLE-US-00003 GS-BsaBI-F Primer (forward primer): (SEQ ID NO. 10)
5'-gatgaggatcatggccacctcagcaag-3' GS-BstBI-R (reverse primer): (SEQ
ID NO. 11) 5'-ttcgaattagtttttgtattggaaggg-3'
[0072] However, the pcDNA3.1-TNFR-Fc-SV40-GS PM vector has no Kozak
sequence prior to the TNFR-Fc gene, unlike the
pcDNA3.1-TNFR-Fc-IRES-GS PM vector. Therefore, a second cloning
step of inserting the Kozak sequence into the vector was performed.
For the Kozak sequence, a Kozak sequence of the previously prepared
pcDNA3.1-Kozak-TNFR-Fc-IRES-DHFR vector was used. Instead of
TNFR-Fc of the pcDNA3.1-TNFR-Fc-SV40-GS PM vector, the
Kozak-TNFR-Fc was inserted to prepare
pcDNA3.1-Kozak-TNFR-FC-SV40-GS PM.
[0073] The restriction enzymes to be used for the cloning may
include NdeI and NheI at N-terminal of TNFR-Fc and BstXI, SgrAI,
and NotI at C-terminal of TNFR-Fc. Available restriction enzymes
were selected from them, so as to construct a
pcDNA-Kozak-TNFR-Fc-SV 40-GS PM vector ("SV40-GS PM vector") (FIG.
3). FIG. 3 is a schematic diagram showing the cloning method of
pcDNA3.1-kozak-TNFR-Fc-SV40-GS or pcDNA3.1-kozak-TNFR-Fc-SV40-GS
PM, which is a recombinant expression vector including the
TNFR-Fc-encoding gene of the present invention.
[0074] The constructed SV40-GS PM vector includes an antibiotic
resistance gene, ampicillin resistance gene for cell line
selection, and thus a third cloning step was performed to replace
the gene with a kanamycin resistance gene. The antibiotic
resistance gene was replaced by an antibiotic with a low frequency
of use on grounds of safety, because ampicillin is one of the
antibiotics frequently used by patients.
[0075] In order to perform the cloning for replacement with the
kanamycin resistance gene at a low frequency of use, the kanamycin
resistance gene was obtained from the pAC-GFP vector (#632483) of
Clontech, and introduced into a SV40-GS PM vector. Furthermore, the
SV40-GS PM vector was used to construct a pcDNA-Kozak-TNFR-Fc-SV
40-GS vector (`SV40-GS vector`) (FIG. 4). FIG. 4 is a cleavage map
showing pcDNA3.1-kozak-TNFR-Fc-SV40-GS or
pcDNA3.1-kozak-TNFR-Fc-SV40-GS PM, which is a recombinant
expression vector including the TNFR-Fc-encoding gene of the
present invention.
[0076] Finally, 4 different expression vectors of IRES-GS, IRES-GS
PM, SV40-GS, and SV40-GS PM were constructed as a protein
expression vector system for expressing a target protein in
mammalian cells.
EXAMPLE 5
Preparation of Transformant
[0077] In order to compare the productivity of TNFR-Fc fusion
protein between the GS and DHFR gene expression systems, each of
the expression vectors constructed in Example 4 was introduced into
CHO K-1 cells so as to prepare each transformant.
[0078] In detail, CHO K-1 cells were cultured in a DMEM/F12 medium
supplemented with 10% FBS, and 3 to 4.times.10.sup.5 cells/well
were inoculated into a 6 well-plate, followed by cultivation
overnight. When the cells reached 80 to 90% confluence, each of the
expression vectors was introduced thereto.
[0079] To achieve this, 4 .mu.g of each of the expression vectors
constructed in Example 4 and 250 .mu.l of Opti-MEM were mixed with
each other, and separately, 10 .mu.l of Lipofectamine 2000
(Invitrogen) and 250 .mu.l of Opti-MEM were mixed with each other.
Then, each mixture was left at room temperature for 5 minutes.
Subsequently, the mixtures were mixed with each other, and left at
room temperature for 20 minutes.
[0080] Thereafter, the culture medium in the 6-well plate was
replaced with 2 ml of Opti-MEM I media, and 500 .mu.l of the final
mixture was added to each well of the 6-well plate, followed by
cultivation at 37.degree. C. for 4 to 6 hours. The medium was
replaced with the original culture medium (DMEM/F12 medium
supplemented with 10% FBS), followed by further cultivation
overnight. Then, the culture medium in the 6-well plate was
replaced with a GS selection medium, and subculture was performed
according to the growth rate of the cells introduced with each of
the expression vectors. At this time, the GS selection medium was
glutamine-free DMEM or glutamine-free IMDM containing 10% FBS,
1.times. GS supplement, and the GS inhibitor, MSX. The 1.times. GS
supplement was prepared by including adenosine (500.times., 15 mM),
cytidine (1000.times., 30 mM), uridine (1000.times., 30 mM),
guanosine (1000.times., 3 mM), thymidine (1000.times., 10 mM),
asparagine (1000.times., 500 mM) and glutamic acid (1000.times.,
500 mM) or a commercially available 50.times. GS supplement (SAFC)
was used after dilution.
EXAMPLE 6
Assessment of TNFR-Fc Expression Level
[0081] The culture broth of each transformant subcultured in
Example 5 was applied to ELISA, in order to measure the expression
level of each protein expressed from the transformant.
[0082] In detail, a 96-well plate was coated with anti-human IgG Fc
antibody (Pierce, 31125), and blocked with 1% BSA. Next, the
culture broth of each subcultured transformant was added to each
well, and reacted. Subsequently, a biotin-conjugated anti-human
TNFR antibody (R&D system) as a detection antibody was added to
each well, and reacted. Each well was treated with HRP-conjugated
streptavidin, and reacted. Finally, each well was treated with TMB
for color development so as to examine the expression level of each
protein.
[0083] First, the expression levels in CHO K-1 were compared
between three types of TNFR-Fc GS vector (IRES-GS PM, IRES-GS,
SV40-GS) and the IRES-DHFR system, and the protein expression
levels were also compared between the GS vectors (FIG. 5). FIG. 5
is a graph showing the expression levels of TNFR-Fc in CHO K-1
cells transfected with three types of TNFR-Fc gene expression
vector (IRES-GS PM, IRES-GS, SV40-GS).
[0084] As a result, as shown in FIG. 5, even at 64 hours after
transfection with the expression vectors, CHO K-1 cells showed no
changes in the expression level of TNFR-FC protein by three types
of GS vectors or in the cell survival rate. Rather, three types of
the GS systems were more excellent in terms of expression level
than the IRES-DHFR system. Moreover, even at 5 days after
transfection of the expression vectors, the cells transfected with
the three types of expression vectors maintained their growth and
continuously expressed the TNFR-Fc protein.
[0085] Next, it was also examined whether the same results can be
obtained when a glutamine-free selection media containing 25 .mu.m
of MSX was used to culture the cells transfected with the three
types of the TNFR-Fc gene expression vectors (IRES-GS PM, IRES-GS,
SV40-GS) (FIG. 6). FIG. 6 is a graph showing the time-dependent
expression level of TNFR-Fc protein according to initial MSX
treatment of CHO K-1 cells, which were transfected with the three
types of TNFR-Fc gene expression vectors (IRES-GS PM, IRES-GS,
SV40-GS).
[0086] As shown in FIG. 6, as the cell culture time increased, the
cell line transfected with the IRES-GS PM vector of the present
invention exhibited higher levels in terms of the total TNFR-Fc
protein expression level and the expression level per an equal
number of cells, compared to the cell lines transfected with other
vectors. Immediately after transfection of the expression vectors,
the cells transfected with the SV40-GS vector exhibited the highest
level. However, after addition of MSX, the cells transfected with
the IRES-GS PM vector of the present invention exhibited the
highest level.
[0087] Meanwhile, the overall analysis of the results of FIG. 5
showed that there is a great difference in the expression level
between the IRES-GS and IRES-GS PM, even though both of them
include the identical IRES. At the beginning of the experiment, it
was expected that there would be no difference in their activities,
because of only one amino acid difference between the two genes.
Actually, there was a difference between GS and GS PM, as the
cultivation maintained in the selection media.
[0088] Therefore, in order to clearly examine whether one amino
acid difference between GS and GS PM greatly affects the expression
level, the SV40-GS PM vector was used to compare the expression
levels of TNFR-Fc protein between the cell lines transfected with a
total of 4 types of vectors (IRES-GS, TRES-GS PM, SV40-GS, SV40-GS
PM) (FIG. 7). FIG. 7 is a graph showing the TNFR-Fc expression
level after a secondary transient transfection (upper graph) and
the time-dependent TNFR-Fc expression level (lower graph).
[0089] As a result, as shown in the upper graph of FIG. 7, the
TNFR-Fc expression levels were mostly 6000-9000 ng/10.sup.6 cells
when the cells were cultured without MSX for 6 days after
transfection, and the TNFR-Fc expression levels were relatively
increased in the cells transfected with the wild type, TRES-GS and
SV40-GS vectors. However, as shown in the lower graph of FIG. 7,
when the cells were treated with 25 .mu.M MSX at 3 days after
transfection and 200 .mu.m MSX at 20 days after transfection, and
then cultured for further 7 days, the TNFR-Fc expression levels
were 1000 ng/10.sup.6 cells or lower, immediately after the
addition of 25 .mu.M MSX, but the expression levels increased
according to time. After the addition of 200 .mu.M MSX, only the
cells transfected with the IRES-GS PM vector of the present
invention showed an increase in the TNFR-Fc expression level.
[0090] The above results suggest that the modified GS protein of
the present invention shows an enhanced sensitivity to GS
inhibitors as compared to that of the wildtype GS protein, and the
vector comprising the polynucleotide encoding the modified GS
protein and the gene encoding the target protein can be effective
to produce the target protein.
EXAMPLE 7
Comparison of TNFR-Fc Protein Expression Level in Stably
Transfected CHO-S cells
[0091] The results of Example 6 showed that the higher protein
expression level per an equal number of cells was observed in the
cell lines transfected with the GS-containing expression vectors
(IRES-GS PM and SV40-GS PM expression vectors) among the four types
of GS expression vectors. Therefore, the expression levels in the
stable CHO-S cell line were compared between the IRES-GS PM and
SV40-GS PM expression vectors. In order to produce a large amount
of recombinant protein, the CHO-S cell adapted for growth in
suspension was used for the stable cell line establishment, instead
of CHO K-1.
[0092] First, TNFR-Fc-expressing cell lines transfected with the
IRES-GS PM and SV40-GS PM expression vectors were established. Each
of the established TNFR-Fc-expressing cell lines was cultured in a
medium containing 25 .mu.M or 250 .mu.M MSX, and the expression
levels of TNFR-Fc protein expressed therefrom were compared (FIG.
8). FIG. 8 is a graph showing the comparison in the stable cell
line between the IRES-GS PM vector and the SV40-GS PM vector. In
this regard, PCD indicates pg/cell/day, and it was calculated from
the following Equation.
PCD=expression amount (ng/mL)/((A-B)*Culture day/LN(A/B))/1000
[0093] A: Harvest cell conc.(.times.10.sup.6 cells/mL)
[0094] B: Seed cell conc.(.times.10.sup.6 cells/mL)
[0095] As a result, as shown in FIG. 8, the cells transfected with
the IRES-GS PM expression vector showed a higher expression level
of TNFR-FC protein than those transfected with the SV40-GS PM
expression vector, irrespective of MSX concentration. In addition,
the maximum PCD at 250 .mu.M MSX was .about.8 PCD, which is higher
than that of a single cell line limiting-diluted using the known
IRES-DHFR system. When single cell lines are selected from the cell
groups of 8 PCD, it was expected to obtain the expression cell
lines of higher than 8 PCD.
[0096] The above results demonstrate that the expression system
using the vector comprising the modified GS gene of the present
invention, specifically, the polycistronic vector comprising the
modified GS gene of the present invention, can produce the target
protein with high efficiency.
[0097] While the present invention has been particularly shown and
described with reference to the foregoing preferred and alternative
embodiments, it should be understood by those skilled in the art
that various alternatives to the embodiments of the invention
described herein may be employed in practicing the invention
without departing from the spirit and scope of the invention as
defined in the following claims. It is intended that the following
claims define the scope of the invention and that the method and
apparatus within the scope of these claims and their equivalents be
covered thereby. This description of the invention should be
understood to include all novel and non obvious combinations of
elements described herein, and claims may be presented in this or a
later application to any novel and non-obvious combination of these
elements.
Sequence CWU 1
1
1111122DNAArtificial Sequencesynthetic-gene encoding a modified GS
1atggccacct cagcaagttc ccacttgaac aaaaacatca agcaaatgta cttgtgcctg
60ccccagggtg agaaagtcca agccatgtat atctgggttg atggtactgg agaaggactg
120cgctgcaaaa cccgcaccct ggactgtgag cccaagtgtg tagaagagtt
acctgagtgg 180aattttgatg gctctagtac ctttcagtct gagggctcca
acagtgacat gtatctcagc 240cctgttgcca tgtttcggga ccccttccgc
agagatccca acaagctggt gttctgtgaa 300gttttcaagt acaaccggaa
gcctgcagag accaatttaa ggcactcgtg taaacggata 360atggacatgg
tgagcaacca gcacccctgg tttggaatgg aacaggagta tactctgatg
420ggaacagatg ggcacccttt tggttggcct tccaatggct ttcctgggcc
ccaaggtccg 480tattactgtg gtgtgggcgc agacaaagcc tatggcaggg
atatcgtgga ggctcactac 540cgcgcctgct tgtatgctgg ggtcaagatt
acaggaacaa atgctgaggt catgcctgcc 600cagtgggaat tccaaatagg
accctgtgaa ggaatccgca tgggagatca tctctgggtg 660gcccgtttca
tcttgcatcg agtatgtgaa gactttgggg taatagcaac ctttgacccc
720aagcccattc ctgggaactg gaatggtgca ggctgccata ccaactttag
caccaaggcc 780atgcgggagg agaatggtct gaagcacatc gaggaggcca
tcgagaaact aagcaagcgg 840caccggtacc acattcgagc ctacgatccc
aaggggggcc tggacaatgc ccgtcgtctg 900actgggttcc acgaaacgtc
caacatcaac gacttttctg ctggtgtcgc caatcgcagt 960gccagcatcc
gcattccccg gactgtcggc caggagaaga aaggttactt tgaagaccgc
1020cgcccctctg ccaattgtga cccctttgca gtgacagaag ccatcgtccg
cacatgcctt 1080ctcaatgaga ctggcgacga gcccttccaa tacaaaaact aa
11222373PRTArtificial Sequencesynthetic-modified GS 2Met Ala Thr
Ser Ala Ser Ser His Leu Asn Lys Asn Ile Lys Gln Met 1 5 10 15 Tyr
Leu Cys Leu Pro Gln Gly Glu Lys Val Gln Ala Met Tyr Ile Trp 20 25
30 Val Asp Gly Thr Gly Glu Gly Leu Arg Cys Lys Thr Arg Thr Leu Asp
35 40 45 Cys Glu Pro Lys Cys Val Glu Glu Leu Pro Glu Trp Asn Phe
Asp Gly 50 55 60 Ser Ser Thr Phe Gln Ser Glu Gly Ser Asn Ser Asp
Met Tyr Leu Ser 65 70 75 80 Pro Val Ala Met Phe Arg Asp Pro Phe Arg
Arg Asp Pro Asn Lys Leu 85 90 95 Val Phe Cys Glu Val Phe Lys Tyr
Asn Arg Lys Pro Ala Glu Thr Asn 100 105 110 Leu Arg His Ser Cys Lys
Arg Ile Met Asp Met Val Ser Asn Gln His 115 120 125 Pro Trp Phe Gly
Met Glu Gln Glu Tyr Thr Leu Met Gly Thr Asp Gly 130 135 140 His Pro
Phe Gly Trp Pro Ser Asn Gly Phe Pro Gly Pro Gln Gly Pro 145 150 155
160 Tyr Tyr Cys Gly Val Gly Ala Asp Lys Ala Tyr Gly Arg Asp Ile Val
165 170 175 Glu Ala His Tyr Arg Ala Cys Leu Tyr Ala Gly Val Lys Ile
Thr Gly 180 185 190 Thr Asn Ala Glu Val Met Pro Ala Gln Trp Glu Phe
Gln Ile Gly Pro 195 200 205 Cys Glu Gly Ile Arg Met Gly Asp His Leu
Trp Val Ala Arg Phe Ile 210 215 220 Leu His Arg Val Cys Glu Asp Phe
Gly Val Ile Ala Thr Phe Asp Pro 225 230 235 240 Lys Pro Ile Pro Gly
Asn Trp Asn Gly Ala Gly Cys His Thr Asn Phe 245 250 255 Ser Thr Lys
Ala Met Arg Glu Glu Asn Gly Leu Lys His Ile Glu Glu 260 265 270 Ala
Ile Glu Lys Leu Ser Lys Arg His Arg Tyr His Ile Arg Ala Tyr 275 280
285 Asp Pro Lys Gly Gly Leu Asp Asn Ala Arg Arg Leu Thr Gly Phe His
290 295 300 Glu Thr Ser Asn Ile Asn Asp Phe Ser Ala Gly Val Ala Asn
Arg Ser 305 310 315 320 Ala Ser Ile Arg Ile Pro Arg Thr Val Gly Gln
Glu Lys Lys Gly Tyr 325 330 335 Phe Glu Asp Arg Arg Pro Ser Ala Asn
Cys Asp Pro Phe Ala Val Thr 340 345 350 Glu Ala Ile Val Arg Thr Cys
Leu Leu Asn Glu Thr Gly Asp Glu Pro 355 360 365 Phe Gln Tyr Lys Asn
370 31122DNACricetulus longicaudatus 3atggccacct cagcaagttc
ccacttgaac aaaaacatca agcaaatgta cttgtgcctg 60ccccagggtg agaaagtcca
agccatgtat atctgggttg atggtactgg agaaggactg 120cgctgcaaaa
cccgcaccct ggactgtgag cccaagtgtg tagaagagtt acctgagtgg
180aattttgatg gctctagtac ctttcagtct gagggctcca acagtgacat
gtatctcagc 240cctgttgcca tgtttcggga ccccttccgc agagatccca
acaagctggt gttctgtgaa 300gttttcaagt acaaccggaa gcctgcagag
accaatttaa ggcactcgtg taaacggata 360atggacatgg tgagcaacca
gcacccctgg tttggaatgg aacaggagta tactctgatg 420ggaacagatg
ggcacccttt tggttggcct tccaatggct ttcctgggcc ccaaggtccg
480tattactgtg gtgtgggcgc agacaaagcc tatggcaggg atatcgtgga
ggctcactac 540cgcgcctgct tgtatgctgg ggtcaagatt acaggaacaa
atgctgaggt catgcctgcc 600cagtgggaat tccaaatagg accctgtgaa
ggaatccgca tgggagatca tctctgggtg 660gcccgtttca tcttgcatcg
agtatgtgaa gactttgggg taatagcaac ctttgacccc 720aagcccattc
ctgggaactg gaatggtgca ggctgccata ccaactttag caccaaggcc
780atgcgggagg agaatggtct gaagcacatc gaggaggcca tcgagaaact
aagcaagcgg 840caccggtacc acattcgagc ctacgatccc aaggggggcc
tggacaatgc ccgtggtctg 900actgggttcc acgaaacgtc caacatcaac
gacttttctg ctggtgtcgc caatcgcagt 960gccagcatcc gcattccccg
gactgtcggc caggagaaga aaggttactt tgaagaccgc 1020cgcccctctg
ccaattgtga cccctttgca gtgacagaag ccatcgtccg cacatgcctt
1080ctcaatgaga ctggcgacga gcccttccaa tacaaaaact aa
11224373PRTCricetulus longicaudatus 4Met Ala Thr Ser Ala Ser Ser
His Leu Asn Lys Asn Ile Lys Gln Met 1 5 10 15 Tyr Leu Cys Leu Pro
Gln Gly Glu Lys Val Gln Ala Met Tyr Ile Trp 20 25 30 Val Asp Gly
Thr Gly Glu Gly Leu Arg Cys Lys Thr Arg Thr Leu Asp 35 40 45 Cys
Glu Pro Lys Cys Val Glu Glu Leu Pro Glu Trp Asn Phe Asp Gly 50 55
60 Ser Ser Thr Phe Gln Ser Glu Gly Ser Asn Ser Asp Met Tyr Leu Ser
65 70 75 80 Pro Val Ala Met Phe Arg Asp Pro Phe Arg Arg Asp Pro Asn
Lys Leu 85 90 95 Val Phe Cys Glu Val Phe Lys Tyr Asn Arg Lys Pro
Ala Glu Thr Asn 100 105 110 Leu Arg His Ser Cys Lys Arg Ile Met Asp
Met Val Ser Asn Gln His 115 120 125 Pro Trp Phe Gly Met Glu Gln Glu
Tyr Thr Leu Met Gly Thr Asp Gly 130 135 140 His Pro Phe Gly Trp Pro
Ser Asn Gly Phe Pro Gly Pro Gln Gly Pro 145 150 155 160 Tyr Tyr Cys
Gly Val Gly Ala Asp Lys Ala Tyr Gly Arg Asp Ile Val 165 170 175 Glu
Ala His Tyr Arg Ala Cys Leu Tyr Ala Gly Val Lys Ile Thr Gly 180 185
190 Thr Asn Ala Glu Val Met Pro Ala Gln Trp Glu Phe Gln Ile Gly Pro
195 200 205 Cys Glu Gly Ile Arg Met Gly Asp His Leu Trp Val Ala Arg
Phe Ile 210 215 220 Leu His Arg Val Cys Glu Asp Phe Gly Val Ile Ala
Thr Phe Asp Pro 225 230 235 240 Lys Pro Ile Pro Gly Asn Trp Asn Gly
Ala Gly Cys His Thr Asn Phe 245 250 255 Ser Thr Lys Ala Met Arg Glu
Glu Asn Gly Leu Lys His Ile Glu Glu 260 265 270 Ala Ile Glu Lys Leu
Ser Lys Arg His Arg Tyr His Ile Arg Ala Tyr 275 280 285 Asp Pro Lys
Gly Gly Leu Asp Asn Ala Arg Gly Leu Thr Gly Phe His 290 295 300 Glu
Thr Ser Asn Ile Asn Asp Phe Ser Ala Gly Val Ala Asn Arg Ser 305 310
315 320 Ala Ser Ile Arg Ile Pro Arg Thr Val Gly Gln Glu Lys Lys Gly
Tyr 325 330 335 Phe Glu Asp Arg Arg Pro Ser Ala Asn Cys Asp Pro Phe
Ala Val Thr 340 345 350 Glu Ala Ile Val Arg Thr Cys Leu Leu Asn Glu
Thr Gly Asp Glu Pro 355 360 365 Phe Gln Tyr Lys Asn 370
51401DNAArtificial Sequencesyntehtic - TNFR-Fc 5ctgcctgccc
aggtggcctt caccccttac gcccctgagc ctggctccac ctgccggctg 60cgggagtact
acgaccagac cgcccagatg tgctgctcca agtgctcccc tggccagcac
120gccaaggtgt tctgcaccaa gacctccgac accgtgtgcg acagctgcga
ggactccacc 180tacacccagc tgtggaactg ggtgcccgag tgcctgtcct
gcggctcccg gtgctcctcc 240gaccaggtgg agacccaggc ctgcacccgg
gagcagaacc ggatctgcac ctgcaggcct 300ggctggtact gcgccctgtc
caagcaggag ggctgccggc tgtgcgcccc tctgcggaag 360tgccggcctg
gcttcggcgt ggccaggcct ggcaccgaga ccagcgacgt ggtgtgcaag
420ccttgcgccc ctggcacctt ctccaacacc acctcctcca ccgacatctg
ccggcctcac 480cagatctgca acgtggtggc catccctggc aacgcctcca
tggacgccgt gtgcacctcc 540acctccccca cccggtctat ggcccctggc
gctgtgcacc tgcctcagcc tgtgtccacc 600cggtcccagc acacccagcc
tacccctgag ccctccaccg ccccttctac cagcttcctg 660ctgcctatgg
gccctagccc tcctgccgag ggctccaccg gcgacgagcc taagtcctgc
720gacaagaccc acacctgccc tccctgccct gcccctgagc tgctgggcgg
accttccgtg 780ttcctgttcc ctcctaagcc taaggacacc ctgatgatct
cccggacccc tgaggtgacc 840tgcgtggtgg tggacgtgtc ccacgaggat
cctgaggtga agttcaattg gtacgtggac 900ggcgtggagg tgcacaacgc
caagaccaag cctcgggagg agcagtacaa cagcacctac 960cgggtggtgt
ccgtgctgac cgtgctgcac caggactggc tgaacggcaa ggaatacaag
1020tgcaaggtgt ccaacaaggc cctgcccgct cctatcgaaa agaccatctc
caaggccaag 1080ggccagcctc gcgagcctca ggtgtacacc ctgcctccct
cccgggagga gatgaccaag 1140aaccaggtgt ccctgacctg cctggtgaag
ggcttctacc cttccgacat cgccgtggag 1200tgggagtcca acggccagcc
tgagaacaac tacaagacca cccctcctgt gctggactcc 1260gacggctcct
tcttcctgta ctccaagctg accgtggaca agtcccggtg gcagcagggc
1320aacgtgttct cctgctccgt gatgcacgag gccctgcaca accactacac
ccagaagtcc 1380ctgtccctga gccccggcaa g 1401628DNAArtificial
Sequencesynthetic-GS SalI-F 6gtcgacatgg ccacctcagc aagttccc
28728DNAArtificial Sequencesynthetic-GS XbaI-R 7tctagattag
tttttgtatt ggaaaggg 28860DNAArtificial Sequencesynthetic-KpnI
F-primer 8caccggtacc acattcgagc ctacgatccc aaggggggcc tggacaatgc
ccgtggtctg 60927DNAArtificial Sequencesynthetic-XbaI R-primer
9tctagattag tttttgtatt ggaaggg 271027DNAArtificial
Sequencesynthetic-GS-BsaBI-F 10gatgaggatc atggccacct cagcaag
271127DNAArtificial Sequencesynthetic-GS-BstBI-R 11ttcgaattag
tttttgtatt ggaaggg 27
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