Description
Title of Invention: A PROTEIN SECRETORY FACTOR WITH
HIGH SECRETORY EFFICIENCY AND AN EXPRESSION
VECTOR COMPRISING THE SAME
Technical Field
[1] The present invention relates to a novel protein secretion factor, a vector including a
nucleic acid sequence encoding the protein secretion factor, and a transformed cell into
which the vector is introduced. Further, the present invention relates to a method of
producing a target protein using a transformed cell including the vector.
[2]
Background Art
[3] A recombinant polypeptide or protein including an antibody is produced using
various kinds of genetically-modified organisms including prokaryotic and eukaryotic
cells. Many of the proteins used for medical treatment, research and the like are not
suitable to be produced by prokaryotic cells such as bacteria because they are glyco
proteins. For this reason, protein expression systems using eukaryotic cells such as
yeast cells, insect cells or mammalian cells have been developed and widely used.
[4] One of the major problems in the biotechnology for producing heterologous proteins
is to produce and recover polypeptides, such as proteins and protein subunits ), not
easily expressed or secreted in geneticallymodified organisms. Since these proteins or
protein subunits are expressed in cells at a very low level or a normal level, the scale of
culturing and purifying tends to become larger in order to obtain a desired amount of
proteins or protein subunits.
[5] A typical method for solving such problems is to induce the proteins or protein
subunits expressed in a cell to be secreted into a culture medium as high a level as
possible. It is very useful even in purification to allow the proteins or protein subunits
expressed in the cell to be secreted into an extracellular medium because these proteins
are easily purified by doing so. In addition, the recombinant proteins or protein
subunits secreted into an extracellular medium are advantageous in that protein decom
position occurring in the cell can be prevented and in that protein products with
accurate folding can be obtained.
[6] For successful secretion of the proteins expressed in a eukaryotic cell to the outside
of the cell, a translocation of a protein traversing an intracellular endoplasmic
reticulum is required. During the translocation, several modification steps required for
protein activation occur concurrently, and thus the protein secreted to the outside of the
cell can be considered as a mature protein which was immediately saccharified or
modified.
[7] Proteins secreted from a cell through a cell membrane are generally produced in the
cell in the form of a precursor, and is referred to as a "preprotein". The preprotein
includes an additional peptide sequence at the amino terminal (NH3-terminal), and this
peptide sequence allows the expressed protein to enter a secretion pathway by targeting
this protein into an intracellular endoplasmic reticulum. This additional peptide
sequence is referred to as a "protein secretion factor" or "signal sequence or signal
peptide".
[8] In the case of a recombinant protein, secretion may not operate as expected because
the natural signal sequence of the recombinant protein does not operate well in a host
cell. Although there are many known signal sequences that can be used for the
secretion of a specific recombinant protein, there is still a need for the discovery of ad
ditional signal sequences capable of promoting the effective secretion of recombinant
proteins, particularly, immunoglobulins in a mammalian host cell.
[9]
Disclosure of Invention
Technical Problem
[10] As such, the present inventors have made numerous efforts to develop a protein
secretion factor capable of more effectively secreting and producing various r e
combinant proteins or target proteins. Accordingly, they developed a protein secretion
factor capable of effectively secreting a target protein from an animal host cell to the
outside thereof. In addition, they also found that an antibody could be effectively
secreted and expressed using the developed protein secretion factor, thereby
completing the present invention.
[11]
Solution to Problem
[12] An object of the present invention is to provide a novel protein secretion factor.
[13] Another object of the present invention is to provide an expression cassette including
a nucleic acid sequence encoding the protein secretion factor, which is linked to a gene
encoding a target protein.
[14] Still another object of the present invention is to provide a recombinant vector
including a nucleic acid sequence encoding the protein secretion factor.
[15] Still another object of the present invention is to provide a vector for secretory ex
pression of target protein, which includes the expression cassette. Still another object
of the present invention is to provide a transformed cell, into which the vector is in
troduced, into a host cell.
[16] Still another object of the present invention is to provide a method of producing a
target protein, including: culturing a transformed cell, into which a vector for ex
pression of target protein secretion including the expression cassette is introduced to
express a target protein and secrete the target protein to the outside of the cell; and r e
covering the target protein from a culture or a culture supernatant of the cell.
[17] Still another object of the present invention is to provide use of the protein secretion
factor for preparing a vector for secretory expression of target protein.
[18] Still another object of the present invention is to provide use of the protein secretion
factor for secreting target protein.
[19]
Advantageous Effects of Invention
[20] When the protein secretion factor according to the present invention was used, the
secretion of a target protein was remarkably increased, and, particularly, a remarkably
excellent secretion effect for antibodies was exhibited, compared to when conventional
protein secretion factors were used. Therefore, the protein secretion factor of the
present invention can be widely used in the field of recombinant protein production,
and particularly, in the field of antibody production.
Brief Description of Drawings
[21] FIG. 1 shows a plasmid map of a luciferase expression vector pCBIN-CLUC having
a protein secretion factor SP6 prepared by the present inventors.
[22] FIG. 2 shows ae plasmid map of a luciferase expression vector pCBIN-CLUCl
having a protein secretion factor SP1 prepared by the present inventors.
[23] FIG. 3 shows a plasmid map of a luciferase expression vector pCBIN-CLUC2 having
a protein secretion factor SP2 prepared by the present inventors.
[24] FIG. 4 shows a plasmid map of a luciferase expression vector pCBIN-CLUC3 having
a protein secretion factor SP3 prepared by the present inventors.
[25] FIG. 5 shows a plasmid map of a luciferase expression vector pCBIN-CLUC4 having
a protein secretion factor SP4 prepared by the present inventors.
[26] FIG. 6 shows a plasmid map of a luciferase expression vector pCBIN-CLUC5 having
a protein secretion factor SP5 prepared by the present inventors.
[27] FIG. 7 shows a plasmid map of a luciferase expression vector pCBIN-CLUC7.2
having a protein secretion factor SP7.2 prepared by the present inventors.
[28] FIG. 8 shows a plasmid map of a luciferase expression vector pCBIN-CLUC7.3
having a protein secretion factor SP7.3 prepared by the present inventors.
[29] FIG. 9 is a graph showing the results of the secretion amount of a luciferase present
in a culture medium, measured on the 2nd, 3rd, 5th, and 6th day after eight different
types of plasmid vectors (pCBIN-CLUCl, pCBIN-CLUC2, pCBIN-CLUC3, pCBINCLUC4,
pCBIN-CLUC5, pCBIN-CLUC, pCBIN-CLUC7.2 and pCBIN-CLUC7.3)
were transformed into a CHO cell line, into each of which a luciferase gene prepared
by the present inventors was inserted.
[30] FIG. 10 is a schematic view showing a process of operably linking a protein
secretion factor (SP) to light-chain and heavy-chain genes of an IgGl-type monoclonal
antibody (Rx antibody) via in-frame.
[31] FIG. 11 is a plasmid map showing the general form of a pCB-Rx_v5.4 plasmid
prepared by linking each protein secretion factor to light-chain and heavy-chain genes
of an Rx antibody. In the present invention, the pCB-Rx_v5.4-based plasmids were
prepared such that only the protein secretion factors inserted in the light chain SP and
heavy chain SP of the plasmid map are different, and other portions of the plasmid map
are identical.
[32] FIG. 12 is a graph showing the antibody secretion ability depending on the com
bination of protein secretion factors linked to light-chain and heavy-chain genes of an
Rx antibody, which was measured by enzyme-linked immunosorbent assay (ELISA).
Best Mode for Carrying out the Invention
[33] In one embodiment, the present invention provides a protein secretion factor.
[34]
[35] In detail, the present invention provides a protein secretion factor having an amino
acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
[36] As used herein, the term "protein secretion factor" means a factor linked to a protein
to induce the protein to be secreted to the outside of a cell. Specifically, the protein
secretion factor may be composed of a polypeptide. In the present invention, the
protein secretion factor can be used together mixed with a signal sequence, a secretion
sequence, a signal peptide (SP) or the like.
[37] Specifically, the protein secretion factor may have an amino acid sequence selected
from the group consisting of SEQ ID NOS: 1 to 8, and, more specifically, may have an
amino acid sequence of SEQ ID NO: 1 or 2, but the present invention is not limited
thereto.
[38] The present inventors have identified a human gene LBFL313 with a pancreatic
cancer marker differentially expressed in pancreatic adenocarcinoma tissues compared
with normal pancreatic tissues through prior research (Korean Patent Application Pub
lication No. 10-2007-0119250). The human gene LBFL313 identified in this way may
have a cDNA sequence of SEQ ID NO: 47, but is not limited thereto. It is known that
this human gene can be used as a diagnostic agent or marker for detecting pancreatic
cancer or identifying normal tissues and pancreatic adenocarcinoma in a sample, but
whether or not this gene has a secretion factor has not been known.
[39] In an exemplary embodiment of the present invention, the present inventors have
selected peptide sequences presumably having a potential to be used as a secretion
factor while analyzing the configuration of the newly-identified gene. As a result, they
determined secretion factor candidates (SP7.2 and SP7.3) having an amino acid
sequence of SEQ ID NO: 1 and SEQ ID NO: 2. After determining the secretion factor
candidates, their secretagogue capabilities were compared with those of six known
secretion factors (SP1 to SP6).
[40] As the result of measuring the luciferase secretion efficacy of each of the secretion
factors, two signal sequences having an amino acid sequence of SEQ ID NO: 1 or SEQ
ID NO: 2 and derived from gene LBFL313 were shown to improve the level of lu
ciferase secretion compared to the conventional signal sequence (SP1) (refer to FIG.
9). Particularly, in the case of SP7.2 and SP7.3 vectors, a very large amount of lu
ciferase was secreted at the early stage of culture (2d and 3d).
[41] The protein secretion factor can be used to promote the secretion of a target protein.
[42] As used herein, the target protein refers to a protein intended to be expressed and
secreted in a desired host cell using the protein secretion factor. A nucleic acid
sequence encoding the target protein can be named "gene of interest".
[43] In the present invention, the target protein may be a protein intrinsically expressed in
a host cell or a protein expressed by a foreign gene introduced thereinto. The kind of
the target protein is not particularly limited as long as extracellular secretion efficiency
is increased by the protein secretion factor.
[44] Examples of the target protein may include an antibody, a human growth hormone, a
serum protein, immunoglobulin, cytokine, -, b- or g -interferon, a colony- stimulating
factor (GM-CSF), a platelet-derived growth factor (PDGF), a phospholipase-activating
protein (PLAP), insulin, a tumor necrosis factor (TNF), a growth factor, a hormone,
calcitonin, a calcitonin gene related peptide (CGRP), enkephalin, somatomedin, ery
thropoietin, a hypothalamic secretion factor, prolactin, chronic gonadotropin, a tissue
plasminogen activator, a growth hormone releasing peptide (GHRP), a thymic humoral
factor (THF), asparaginase, arginase, arginine deaminase, adenosine deaminase,
aminase, peroxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, uricase,
adenosine diphosphatase, tyrosinase, bilirubin oxidase, glucose oxidase, glucosidase,
galactosidase, glucocerebrosidase, and glucourodinase. Specific examples thereof may
include heavy-chain and light-chain proteins, but are not limited thereto. Here, the
antibody is a concept including full-length antibodies, Fc fragments, and antibody
fragments such as Fab, Fab', F(ab')2, and Fv. In addition, the antibody light chain may
have an amino acid sequence of SEQ ID NO: 48, and the antibody heavy chain may
have an amino acid sequence of SEQ ID NO: 49, but are not limited thereto.
[45]
[46] The protein secretion factor can be linked to a target protein. Specifically, the protein
secretion factor is designed to be linked to a target protein in frame, thereby causing
the secretory expression of a target protein in a host cell.
[47] Meanwhile, a nucleic acid sequence encoding the protein secretion factor linked to a
gene encoding a target protein is a concept that includes the direct linkage of the
nucleic acid sequence and the gene and/or the linkage thereof through a linker.
[48] The example of linker may include an affinity tag and/or a protease recognition
sequence.
[49] Examples of the affinity tag may include GST, MBP, NusA, thioredoxin, ubiquitin,
FLAG, BAP, 6HIS, STREP, CBP, CBD, and S-tag, but are not limited to, and various
affinity tags known in the art may be used.
[50] Examples of the protease recognition sequence may include sequences recognized by
mammal purine, factor Xa, enterokinase, subtilisin, tobacco etch virus protease, and
ubiquitin hydrolase, but are not limited to, and various protease recognition sequences
known in the art may be used.
[51]
[52] In another embodiment, the present invention provides an expression cassette
including a nucleic acid sequence encoding the protein secretion factor which is linked
to a gene encoding a target protein.
[53] In the present invention, the protein secretion factor, target protein, and the like are
the same as those described above.
[54] As used herein, the term "expression cassette" refers to a sequence regulating one or
more genes and expression thereof, that is, a nucleic acid sequence including any com
bination of various czs-acting transcription regulating elements. The expression
cassette of the present invention may further include various elements, for example,
nucleic acid sequences such as a promoter and an enhancer, which are recognized in
the art to be necessary for expression regulation, as well as the nucleic acid sequence
encoding a protein secretion factor and a target protein. The sequence regulating the
expression of a gene, that is, the sequence regulating the transcription of a gene and the
expression of the transcription product thereof, is generally referred to as a "regulatory
unit". Most of the regulatory unit is located upstream of a coding sequence of a target
gene such that it is operably linked thereto. In addition, the expression cassette may
include a 3' non-transcriptional region including a poly-adenylation site at a 3'
terminal.
[55] The expression cassette includes a promoter sequence and a nucleic acid sequence
encoding a fusion protein in which the protein secretion factor and the target protein
are linked, and is configured such that the promoter sequence is functionally linked to
the nucleic acid sequence encoding the fusion protein.
[56] Here, the term "functionally linked" means that one DNA region is functionally
linked to another DNA region. For example, a desired gene sequence is functionally
linked to an expression regulating sequence such as a promoter to allow the desired
gene to be expressed by the activation of the promoter.
[57] In the present invention, the expression cassette includes a promoter sequence, and a
nucleic acid sequence encoding a protein secretion factor having an amino acid
sequence of SEQ ID NO: 1 or SEQ ID NO: 2, which is linked to a gene encoding a
target protein, and is designed to realize the extracellular secretory expression of a
target protein in a host cell, and particularly, in an animal host cell.
[58]
[59] In still another embodiment, the present invention provides a recombinant vector
including a nucleic sequence encoding the protein secretion factor.
[60] More specifically, the present invention provides a vector for expression of target
protein secretion, including a nucleic acid sequence encoding a protein secretion factor
which is linked to a gene encoding the target protein.
[61] The protein secretion factor, the target protein, and the linkage of the protein section
factor and the target protein are the same as described above.
[62] Further, the vector for expression of target protein secretion according to the present
invention may further include an expression cassette including a nucleic acid sequence
encoding a protein secretion factor, which is linked to a gene encoding a target protein,
selected from the group consisting of SEQ ID NOS: 1 to 8.
[63] Moreover, the vector for expression of target protein secretion according to the
present invention may be a vector for secretory expression of antibody.
[64] For example, the vector for expression of target protein secretion may include: a) a
first expression cassette including a nucleic acid sequence encoding a protein secretion
factor, which is linked to a gene encoding an antibody light chain; and b) a second ex
pression cassette including a nucleic acid sequence encoding a protein secretion factor,
which is linked to a gene encoding an antibody heavy chain.
[65] Specifically, the vector for secretory expression of an antibody may include: a) a first
expression cassette including a nucleic acid sequence encoding a protein secretion
factor having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, which is
linked to a gene encoding an antibody light chain; and b) a second expression cassette
including a nucleic acid sequence encoding a protein secretion factor having an amino
acid sequence selected from the group consisting of SEQ ID NOS: 1 to 8, which is
linked to an antibody heavy chain. For example, the protein secretion factor of b) may
be a protein secretion factor having an amino acid sequence of SEQ ID NO: 3.
[66] More specifically, the protein secretion factor of a) may be a protein secretion factor
having an amino acid sequence of SEQ ID NO: 1, and the protein secretion factor of b)
may be a protein secretion factor having an amino acid sequence of SEQ ID NO: 3.
Here, the antibody light chain may be composed of an amino acid sequence of SEQ ID
NO: 48, and the antibody heavy chain may be composed of an amino acid sequence of
SEQ ID NO: 49, but the present invention is not limited thereto.
[67] As used herein, the term "vector for secretory expression of target protein" refers to
an expression vector, which includes a nucleic acid encoding a protein secretion factor,
which is linked to a gene encoding a target protein to cause the extracellular secretion
of a target protein at the time of introducing the vector into a host cell and expressing
this vector.
[68] As used herein, the term "expression vector" refers to a double-stranded DNA
fragment as a carrier into which a target DNA fragment is inserted. The expression
vector used in expressing a protein in the art may be used without limitation. Here, the
target DNA refers to a DNA encoding a target protein intended to be expressed. Once
the expression vector is in a host cell, this expression vector can be replicated re
gardless of a host chromosomal DNA, and the inserted target DNA can be expressed.
As well known in the art, in order to increase the expression level of a transfected gene
in a host cell, the transfected gene must be operably linked to a transcription and
decoding expression regulating sequence allowing the gene to exhibit a function in the
selected host cell.
[69] In an exemplary embodiment of the present invention, based on the pTOPBA-
RL-pA vector having 'CMVe', 'CB' and 'Beta-actin Intron' (Korean Patent Ap
plication Publication No. 10-2012-0059222), a vector for expression of target protein
secretion was prepared by operably liking a nucleic acid sequence encoding a protein
secretion factor composed of amino acid sequences of SEQ ID NOS: 1 to 8 with a gene
encoding a protein to be produced.
[70] In the specific embodiment of the present invention, the present inventors prepared
an antibody expression vector (Example 5) by selecting SP2 (SEQ ID NO: 4), SP6
(SEQ ID NO: 8) and SP7.2 (SEQ ID NO: 1) from among the signal sequences ex
hibiting excellent secretion inducing effects in the luciferase secretion measurement
test in order to confirm whether the prepared antibody expression vector exhibit
excellent secretion inducing ability even to a monoclonal antibody, for which an in
dustrial large-scale production is required. In this test, an Rx antibody was used as the
monoclonal antibody, and the Rx antibody includes an antibody light chain composed
of an amino acid sequence of SEQ ID NO: 48 and an antibody heavy chain composed
of an amino acid sequence of SEQ ID NO: 49.
[71] In order to determine the optimal configuration of an antibody secretion factor, the
secretion factors of the antibody light chain and antibody heavy chain were differently
combined, and the secretion efficiency thereof was examined.
[72] That is, the antibody light chain and antibody heavy chain were expressed from the
vector prepared by linking the signal sequences selected from the group consisting of
SPl (SEQ ID NO: 3), SP2 (SEQ ID NO: 4), SP6 (SEQ ID NO: 8), and SP7.2 (SEQ ID
NO: 1) to the antibody light chain and antibody heavy chain, respectively, and the
secretion efficiency thereof was examined.
[73] In order to examine the extracellular secretion efficiency of the signal sequence in invitro
cell culture system, the signal sequence was transformed into a CHO cell, and
then the secretion level of a monoclonal antibody was examined via ELISA.
[74] As a result of measuring the secretion level of an antibody via ELISA assay, high
secretion level was confirmed from expression vector pCB-Rx71_v5.4 including the
SP7.2 signal sequence encoding an amino acid sequence of SEQ ID NO: 1 derived
from the LBFL313 gene (refer to FIG. 12). Particularly, a significantly high level of
secretion was observed from the combination of the SP7.2 signal sequence linked to an
antibody light chain and the SPl signal sequence encoding an amino acid sequence of
SEQ ID NO: 3 and linked to the antibody heavy chain. Further, it was confirmed that
the secretion level increased further along with the increase in culture period. Con
sequently, it was confirmed that, when the SP7.2 signal sequence was used, the
secretion level was remarkably increased even in a long-term culture, compared to the
results of the luciferase secretion test, in which the secretion level was remarkably
increased in a short-term culture.
[75]
[76] In still another embodiment, the present invention provides a transformed cell in
which the vector is introduced into a host cell.
[77] As used herein, the term "transformation" means that DNA is introduced into a host
cell, and thus the DNA is made replicable by chromosomal integration. In the present
invention, the host cell that can be used in the transformation in the present invention
may include a prokaryotic or/and a eukaryotic cell.
[78] In the present invention, examples of the host cell may include bacteria; generally
known prokaryotic and eukaryotic hosts such as Escherichia, Pseudomonas, Bacillus,
Streptomyces, fungi, and yeasts; insect cells such as Spodoptera frugiperda (SF9); and
animal cells such as CHO, COS 1, COS 7, BSC 1, BSC 40, and BMT 10. In the
present invention, the host cell may be an animal host cell, and particularly a Chinese
Hamster Ovary Cell (CHO) cell, but is not limited thereto.
[79] In an exemplary embodiment of the present invention, a Chinese Hamster Ovary
(CHO) cell, which is widely used in the production of a recombinant protein, was as
the host cell.
[80]
[81] In still another embodiment, the present invention provides a method of producing a
target protein, including: i) culturing a transformed cell, into which the vector for
secretory expression of target protein is introduced, to express a target protein and
secrete the target protein to the outside of the cell; and ii) recovering the target protein
from a culture or a culture supernatant of the cell.
[82] The method of producing a target protein may further include purifying the recovered
target protein. If necessary, the purification of the target protein may be performed by a
protein purification method generally used in the art. For example, the target protein
can be separated from the culture or culture supernatant of the host cell by a con
ventional chromatography method, such as immunoaffinity chromatography, receptor
affinity chromatography, hydrophobic interaction chromatography, lectin affinity chro
matography, size exclusion chromatography, cation or anion exchange chro
matography, high performance liquid chromatography (HPLC) or reversed-phase highperformance
liquid chromatography. Meanwhile, when the target protein is a fusion
protein having an idiosyncratic tag, label or chelate moiety, this target protein may be
purified using an idiosyncratic binding partner or agent. The purified protein may be
cleaved into desired protein parts by removing a protein secretion factor or may remain
in itself. In the process of cleaving a fusion protein, a desired protein having additional
amino acid can be made.
[83] In the present invention, the protein secretion factor, protein, expression cassette,
target protein, vector for secretory expression, transformation, host cell, and the like
are the same as described above.
[84] The host cell used in the method may be an animal host cell, and particularly, a
Chinese Hamster Ovary (CHO) cell. Further, the transformed host cell, if necessary,
may be cultured by a general culture method known in the art.
[85]
[86] In still another embodiment, the present invention provides use of the protein
secretion factor for preparing a vector for secretory expression of target protein.
[87] The protein secretion factor, the vector and target protein are the same as described
above.
[88]
[89] In still another embodiment, the present invention provides use of the protein
secretion factor for secreting target protein.
[90] The protein secretion factor, the vector and target protein are the same as described
above.
Mode for the Invention
[91] Hereinafter, the present invention will be described in more detail with reference to
Examples below. However, these Examples are set forth only to illustrate the present
invention, and the scope of the present invention is not limited to these Examples.
[92]
[93] Example 1: Molecular biology technique
[94] Methods generally used in molecular biology, such as restriction enzyme treatment,
agarose gel electrophoresis, Gel Extraction Kit (QIAGEN), plasmid DNA purification,
polymerase chain reaction (PCR), ligation of DNA fragments, and transformation of E.
coli, were performed according to the methods described in the literature (Sambrook J
et al., 2001 Molecular cloning: A laboratory manual, 2nd edition. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York) with minimum modifications.
[95]
[96] Example 2: Selection of signal sequences
[97]
[98] 2-1: Test method
[99] In order to identify a signal sequence for enhancing secretion during the process of
expressing a heterologous protein using an animal host cell, the possibility for a highefficiency
secretory signal sequence was intended to be examined from the literature
"novel gene LBFL313 related to pancreatic cancer" disclosed in Korean Patent No.
10-0954322.
[100] Specifically, peptide sequences, presumed to have a potential as signal sequences,
were selected from an LBFL313 gene, and these selected peptide sequences were
compared with the conventional six signal sequences generally used as signal
sequences in animal cells. In this regard, as a first comparison test, a Chinese hamster
ovary (CHO) cell line widely used in the production of a recombinant protein was used
as a host cell, and a secretory luciferase gene was used as a target gene. The secretion
level was determined by measuring the amount of the light emitted by the oxidation of
luciferin (used as a substrate) by a luciferase secreted to the outside of a cell using a luminometer.
[101] Thereafter, as a second comparison test for comparing the secretion of a monoclonal
antibody, which is an industrially available protein, instead of a luciferase, to that of
the signal sequences selected in the first comparison test, the amount of the antibodies
secreted by various combinations of the signal sequence of the light chain and the
signal sequence of the heavy chain of the antibodies was measured via ELISA using a
CHO cell line as a host cell. Here, the antibody secreted to the outside of cell was fixed
by covering an ELISA plate with F(ab')2 recognizing the Fc portion of the heavy chain,
and the antibody bonded to the kappa portion of the light chain was marked with a
horseradish peroxidase (HRP), and the oxidation of TMB used as a substrate was
measured using a spectrophotometer, thereby determining the secretion level.
[102]
[103] 2-2: Signal sequence used in test
[104] The peptide sequences expected as signal sequences were presumed from the
LBFL313 gene in Example 2-1. As a result, SP7.2 and SP7.3 were selected.
[105]
[106] * SP7.2 (SEQ ID NO: 1)
[107] NH3-MHRPEAMLLLLTLALLGGPTWA-C0 2H
[108] ¾ SP7.3 (SEQ ID NO: 2)
[109] NH3-MWRVPGTTRRPVTGESPGMHRPEAMLLLLTLALLGGPTWA-CO 2H
[110]
[111] The six conventional signal sequences used for comparison test with the aboveselected
signal sequences were named SP1 to SP6. These signal sequences are as
follows.
[112] v¾SPl (SEQ ID NO: 3)
[113] NH3-MGWSYIILFLVATATDVHS-CO 2H
[114] v¾SP2 (SEQ ID NO: 4)
[115] NH3-MKWVTFISLLFLFSSAYSRGVFRR-CO 2H
[116] v¾SP3 (SEQ ID NO: 5)
[117] NH3-MDFQVQIISFLLISASVIMSRG-CO 2H
[118] v¾SP4 (SEQ ID NO: 6)
[119] NH3-MGWSLILLFLVAVATRVLS-CO 2H
[120] v¾SP5 (SEQ ID NO: 7)
[121] NH3-MLLLLLLLGLRLQLSLG-CO 2H
[122] v¾SP6 (SEQ ID NO: 8)
[123] NH3-MKTLILAVALVYCATVHC-CO 2H
[124]
[125] In this test, SP1 is a signal sequence derived from mouse IgG2; SP2 is a signal
sequence derived from human serum albumin (HSA); SP3 is a signal sequence derived
from mouse IkC; SP4 is an artificially synthesized signal sequence (not a natural signal
sequence) and is a signal sequence used in U.S. Pat. No. 7,381,560; SP5 is a signal
sequence derived from a secretory alkaline phosphatase (SEAP); and SP6 is a signal
sequence derived from Cypridina noctiluca lucif erase (CLUC), which is a secretory luciferase.
[126]
[127] In this test, in order to select plasmid vectors exhibiting high target protein secretion
from among such plasmid vectors by optimal combination, typically, a Cypridina
noctiluca luciferase (CLUC) gene, which is an easily-measurable secretory luciferase,
and an Rx antibody gene, which is an IgGl type antibody gene, were used as a
reporter.
[128] The following various combinations of plasmid vectors were prepared by linking
DNA sequences encoding the eight signal sequences with gene sequences (Cypridina
noctiluca luciferase (CLUC) gene or light chain and heavy chain genes of an Rx
antibody, which is an IgGl type antibody) in frame. The combinations and components
of the thus prepared plasmid vectors are summarized in Table 1 below.
Table 1
[Table 1]
Plasmid name Components
pCBIN-CLUCl SP1 + CLUC
pCBIN-CLUC2 SP2 + CLUC
pCBIN-CLUC3 SP3 + CLUC
pCBIN-CLUC4 SP4 + CLUC
pCBIN-CLUC5 SP5 + CLUC
pCBIN-CLUC SP6 + CLUC
pCBIN-CLUC7.2 SP7.2 + CLUC
pCBIN-CLUC7.3 SP7.3 + CLUC
pCB-Rxll_v5.4 (SP1 + antibody light chain) + (SP1 + antibody heavy chain)
pCB-Rxl2_v5.4 (SP1 + antibody light chain) + (SP2 + antibody heavy chain)
pCB-Rxl6_v5.4 (SP1 + antibody light chain) + (SP6 + antibody heavy chain)
pCB-Rxl7_v5.4 (SP1 + antibody light chain) + (SP7.2 + antibody heavy chain)
pCB-Rx21_v5.4 (SP2 + antibody light chain) + (SP1 + antibody heavy chain)
pCB-Rx22_v5.4 (SP2 + antibody light chain) + (SP2 + antibody heavy chain)
pCB-Rx26_v5.4 (SP2 + antibody light chain) + (SP6 + antibody heavy chain)
pCB-Rx27_v5.4 (SP2 + antibody light chain) + (SP7.2 + antibody heavy chain)
pCB-Rx31_v5.4 (SP3 + antibody light chain) + (SP1 + antibody heavy chain)
pCB-Rx32_v5.4 (SP3 + antibody light chain) + (SP2 + antibody heavy chain)
pCB-Rx36_v5.4 (SP3 + antibody light chain) + (SP6 + antibody heavy chain)
pCB-Rx37_v5.4 (SP3 + antibody light chain) + (SP7.2 + antibody heavy chain)
pCB-Rx61_v5.4 (SP6 + antibody light chain) + (SP1 + antibody heavy chain)
pCB-Rx62_v5.4 (SP6 + antibody light chain) + (SP2 + antibody heavy chain)
pCB-Rx66_v5.4 (SP6 + antibody light chain) + (SP6 + antibody heavy chain)
pCB-Rx67_v5.4 (SP6 + antibody light chain) + (SP7.2 + antibody heavy chain)
pCB-Rx71_v5.4 (SP7.2 + antibody light chain) + (SP1 + antibody heavy chain)
pCB-Rx72_v5.4 (SP7.2 + antibody light chain) + (SP2 + antibody heavy chain)
pCB-Rx76_v5.4 (SP7.2 + antibody light chain) + (SP6 + antibody heavy chain)
pCB-Rx77_v5.4 (SP7.2 + antibody light chain) + (SP7.2 + antibody heavy chain)
[130]
[131] In the test using CLUC, the extracellular secretion level was measured via luciferase
assay, and in the test using the Rx antibody, the extracellular secretion level was
measured via ELISA assay.
[132]
[133] Example 3: Preparation of luciferase plasmid vectors
[134] Plasmid vectors having the secretory sequences designed in Example 2-2 and having
a secretory luciferase (CLUC) as a reporter gene were prepared.
[135]
[136] 3-1: Preparation of pCBIN-CLUC6
[137] In order to construct a reporter vector having a CMV enhancer (CMVe) and a CMW
beta-actin fusion promoter (CB), a DNA fragment (1762 bp), which was obtained by
treating a pTOP-BA-RL-pA vector having 'CMVe', 'CB' and 'beta-actin intron'
(disclosed in Korean Patent Application Publication No. 10-2012-0059222) with EcoR
I and BamHl, was inserted into a pCLuc-Basic2 vector (NEB, Cat# : N0317S) digested
by the same restriction enzyme. The reporter vector constructed in this way has a
signal sequence 'SP6' (pCBIN-CLUC) (refer to FIG. 1).
[138]
[139] 3-2: Preparation of pCBIN-CLUCl
[140] A DNA fragment (80 bp), which was obtained via PCR amplification of a DNA
sequence encoding a peptide sequence of a mouse IgG2 signal sequence (SP1: SEQ ID
NO: 3) using two primers of SEQ ID NOS: 9 and 10 using pCB-Ix6_v5.4 as a template
and then digesting the PCR-amplified product with BamHl and Ndel, and a DNA
fragment (1654 bp), which was obtained via PCR amplification a CLUC gene using
two primers (SEQ ID NOS: 11 and 12) and the pCLuc-Basic2 vector as a template and
then digesting the PCR-amplified product with Ndel and Xbal, were inserted into the
site of a DNA fragment (6049bp), which was obtained by digesting the pCBIN-CLUC
vector with BamHl and Xbal, so as to prepare a pCBIN-CLUCl vector (refer to FIG.
2).
[141]
[142] The primers used are as follows.
[143] * oSPl-f (SEQ ID NO: 9)
[144] 5'-tt GGATCC gcc acc atg gga tgg age tat-3'
[145] * oSPl-r (SEQ ID NO: 10)
[146] 5'-ttC ATA TGg aca gtc ctg gga gtg gac ate tgt-3'
[147] * oCLUC-Nl-f (SEQ ID NO: 11)
[148] 5'-tt c CATATG aa cct gat cca cca aa-3'
[149] * oBasic-r (SEQ ID NO: 12)
[150] 5'-tca gaa gcc ata gag ccc acc gca t-3'
[151]
[152] 3-3: Preparation of pCBIN-CLUC2
[153] A DNA fragment (95 bp), which was obtained by annealing a DNA sequence
encoding a peptide sequence of a human serum albumin (HAS) signal sequence to two
oligonucleotides (SEQ ID NOS: 13 and 14) to use the annealed DNA sequence as a
template, amplifying the resulting DNA sequence via PCR using two primers (SEQ ID
NOS: 15 and 16) and then digesting the PCR-amplified product with BamHl and Ndel,
and a DNA fragment (1654 bp), which was obtained via PCR amplification a CLUC
gene using two primers (SEQ ID NOS: 11 and 12) using the pCLuc-Basic2 vector as a
template and then cleaving the PCR-amplified product using Nde and Xbal were
inserted into the cleft site of a DNA fragment (6049bp), which was obtained by
digesting the pCBIN-CLUC vector with BamHl and Xbal, so as to prepare a pCBINCLUC2
vector (refer to FIG. 3).
[154]
[155] The primers used are as follows.
[156] * oHSAL-U (SEQ ID NO: 13)
[157] 5'-atg aag tgg gtg acc ttc ate tec ctg ctg ttc ctg ttc tec tec gcc tac tec agg ggc gtg ttc
agg agg-3'
[158] * oHSAL-L (SEQ ID NO: 14)
[159] 5'-cct cct gaa cac gcc cct gga gta ggc gga gga gaa cag gaa cag cag gg-3'
[160] * oSP2-f (SEQ ID NO: 15)
[161] 5'-tt GGATCC gcc acc atg aag tgg gtg acc-3'
[162] * oSP2-r (SEQ ID NO: 16)
[163] 5'-ttC ATA TGg aca gtc ctg cct cct gaa cac gcc -3'
[164]
[165] 3-4: Preparation of pCBIN-CLUC3
[166] A DNA fragment (89 bp), which was obtained via PCR amplification using two
primers of SEQ ID NOS: 17 and 18 using a pCB-Rx vector (expression vector retained
by our company, in which 'SP3' and 'SP4' were used as signal sequences) expressing a
mouse-human chimeric IgGl monoclonal antibody as a template and then digesting the
PCR-amplified product with BamHl and Ndel, and a DNA fragment (1654 bp), which
was obtained via PCR amplification ofvia PCR amplification a CLUC gene using two
primers (SEQ ID NOS: 11 and 12) and the pCLuc-Basic2 vector as a template, and
then digesting the PCR-amplified product with Ndel and Xbal were inserted into the
restriction site of a DNA fragment (6049 bp), which was obtained by digesting the
pCBIN-CLUC vector with BamHl and Xbal, so as to prepare a pCBIN-CLUC3 vector
(refer to FIG. 4).
[167]
[168] The primers used are as follows.
[169] * oSP3-f (SEQ ID NO: 17)
[170] 5'-tt GGATCC gcc acc atg gac ttc cag gtg-3'
[171] * oSP3-r (SEQ ID NO: 18)
[172] 5'-ttC ATA TGg aca gtc ctg gcc cct gga cat gat -3'
[173]
[174] 3-5: Preparation of pCBIN-CLUC4
[175] A DNA fragment (80 bp), which was obtained via PCR amplification using two
primers of SEQ ID NOS: 19 and 20 and a pCB-Rx vector expressing a mouse-human
chimeric IgGl monoclonal antibody as a template and then digesting the PCRamplified
product with BamHl and Ndel, and a DNA fragment (1654 bp), which was
obtained via PCR amplification a CLUC gene using two primers (SEQ ID NOS: 11
and 12) and the pCLuc-Basic2 vector as a template and then digesting the PCRamplified
product with Nde and Xbal were inserted into the restriction site of a DNA
fragment (6049bp), which was obtained by digesting the pCBIN-CLUC vector with
BamHl and Xbal, so as to prepare a pCBIN-CLUC4 vector (refer to FIG. 5).
[176]
[177] The primers used are as follows.
[178] * oSP4-f (SEQ ID NO: 19)
[179] -tt GGATCC gcc acc atg ggc tgg age ctg-3'
[180] * oSP4-r (SEQ ID NO: 20)
[181] 5'-ttC ATA TGg aca gtc ctg gga cag cac cct ggt -3'
[182]
[183] 3-6: Preparation of pCBIN-CLUC5
[184] A DNA fragment (74 bp), which was obtained via PCR amplification using two
primers of SEQ ID NOS: 2 1 and 22 and a pSEAP-Basic2 vector, which is a reporter
vector using secretory alkaline phosphatase (SEAP), as a template and then digesting
the PCR-amplified product with BamHl and Ndel, and a DNA fragment (1654 bp),
which was obtained via PCR amplification of a CLUC gene using two primers (SEQ
ID NOS: 11 and 12) and the pCLuc-Basic2 vector as a template and then digesting the
PCR-amplified product with Ndel and Xbal were inserted into the restriction site of a
DNA fragment (6049bp), which was obtained by digesting the pCBIN-CLUC vector
with BamHl and Xbal, so as to prepare a pCBIN-CLUC5 vector (refer to FIG. 5).
[185] The primers used are as follows.
[186] * oSP5-f (SEQ ID NO:21)
[187] -tt GGATCC gcc acc atg ctg ctg ctg ctg ctg ctg ctg g-3'
[188] * oSP5-r (SEQ ID NO: 22)
[189] 5'-ttC ATA TGg aca gtc ctg gcc cag gga gag ctg-3'
[190]
[191] 3-7: Preparation of pCBIN-CLUC7.2
[192] A DNA fragment (89 bp) obtained via PCR amplification using two primers of SEQ
ID NOS: 23 and 24 and pLFG250 (Korean Patent Application Publication No.
10-0954322), which has a LBFL313 gene, as a template and then digesting the PCRamplified
product with BamHl and Ndel, and a DNA fragment (1654 bp), which was
obtained via PCR amplification a CLUC gene using two primers (SEQ ID NOS: 11
and 12) and the pCLuc-Basic2 vector as a template and then digesting the PCRamplified
product with Nde and Xbal were inserted into the restriction site of a DNA
fragment (6049bp), which was obtained by digesting the pCBIN-CLUC vector with
BamHl and Xbal, so as to prepare a pCBIN-CLUC7.2 vector (refer to FIG. 7).
[193]
[194] The primers used are as follows.
[195] * oSP7-Bl-f2 (SEQ ID NO: 23)
[196] 5'-tt GGATCC gcc acc atg cac egg cca gag-3'
[197] * oSP7-Nl-r (SEQ ID NO: 24)
[198] 5'-ttC ATA TGg aca gtc ctg tgc cca ggt ggg gcc-3'
[199]
[200] 3-8: Preparation of pCBIN-CLUC7.3
[201] A DNA fragment (143 bp), which was obtained via PCR amplification using two
primers of SEQ ID NOS: 24 and 25 and pLFG250 (Korean Patent No. 10-0954322),
which has a LBFL313 gene, as a template and then digesting the PCR-amplified
product with BamHl and Ndel, and a DNA fragment (1654 bp), which was obtained
via PCR amplification a CLUC gene using two primers (SEQ ID NOS: 11 and 12) and
the pCLuc-Basic2 vector as a template and then digesting the PCR-amplified product
with Ndel and Xbal were inserted into the cleft site of a DNA fragment (6049bp),
which was obtained by digesting the pCBIN-CLUC vector with BamHl and Xbal, so as
to prepare a pCBIN-CLUC7.3 vector (refer to FIG. 8).
[202]
[203] The used primer is as follows.
[204] * oSP7-Bl-f3 (SEQ ID NO: 25)
[205] -tt GGATCC gcc acc atg tgg agg gtg ccc-3'
[206]
[207] Example 4: In vitro secretion efficacy test of luciferase plasmid vectors
[208]
[209] Each of the luciferase plasmid vectors prepared in Example 3 is configured such that
a secretory luciferase derived from Cypridina noctiluca is inserted as a reporter. In
order to examine the extracellular secretion efficacy of a signal sequence in an in vitro
cell culture system, the signal sequence was transformed in a CHO cell, and then the
secretion inducing level of the signal sequence was examined through luciferase assay.
[210] Specifically, each of the luciferase plasmid vectors prepared in Example 3 was
transformed in a CHO cell, which was cultured in a Dulbecco's modified Eagle's
medium (DMEM, manufactured by GIBCO-BRL Corporation) containing 10% of
heat-inactivated fetal bovine serum (FBS, manufactured by GIBCO-BRL Corporation),
using Lipofectamine™ 2000 (Invitrogen, Cat.#: 11668-019). One day before the trans
formation, 6 x 104 CHO cells per each well of a 24-well plate (Falcon Corporation)
were cultured, and, on the next day, tube 1 ( 1 well reaction amount) filled with 500 ng
of eight different types plasmid vectors (pCBIN-CLUCl, pCBIN-CLUC2, pCBINCLUC4,
pCBIN-CLUC5, pCBIN-CLUC, pCBIN-CLUC7.2, and pCBIN-CLUC7.3),
in each of which a luciferase gene is inserted, and 50 of Opti-MEM ®I (invitrogen,
Cat.# 31985-070), and tube 2 ( 1 well reaction amount), filled with 2 L· of Lipo
fectamine™ 2000 and 48 of Opti-MEM ®I, were respectively left at room tem
perature for 5 minutes, and then the two tubes were mixed to react at room temperature
for 20 minutes. The mixture was added to the CHO cells in 250 of Opti-MEM ®I in
a volume of 100 and cultured in an incubator (5% C0 2) at 37 °C, and then the
DMEM containing 20% FBS was put into each well and cultured for 6 days. On the
2nd, 3rd, 5th, and 6th day after the transformation, the culture medium of each well
was collected as a sample in the amount of 100 m , stored at 20°C, completely
dissolved, and on the 6th day, 20 m each of the resultant was transferred into an assay
plate, respectively, and subjected to luciferase assay.
[211]
[212] As a result of the measurement of the luciferase secretion efficacy, as shown in FIG.
9, the secretion level of luciferase was improved in the total four signal sequences
(SP2, SP6, SP7.2, and SP7.3) of the two signal sequences derived from a LBFL313
and the existing two signal sequences compared to the existing signal sequence (SP1).
Particularly, it was confirmed that, in the case of SP7.2 and SP7.3 vectors, a large
amount of luciferase is secreted at the early stage of culture (2d and 3d).
[213]
[214] Examples 5: Preparation of antibody expression plasmid vectors
[215] The following various antibody expression vectors were prepared by selecting SP2,
SP6 and SP7.2 from among the signal sequences exhibiting effects in Example 4 in
order to examine whether each of the prepared antibody expression vectors exhibits
excellent secretion inducing ability even to a monoclonal antibody, for which an in
dustrial large-scale production is required.
[216]
[217] 5-1: Preparation of pCB-Rxl 1 v5.4
[218] A DNA fragment, which was obtained by digesting with BamHl and Xho of a PCR
product, in which SPl and an antibody light chain are linked, obtained via PCR using
four primers (SEQ ID NOS: 9, 26, 27, and 28) and the pCBIN-CLUCl vector and
pCB-Rx_v5.4 vector as templates, and a DNA fragment (refer to FIG. 10 and Table 2),
which was obtained by digesting with Asc and N il of a PCR product, in which SPl
and an antibody heavy chain are linked, obtained via PCR using four primers (SEQ ID
NOS: 29, 30, 31, and 32) and the pCBIN-CLUCl vector and pCB-Rx_v5.4 vector as
templates, were inserted into the BamHl and Xho sites of pCB-Rx_v5.4 and the Asc
and No l sites of pCB-Rx_v5.4, respectively, so as to prepare a pCB-Rxl l_v5.4 vector
(refer to FIG. 11 and Table 3).
[219]
[220] The primers used are as follows.
[221] * oIxLs-rl (SEQ ID NO: 26)
[222] 5'-cag cag gat gtc gcc cct gga cat gat cac -3'
[223] * oRx-LFl (SEQ ID NO: 27)
[224] 5'-cag ate gtg ctg tct cag tct-3'
[225] oIkC-MlXl-r (SEQ ID NO: 28)
[226] -tt ACGCGT CTCGAG tea aca etc tec c-3'
[227] * oRHn-f (SEQ ID NO: 29)
[228] -tt GGCGCGCC atg gga tgg age tat-3'
[229] * oIxLs-r2 (SEQ ID NO: 30)
[230] 5'-cag cag gat gtc gga cag cac cct ggt ggc cac ggc-3'
[23 1] * oRx_HFl (SEQ ID NO: 31)
[232] 5'-cag gtg cag ctg cag cag ccc-3'
[233] * olgGl-XINl-r (SEQ ID NO: 32)
[234] 5'-aa CTCGAG GCGGCCGC tea ttt acc egg aga c-3'
[235]
[236] Table 2
[Table 2]
[237]
[238] Table 3
[Table 3]
[239]
[240] 5-2: Preparation of pCB-Rx!2 v5.4
[241] A DNA fragment, which was obtained by digesting with BamHl and Xho of a PCR
product, in which SPl and an antibody light chain are linked, obtained via PCR ampli
fication using four primers (SEQ ID NOS: 9, 26, 27, and 28) and the pCBIN-CLUCl
vector and pCB-Rx_v5.4 vector as templates, and a DNA fragment (refer to FIG. 10
and Table 4), which was obtained by digesting with Asc and N il of a PCR product, in
which SP2 and an antibody heavy chain are linked, obtained via PCR amplification
using four primers (SEQ ID NOS: 33, 34, 35, and 32) and the pCBIN-CLUC2 vector
and pCB-Rx_v5.4 vector as templates, were inserted into the BamHl and Xho sites of
pCB-Rx_v5.4 and the Asc and No l sites of pCB-Rx_v5.4, respectively, so as to
prepare a pCB-Rxl2_v5.4 vector (refer to FIG. 11 and Table 5).
[242] The primers used are as follows.
[243] * oAscI_SP2-f (SEQ ID NO: 33)
[244] 5'-ctg gcg cgc cat gaa gtg ggt gac c-3'
[245] * oSP2_RH-r (SEQ ID NO: 34)
[246] 5'-gca get gca cct gec tec tga aca c-3'
[247] * oSP2_RH-f (SEQ ID NO: 35)
[248] 5'-ctg ttc att gec agg tgc age tgc-3'
[249]
[250] Table 4
[Table 4]
[251]
[252] Table 5
[Table 5]
[253]
[254] 5-3: Preparation of pCB-Rx 16 v5.4
[255] A DNA fragment, which was obtained by digesting with BamHl and Xho of a PCR
product, in which SPl and an antibody light chain are linked, obtained via PCR ampli
fication using four primers (SEQ ID NOS: 9, 26, 27, and 28) using the pCBIN-CLUCl
vector and pCB-Rx_v5.4 vector as templates, and a DNA fragment (refer to FIG. 10
and Table 6), which was obtained by digesting with Asc and N il of a PCR product, in
which SP6 and an antibody heavy chain are linked, obtained via PCR amplification
using four primers (SEQ ID NOS: 36, 37, 38, and 32) and the pCBIN-CLUC3 vector
and pCB-Rx_v5.4 vector as templates, were inserted into the BamHl and Xho sites of
pCB-Rx_v5.4 and the Asc and No l sites of pCB-Rx_v5.4, respectively, so as to
prepare a pCB-Rxl6_v5.4 vector (refer to FIG. 11 and Table 7).
[256] The primers used are as follows.
[257] * oAscI_SP6-f (SEQ ID NO: 36)
[258] 5'-CAG GCG CGC CAT GAA GAC CTT AAT TC-3'
[259] * oSP6_RH-r (SEQ ID NO: 37)
[260] 5'-GCA GCT GCA CCT GGC AAT GAA CAG-3'
[261] * oSP6_RH-f (SEQ ID NO: 38)
[262] 5'-CTG TTC ATT GCC AGG TGC AGC TGC-3'
[263]
[264] Table 6
[Table 6]
[265]
[266] Table 7
[Table 7]
[267]
[268] 5-4: Preparation of pCB-Rxl7 v5.4
[269] A DNA fragment, which was obtained by digesting with BamHl and Xho of a PCR
product, in which SPl and an antibody light chain are linked, obtained via PCR ampli
fication using four primers (SEQ ID NOS: 9, 26, 27, and 28) and the pCBIN-CLUCl
vector and pCB-Rx_v5.4 vector as templates, and a DNA fragment (refer to FIG. 10
and Table 8), which was obtained by digesting with AscI and No l of a PCR product, in
which SP7.2 and an antibody heavy chain are linked, obtained via PCR amplification
using four primersvia PCR amplification using four primers (SEQ ID NOS: 39, 40, 41,
and 32) and the pCBIN-CLUC7.2 vector and pCB-Rx_v5.4 vector as templates, were
inserted into the BamHl and Xho sites of pCB-Rx_v5.4 and the AscI and No l sites of
pCB-Rx_v5.4, respectively, so as to prepare a pCB-Rxl7_v5.4 vector (refer to FIG. 11
and Table 9).
[270] The primers used are as follows.
[271] * oAscI_SP7.2-f (SEQ ID NO: 39)
[272] 5'-cag gcg cgc cat gca ccg gcc aga g-3'
[273] * oSP7.2_RH-r (SEQ ID NO: 40)
[274] 5'-gca get gca cct gtg ccc agg tgg g-3'
[275] * oSP7.2_RH-f (SEQ ID NO: 41)
[276] 5'-ccc acc tgg gca cag gtg cag ctg c-3'
[277]
[278] Table 8
[Table 8]
[279]
[280] Table 9
[Table 9]
[281]
[282] 5-5: Preparation of pCB-Rx21 v5.4
[283] A DNA fragment, which was obtained by digesting with BamHl and Xho of a PCR
product, in which SP2 and an antibody light chain are linked, obtained via PCR ampli
fication using four primers (SEQ ID NOS: 15, 42, 43, and 28) and the pCBIN-CLUC2
vector and pCB-Rx_v5.4 vector as templates, and a DNA fragment (refer to FIG. 10
and Table 10), which was obtained by digesting with Asc and N il of a PCR product,
in which SP1 and an antibody heavy chain are linked, obtained via PCR amplification
using four primers s (SEQ ID NOS: 39, 40, 41, and 32) and the pCBIN-CLUCl vector
and pCB-Rx_v5.4 vector as templates, were inserted into the BamHl and Xho sites of
pCB-Rx_v5.4and the Asc and No l sites of pCB-Rx_v5.4, respectively, so as to
prepare a pCB-Rx21_v5.4 vector (refer to FIG. 10 and Table 11).
[284] The primers used are as follows.
[285] * oSP7.2_RH-r (SEQ ID NO: 42)
[286] 5'-gca get gca cct gtg ccc agg tgg g-3'
[287] * oSP7.2_RH-f (SEQ ID NO: 43)
[288] 5'-ccc acc tgg gca cag gtg cag ctg c-3'
[289]
[290] Table 10
[Table 10]
[291]
[292] Table 1 1
[Table 11]
[293]
[294] 5-6: Preparation of pCB-Rx22 v5.4
[295] A DNA fragment, which was obtained by digesting with BamHl and Xhol of a PCR
product, in which SP2 and an antibody light chain are linked, obtained via PCR ampli
fication using four primers (SEQ ID NOS: 15, 42, 4, and 28) and the pCBIN-CLUC2
vector and pCB-Rx_v5.4 vector as templates, and a DNA fragment (refer to FIG. 10
and Table 12), which was obtained by digesting with Ascl and N il of a PCR product,
in which SP2 and an antibody heavy chain are linked, obtained via PCR amplification
using four primers (SEQ ID NOS: 33, 34, 35, and 32) and the pCBIN-CLUC2 vector
and pCB-Rx_v5.4 vector as templates, were inserted into the BamHl and Xhol sites of
pCB-Rx_v5.4and the Ascl and No l sites of pCB-Rx_v5.4, respectively, so as to
prepare a pCB-Rx22_v5.4 vector (refer to FIG. 10 and Table 13).
[296]
[297] Table 12
[Table 12]
[298]
[299] Table 13
[Table 13]
[300]
[301] 5-7: Preparation of pCB-Rx26 v5.4
[302] A DNA fragment, which was obtained by digesting with BamHl and Xhol of a PCR
product, in which SP2 and an antibody light chain are linked, obtained via PCR ampli
fication using four primers (SEQ ID NOS: 15, 42, 43, and 28) and the pCBIN-CLUC2
vector and pCB-Rx_v5.4 vector as templates, and a DNA fragment (refer to FIG. 10
and Table 14), which was obtained by digesting with Ascl and No l of a PCR product,
in which SP6 and an antibody heavy chain are linked, obtained via PCR amplification
using four primerss (SEQ ID NOS: 36, 37, 38, and 32) and the pCBIN-CLUC vector
and pCB-Rx_v5.4 vector as templates, were inserted into the BamHl and Xhol sites of
pCB-Rx_v5.4 and the Ascl and Noil sites of pCB-Rx_v5.4, respectively, so as to
prepare a pCB-Rx26_v5.4 vector (refer to FIG. 11 and Table 15).
[303]
[304] Table 14
[Table 14]
[305]
[306] Table 15
[Table 15]
[307]
[308] 5-8: Preparation of pCB-Rx27 v5.4
[309] A DNA fragment, which was obtained by digesting with BamHl and Xho of a PCR
product, in which SP2 and an antibody light chain are linked, obtained via PCR ampli
fication using four primerss (SEQ ID NOS: 15, 42, 43, and 28) and the pCBIN-CLUC2
vector and pCB-Rx_v5.4 vector as templates, and a DNA fragment (refer to FIG. 10
and Table 16), which was obtained by digesting with Asc and N il of a PCR product,
in which SP6 and an antibody heavy chain are linked, obtained via PCR amplification
using four primersvia PCR amplification using four primers (SEQ ID NOS: 39, 40, 41,
and 32) and the pCBIN-CLUC7.2 vector and pCB-Rx_v5.4 vector as templates, were
inserted into the BamHl and Xho sites of pCB-Rx_v5.4 and the Asc and NotI sites of
pCB-Rx_v5.4, respectively, so as to prepare a pCB-Rx27_v5.4 vector (refer to FIG. 11
and Table 17).
[310]
[311] Table 16
[Table 16]
[312]
[313] Table 17
[Table 17]
[314]
[315] 5-9: Preparation of pCB-Rx32 v5.4
[316] A DNA fragment (refer to FIG. 10 and Table 18), which was obtained by digesting
with AscI and No l of a PCR product, in which SP2 and an antibody heavy chain are
linked, obtained via PCR amplification using four primers (SEQ ID NOS: 33, 34, 35,
and 32) and the pCBIN-CLUC2 vector and pCB-Rx_v5.4 vector as templates, was
inserted into the AscI and No l sites of pCB-Rx_v5.4, so as to prepare a pCBRx32_
v5.4 vector (refer to FIG. 11 and Table 19).
[317] Table 18
[Table 18]
[318]
[319] Table 19
[Table 19]
[320]
[321] 5-10: Preparation of pCB-Rx36 v5.4
[322] A DNA fragment (refer to FIG. 10 and Table 20), which was obtained by digesting
with AscI and No l of a PCR product, in which SP6 and an antibody heavy chain are
linked, obtained via PCR amplification using four primers (SEQ ID NOS: 36, 37, 38,
and 32) and the pCBIN-CLUC vector and pCB-Rx_v5.4 vector as templates, was
inserted into the AscI and No l sites of pCB-Rx_v5.4, so as to prepare a pCBRx36_
v5.4 vector (refer to FIG. 11 and Table 21).
[323] Table 20
[Table 20]
[324]
[325] Table 2 1
[Table 21]
[326]
[327] 5-11: Preparation of pCB-Rx37 v5.4
[328] A DNA fragment (refer to FIG. 10 and Table 22), which was obtained by digesting
with AscI and Noil of a PCR product, in which SP7.2 and an antibody heavy chain are
linked, obtained via PCR amplification using four primers (SEQ ID NOS: 39, 40, 41,
and 32) and the pCBIN-CLUC7.2 vector and pCB-Rx_v5.4 vector as templatess, was
inserted into the AscI and Noil sites of pCB-Rx_v5.4, so as to prepare a pCBRx37_
v5.4 vector (refer to FIG. 11 and Table 23).
[329] Table 22
[Table 22]
Primer SEQ ID NO:
E 39
F 40
G 4 1
H 32
[331] Table 23
[Table 23]
[332]
[333] 5-12: Preparation of pCB-Rx61 v5.4
[334] A DNA fragment, which was obtained by digesting with BamHl and Xhol of a PCR
product, in which SP6 and an antibody light chain are linked, obtained via PCR ampli
fication using four primers (SEQ ID NOS: 44, 45, 46, and 28) and the pCBIN-CLUC
vector and pCB-Rx_v5.4 vector as templates, and a DNA fragment (refer to FIG. 10
and Table 24), which was obtained by digesting with AscI and No l of a PCR product,
in which SPl and an antibody heavy chain are linked, obtained via PCR amplification
using four primers (SEQ ID NOS: 29, 30, 31, and 32) and the pCBIN-CLUCl vector
and pCB-Rx_v5.4 vector as templates, were inserted into the BamHl and Xho sites of
pCB-Rx_v5.4 using BamHl and Xhol and the AscI and No l sites of pCB-Rx_v5.4, r e
spectively, so as to prepare a pCB-Rx61_v5.4 vector (refer to FIG. 11 and Table 25).
[335]
[336] Table 24
[Table 24]
[337]
[338] Table 25
[Table 25]
[339]
[340] The primers used are as follows.
[341] * oSP6-f (SEQ ID NO: 44)
[342] 5'-tt GGATCC gcc acc atg aag acc tta att-3'
[343] * oSP6_RL-r (SEQ ID NO: 45)
[344] 5'-ACA GCA CGA TCT GGC AAT GAA CAG-3'
[345] * oSP6_RL-f (SEQ ID NO: 46)
[346] 5'-CTG TTC ATT GCC AGA TCG TGC TGT-3'
[347]
[348] 5-13: Preparation of pCB-Rx62 v5.4
[349] A DNA fragment, which was obtained by digesting with BamHl and Xhol of a PCR
product, in which SP6 and an antibody light chain are linked, obtained via PCR ampli
fication using four primersvia PCR amplification using (SEQ ID NOS: 44, 45, 46, and
28) and the pCBIN-CLUC vector and pCB-Rx_v5.4 vector as templates, and a DNA
fragment (refer to FIG. 10 and Table 26), which was obtained by digesting with Ascl
and N il of a PCR product, in which SP2 and an antibody heavy chain are linked,
obtained via PCR amplification using four primers via PCR amplification using (SEQ
ID NOS: 33, 34, 35 and 32) and the pCBIN-CLUC2 vector and pCB-Rx_v5.4 vector
as templates, were inserted into the BamHl and Xho sites of pCB-Rx_v5.4 and the Asc
I and No l sites of pCB-Rx_v5.4, respectively, so as to prepare a pCB-Rx62_v5.4
vector (refer to FIG. 11 and Table 27).
[350]
[351] Table 26
[Table 26]
[352]
[353] Table 27
[Table 27]
[354]
[355] 5-14: Preparation of pCB-Rx66 v5.4
[356] A DNA fragment, which was obtained by digesting with BamHl and Xhol of a PCR
product, in which SP6 and an antibody light chain are linked, obtained via PCR ampli
fication using four primersvia PCR amplification using (SEQ ID NOS: 44, 45, 46, and
28) and the pCBIN-CLUC vector and pCB-Rx_v5.4 vector as templates, and a DNA
fragment (refer to FIG. 10 and Table 28), which was obtained by digesting with Ascl
and N il of a PCR product, in which SP6 and an antibody heavy chain are linked,
obtained via PCR amplification using four primersvia PCR amplification using (SEQ
ID NOS: 36, 37, 38, and 32) and the pCBIN-CLUC vector and pCB-Rx_v5.4 vector as
templates, were inserted into the BamHl and Xhol sites of pCB-Rx_v5.4 using BamHl
and Xhol and the Ascl and No l sites of pCB-Rx_v5.4, respectively, so as to prepare a
pCB-Rx66_v5.4 vector (refer to FIG. 11 and Table 29).
[357]
[358] Table 28
[Table 28]
[359]
[360] Table 29
[Table 29]
[361]
[362] 5-15: Preparation of pCB-Rx67 v5.4
[363] A DNA fragment, which was obtained by digesting with BamHl and Xhol of a PCR
product, in which SP6 and an antibody light chain are linked, obtained via PCR ampli
fication using four primersvia PCR amplification using four primers (SEQ ID NOS:
44, 45, 46, and 28) and the pCBIN-CLUC vector and pCB-Rx_v5.4 vector as
templates, and a DNA fragment (refer to FIG. 10 and Table 30), which was obtained
by digesting with Ascl and No l of a PCR product, in which SP7.2 and an antibody
heavy chain are linked, obtained via PCR amplification using four primersvia PCR am
plification using (SEQ ID NOS: 39, 40, 41, and 32) and the pCBIN-CLUC7.2 vector
and pCB-Rx_v5.4 vector as templates, were inserted into the BamHl and Xhol sites of
pCB-Rx_v5.4 and the Ascl and Notl sites of pCB-Rx_v5.4, respectively, so as to
prepare a pCB-Rx67_v5.4 vector (refer to FIG. 11 and Table 31).
[364]
[365] Table 30
[Table 30]
[366]
[367] Table 3 1
[Table 31]
[368]
[369] 5-16: Preparation of pCB-Rx71 v5.4
[370] A DNA fragment, which was obtained by digesting with BamHl and Xhol of a PCR
product, in which SP7.2 and an antibody light chain are linked, obtained via PCR am
plification using four primersvia PCR amplification using (SEQ ID NOS: 23, 42, 43,
and 28) and the pCBIN-CLUC7.2 vector and pCB-Rx_v5.4 vector as templates, and a
DNA fragment (refer to FIG. 10 and Table 32), which was obtained by digesting with
AscI and No l of a PCR product, in which SPl and an antibody heavy chain are linked,
obtained via PCR amplification using four primersvia PCR amplification using (SEQ
ID NOS: 29, 30, 31, and 32) and the pCBIN-CLUCl vector and pCB-Rx_v5.4 vector
as templates, were inserted into the BamHl and Xhol sites of pCB-Rx_v5.4 using
BamHl and Xhol and the AscI and No l sites of pCB-Rx_v5.4, respectively, so as to
prepare a pCB-Rx71_v5.4 vector (refer to FIG. 11 and Table 33).
[371]
[372] Table 32
[Table 32]
Primer SEQ ID NO: Primer SEQ ID NO:
A 23 E 29
B 42 F 30
C 43 G 3 1
[373]
[374] Table 33
[Table 33]
[375]
[376] 5-17: Preparation of pCB-Rx72 v5.4
[377] A DNA fragment, which was obtained by digesting with BamHl and Xhol of a PCR
product, in which SP7.2 and an antibody light chain are linked, obtained via PCR am
plification using four primersvia PCR amplification using (SEQ ID NOS: 23, 42, 43,
and 28) and the pCBIN-CLUC7.2 vector and pCB-Rx_v5.4 vector as templates, and a
DNA fragment (refer to FIG. 10 and Table 34), which was obtained by digesting with
AscI and No l of a PCR product, in which SP2 and an antibody heavy chain are linked,
obtained via PCR amplification using four primersvia PCR amplification using (SEQ
ID NOS: 33, 34, 35, and 32) and the pCBIN-CLUC7.2 vector and pCB-Rx_v5.4 vector
as templates, were inserted into the BamHl and Xho sites of pCB-Rx_v5.4 and the Asc
I and No l sites of pCB-Rx_v5.4, respectively, so as to prepare a pCB-Rx72_v5.4
vector (refer to FIG. 11 and Table 35).
[378]
[379] Table 34
[Table 34]
[380]
[381] Table 35
[Table 35]
[382]
[383] 5-18: Preparation of pCB-Rx76 v5.4
[384] A DNA fragment, which was obtained by digesting with BamHl and Xhol of a PCR
product, in which SP7.2 and an antibody light chain are linked, obtained via PCR am
plification using four primersvia PCR amplification using (SEQ ID NOS: 23, 42, 43,
and 28) and the pCBIN-CLUC7.2 vector and pCB-Rx_v5.4 vector as templates, and a
DNA fragment (refer to FIG. 10 and Table 36), which was obtained by digesting with
AscI and No l of a PCR product, in which SP6 and an antibody heavy chain are linked,
obtained via PCR amplification using four primersvia PCR amplification using (SEQ
ID NOS: 36, 37, 38, and 32) and the pCBIN-CLUC vector and pCB-Rx_v5.4 vector as
templates, were inserted into the BamHl and Xhol sites of pCB-Rx_v5.4I and the AscI
and No l sites of pCB-Rx_v5.4, respectively, so as to prepare a pCB-Rx76_v5.4 vector
(refer to FIG. 11 and Table 37).
[385]
[386] Table 36
[Table 36]
[387]
[388] Table 37
[Table 37]
[389]
[390] 5-19: Preparation of pCB-Rx77 v5.4
[391] A DNA fragment, which was obtained by digesting with BamHI and Xhol of a PCR
product, in which SP7.2 and an antibody light chain are linked, obtained via PCR am
plification using four primersvia PCR amplification using (SEQ ID NOS: 23, 42, 43,
and 28) and the pCBIN-CLUC7.2 vector and pCB-Rx_v5.4 vector as templates, and a
DNA fragment (refer to FIG. 10 and Table 38), which was obtained by digesting with
AscI and Noil of a PCR product, in which SP7.2 and an antibody heavy chain are
linked, obtained via PCR amplification using four primersvia PCR amplification using
(SEQ ID NOS: 39, 40, 41, and 32) and the pCBIN-CLUC7.2 vector and pCB-Rx_v5.4
vector as templates, were inserted into the BamHI and Xhol sites of pCB-Rx_v5.4 and
the AscI and Noil sites of pCB-Rx_v5.4, respectively, so as to prepare a pCBRx77_
v5.4 vector (refer to FIG. 11 and Table 39).
[392] Table 38
[Table 38]
[393]
[394] Table 39
[Table 39]
[395]
[396] Example 6: In vitro secretion efficacy test of antibody expression plasmid vectors
[397]
[398] Each of the antibody expression plasmid vectors prepared in Example 5 is con
structed such that a mouse-human chimeric IgGl type monoclonal antibody is secreted
to the outside of a cell. In order to examine the extracellular secretion efficacy of a
signal sequence in an in vitro cell culture system, the monoclonal antibody was
transformed in a CHO cell, and then the secretion level of the monoclonal antibody
was examined via ELISA.
[399] Specifically, each of the antibody expression plasmid vectors prepared in Example 5
was transformed into a CHO cell, which was cultured in a Dulbecco's modified Eagle's
medium (DMEM, manufactured by GIBCO-BRL Corporation) containing 10% of
heat-inactivated fetal bovine serum (FBS, manufactured by GIBCO-BRL Corporation),
using Lipofectamine™ 2000 (Invitrogen, Cat.#: 11668-019). One day before the trans
formation, 5 x 106 CHO cells per each dish were cultured using phi- 100 dishes (Falcon
Corporation), and on the next day, tube 1 ( 1 dish reaction amount) filled with 36 ng of
16 different types of plasmid vectors, in each of which was inserted with a lucif erase
gened, and 1.5 mL of Opti-MEM ®I (invitrogen, Cat.# 31985-070), and tube 2 ( 1 dish
reaction amount) filled with 90 of Lipofectamine™ 2000 and 1410 of Opti-
MEM®I, were respectively left at room temperature for 5 minutes, and then the two
tubes were mixed to react at room temperature for 20 minutes. The mixture was added
to the CHO cells in 5 mL of Opti-MEM ®I in a volume of 3 mL and cultured in an
incubator (5% C0 2) at 37°C for 3 hours, and then the DMEM culture medium
containing 20% FBS was put into each dish by 5 niL and cultured for 8 days. On the
2nd, 4th, 6th, and 8th day after the transformation, the culture medium in each dish
was collected as a sample in a volume of 500 ul, respectively, stored at 20°C, and then
all dissolved on the 8th day, transferred into an assay plate in a volume of 100 and
subjected to ELISA assay.
[400] The ELISA assay was performed at 4°C using an O/N-coated 96-well plate and an
anti-human Kappa Light chains-peroxidase (A7164-lmL, sigma) under the condition
that F(ab')2 fragments of goat anti-human IgG and Fc gamma fragment specific (Pierce,
3 1163) were set to 0.2 ug/mL, respectively.
[401]
[402] As a result of measurement of antibody secretion level via ELISA assay, as shown in
FIG. 12, it was found that the secretion level of the expression vector pCB-Rx71_v5.4
including signal sequence SP7.2 encoding an amino acid sequence of SEQ ID NO: 1
derived from an LBFL313 gene was high. Particularly, it was found that the amount of
secretion of a combination, in which signal sequence SP7.2 is linked to an antibody
light chain and a signal sequence SP1 encoding an amino acid sequence of SEQ ID
NO: 3 is linked to an antibody heavy chain, was significantly high, and that the amount
of secretion thereof increased with the increase in culture time. Accordingly, it was
found that, when signal sequence SP7.2 was used, the secretion level of the com
bination was very high even in a long-term culture compared to the result of the luciferase
secretion test, in which the secretion level of luciferase was high in a shortterm
culture.
[403] From the foregoing, those skilled in the art will appreciate that many variations and
modifications can be made to the exemplary embodiments without substantially
departing from the principles of the present invention. Therefore, the disclosed
preferred embodiments of the invention are used in a generic and descriptive sense
only and not for purposes of limitation.

Claims
A protein secretion factor having an amino acid sequence of SEQ ID
NO: 1 or SEQ ID NO: 2.
The protein secretion factor according to claim 1, wherein the protein is
an intrinsic protein or a foreign protein.
The protein secretion factor according to claim 1, wherein the protein is
selected from the group consisting of human growth hormones, serum
proteins, antibodies, immunoglobulins, cytokines, -, b- and g -
interferons, colony-stimulating factors (GM-CSF), platelet-derived
growth factors (PDGF), phospholipase-activating protein (PLAP)s,
insulins, tumor necrosis factors (TNF), growth factors, hormones, cal
citonins, calcitonin gene related peptides (CGRP), enkephalins, so
matomedins, erythropoietins, hypothalamic secretion factors,
prolactins, chronic gonadotropins, tissue plasminogen activators,
growth hormone releasing peptides (GHRP), thymic humoral factors
(THF), asparaginases, arginases, arginine deaminases, adenosine
deaminases, peroxide dismutases, endotoxinase, catalases, chymotrypsins,
lipases, uricases, adenosine diphosphatases, tyrosinases,
bilirubin oxidases, glucose oxidases, glucosidases, galactosidases, glucocerebrosidases,
and glucourodinases.
An expression cassette, comprising (i) promoter and (ii) a nucleic acid
sequence encoding a protein secretion factor having an amino acid
sequence of SEQ ID NO: 1 or SEQ ID NO: 2, which is linked to a gene
encoding a target protein.
A vector for secretory expression of target protein, comprising an ex
pression cassette comprising a nucleic acid sequence encoding a protein
secretion factor having an amino acid sequence of SEQ ID NO: 1 or
SEQ ID NO: 2, which is linked to a gene encoding a target protein.
The vector according to claim 5, wherein the protein is selected from
the group consisting of human growth hormones, serum proteins, an
tibodies, immunoglobulins, cytokines, -, b- and g -interferons, colonystimulating
factors (GM-CSF), platelet-derived growth factors (PDGF),
phospholipase-activating protein (PLAP)s, insulins, tumor necrosis
factors (TNF), growth factors, hormones, calcitonins, calcitonin gene
related peptides (CGRP), enkephalins, somatomedins, erythropoietins,
hypothalamic secretion factors, prolactins, chronic gonadotropins,
tissue plasminogen activators, growth hormone releasing peptides
WO 2015/167278 PCT/KR2015/004389
(GHRP), thymic humoral factors (THF), asparaginases, arginases,
arginine deaminases, adenosine deaminases, peroxide dismutases, endotoxinase,
catalases, chymotrypsins, lipases, uricases, adenosine
diphosphatases, tyrosinases, bilirubin oxidases, glucose oxidases, glucosidases,
galactosidases, glucocerebrosidases, and glucourodinases.
[Claim 7] The vector according to claim 5, further comprising an expression
cassette comprising a nucleic acid sequence encoding a protein
secretion factor having an amino acid sequence selected from the group
consisting of SEQ ID NOS: 1 to 8, which is linked to a gene encoding a
target protein.
[Claim 8] The vector according to claim 5, wherein the vector is for secretory ex
pression of antibody.
[Claim 9] The vector according to claim 8, wherein the vector comprises:
a) a first expression cassette comprising a nucleic acid sequence
encoding a protein secretion factor having an amino acid sequence of
SEQ ID NO: 1 or SEQ ID NO: 2, which is linked to a gene encoding an
antibody light chain; and
b) a second expression cassette comprising a nucleic acid sequence
encoding a protein secretion factor having an amino acid sequence
selected from the group consisting of SEQ ID NOS: 1 to 8, which is
linked to an antibody heavy chain.
[Claim 10] The vector according to claim 9, wherein the protein secretion factor of
b) has an amino acid sequence of SEQ ID NO: 3.
[Claim 11] The vector according to claim 9, wherein the protein secretion factor of
a) has an amino acid sequence of SEQ ID NO: 1, and the protein
secretion factor of b) has an amino acid sequence of SEQ ID NO: 3.
[Claim 12] The vector according to claim 9, wherein the antibody light chain has
an amino acid sequence of SEQ ID NO: 48, and the antibody heavy
chain has an amino acid sequence of SEQ ID NO: 49.
[Claim 13] A transformed cell, comprising the vector according to any one of
claims 5 to 12.
[Claim 14] The transformed cell according to claim 13, wherein the cell is an
animal cell.
[Claim 15] The transformed cell according to claim 14, wherein the animal cell is a
Chinese Hamster Ovary (CHO) cell.
[Claim 16] A method of producing a target protein, comprising:
i) culturing a transformed cell, into which the vector according to any
one of claims 5 to 12 is introduced, to express a target protein and
WO 2015/167278 PCT/KR2015/004389
secrete the target protein to the outside of the cell; and
ii) recovering the target protein from a culture or culture supernatant of
the cell of step i).
[Claim 17] The method according to claim 16, further comprising purifying the
recovered target protein.
[Claim 18] The method according to claim 16, wherein the cell is a Chinese
Hamster Ovary (CHO) cell.