Note: Descriptions are shown in the official language in which they were submitted.
'91-09-~ 17:41 ION FRTEhlT 03-854-6280 T-156 P.03
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1
i.,~ ',.. ,.. __ . v~ ~-~
TITLE OF THE INVENTION
Expression Plasmids
BACKGROUND Of THE INVENTION
This invention relates to novel expression plasmids
containing SV9() replication origin and DNA fragment having
tha_ promoter. region of a human pol,ypeptide chain elongation
factor gene.
With the advance of gene engineering studies, production
of substances by recombinant DNA technology has come into a
common rneans. Methods hzwe been established almost
Completely for the production of foreign proteins by means of
recombinant DNA technology using E, coli as the host cells.
The use of E. Gol.i, however, is still inappropriate for the
proCluction of some kinds of proteiris which essentially
require the addition of sugar chains or proteins whose
physiological activities or antigenicities are altered when
they are produced in a different cell type.
1r''or the purpose of solving such problems, various host-
vector systems have been developed using animal cells. In
general, three signals are required for the gene expression
in ana.mal c~rlls; that is, promoter, RNA splicing signals and
polyadenilation signals. It is important to select efficient
promoter for high expression of the gene for a protein of
interest, Promoters which are being used incJ.ude the SV40 (a
papovavirus) early promoter, adenovinus major late promoter
and metallothionein promoter originated from mouse and the
like. The SV40 early promoter is being used frequently, but
' 91-09-0E ~\': 42 I OPJ FRTENT 03-864-6280 T-156 P. 04
na ,.~ _. ~ ',' .,' r.
2
this promoter still has the disadvantage of low expression
efficiency anti narrow host cell range. In other words,
tissue-specific expression and cell type-dopandent chsnc~es in
the expression efficiency are unavoidable even if the SV90
early promoter iN used. For oxample, the expression
efficiency is remarkably low in lymphoid cells and nerve
Gells Compared t0 Other Cell types.
Recently, Y. Takebe et al. (Mol. Cell. Biol., vol. 8, p.
966, 1988) have constructed an SROC promoter by incorporating
a portion of the terminal repeat sequence of human T-Cell
leukemia virus type 1 into downstream of the SV40 early
promotez. Accordirig to the report, expression of the
downstream gene of the SR(7C promoter was 1 or 2 orders of
magnitude more ef~ic.ient than that of the SV40 early promoter
when a certain kind of lymphoid cells was used as the host
Dells. However, it is still unclear whether the SROI, promoter
Gan maintain its high expression efficiency in other host
cells. Tf the diversity o.f useful physiologically active
substances which will be produced in the future by means of
recombinant DNA technology is takQn into consideration, it is
necessary to obtain a new promoter that shows high expression
efficiency in wider range of host cells and to develop
expression plasmids containing such a promoter.
Taking the above-described situation of the prior art in
con:cideration, one of the present inventors has performed
studies on the sc_reenang of novel expression plasmids which
could show high expression efficiency in wide range of host
'91-09-0( 7:44 ION FRTEhlT 03-864-6280 T-156 P.05
___. .....
3
cells. Through these studies, one of the present inventors
has isolated a chromosomal gene encoding human polypQptidc~
chain elongation factor-1cx (to be referred to as human Ef-10t
hereinafter.) which is constitutively produced in all human
cells and has determined its nucleotide sequence fox the
first time(T Uetsuki, et al. ~J. Biol. Chem. Vol. 264,
p.5791, 19$9) .
After that, one of the present inventors found that the
DN1~ fragment containing the human EF-1Cc promoter region
stimulated the expression of its downstream gene, constructed
high expression plasmids containing said DNA fragments
(D.w,Kim, et,al.jGene, vo1.91, p.217, 1990) and have filed
the patent application in Canada Serial No. 2,005,016.
A tz::;nsient expression system, especially in which COS
cells are used, is commonly employed when an attempt is made
to clone a new gene by means of a biological assay using its
activity as a marker. Such a transient expression system has
an advantage in that a protein of interest can be obtained
easily within a short period of time for use in the analysis
o~ function and structure of the expressed protein. In spite
of such an advantage, however, only a few expression plasmids
are available from which, when used in the transient
ex~~ressi,c~ri system, an expressed product can be obtained in
such a large amount that the product can be detected even by
a low sensitivity biological assay system, zn addition to
that. very little actually is known about an expression
' 91-09--(\.7: 45 I ON PRTENT 03-864-6280 T-156 P. 06
r
.-. .. . .._ ,
9
plasmid which shows high caxpression efficiency in broad range
of host cells.
SUMMARY OF THE INVENTION
Taking snCh prior art problems into consideration, the
inventors o:~ the present invention have conduct~ad studies on
the combined effects of various promoters with DNA fragments
which impart effects of improving expression efficiency, with
the aim of developing expression plasmids that show high
expression efficiency in transient expression systems. As
the results, the present inventors have found for the first
time that novHl expression plasmids containing a human EF-lOc
promoter region and simian virus 40 (SV40> replication origin
can show high expression efficiency in broad range of host
cells in the transient expression systems in comparison with
prior art ~xpression plasrnids. The present invention has
been accomplished as a result of these efforts.
First aspect of the present invention provides
expression plasmids having at least SV40 replication origin
and a DNA fragment containing a promoter region for a human
polypeptide chain elongation factor gene.
The human polypeptide chain elongation factor gene. may
be the human polypeptide chaitl elongation factor--ltx gene .
7.'he DNA fragment may comprise at least a portion or a
whole of a region of about 2.5 kilo base pairs which is
located immediately upstream of the translation initiation
colon of the human polypeptide Cha3,n eJ.ongation factor gene.
' 92-09-0E ' /: 46 I ON FpTEtJT 03-864-6280 T-156 P. 07
The DNA fragment preferably contains a
promoter region
of human. EF'-10c gene comprising at least
a portion including
TATA bax [underlined position in sequencethe UNA
(1)]of
fragment represented the following sequence
(T):
CCCGGGCTGGGCTGAGACCCGCAGAGGAAGACGCTCTAGG 90
GATTTGTCCCGGACTAGCGAGATGGCAAGGCTGAGGACGG 80
GAGGCTGATTGAGAGGCGAAGGTACACCCTAATCTCAATA 120
CAACCTTTGGAGCTAAGCCAGCAATGGTAGAGGGAAGATT 160
CTGCACGTCCCTTCCAGGCGGCCTCCCCGTCACCACCCCC 200
CCCAACCCGCCCCGACCGGAGCTGAGAGTAATTCATACAA 240
AAGGACTCGCCCCTGCCTTGGGGAATCCCAGGGACCGTCG 280
TTAAACTCCCACTAACGTAGAACCCAGAGATCGCTGCGTT 320
CCCGCCCCCTCACCCGCCCGCTCTCGTCATCACTGAGGTG 360
GAGAAGAGCATGCGTGAGGCTCCGGTGCCCGTCAGTGGGC 900
AGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGA 440
GGGGTCGGCAATTGAACCGGTGCCTAGAGAAUGTGGCGCG 980
GGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCT 520
TTTTCCCGAGGGTGGGGGAGAACCGTATAT~AGTGCAGTA 560
GTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCA 600
GAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGG 690
CCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACT 680
TCCACGCCCCTGGCTGCAGTACGTGATTCTTGATCCCGAG 720
CTTCGUGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCG 760
' 91-09-0E ~.~~ ~' : 47 I OIJ FRTENT 03-854-6280 T-156 P. 08
,. ,_. . _ .. ,. ..'
6
CTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTG 800
GCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCAC 840
CTTCGCGCCTGTGTCGCTGCTTTCGATAAGTCTCTAGCCA 880
TTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTG 920
GCAAGATAGTC~'TGTAAATGCGGGCCAAGATCTGCACACT 960
GGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCC1000
GTGCGTCCCAGCGCACA~1~GTTCGGCGAGGCGGGGCCTGCGi090
AGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCT1080
GGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTAl120
TCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACC1160
AGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCT1200
GCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGC1240
GGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCC1280
GTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGG1320
GCGCCGTCCAGGCACCTCGA'rTAGTTCTCGAGCTTTTGGA1360
GTACG~!'CGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGAT1400
GGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGG1990
CCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCC1480
TT'~TTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGAC~52O
AGTGGT'~CAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA1560
A 1.561 _-
It is known commonly that chr.pmosomal nucleotide
sequences, exeludinc~ stxu4tural gene, vary from one another
CCTCTTTACGGGTTATGGCC
CA 02051085 1999-02-15
7
slightly depending on the host cells, mutation and the like,
without altering the main activity. Accordingly, it is
intended to include within the scope of the present invention
all variations of the nucleotide sequence of SV40 replication
origin or the DNA fragment represented in sequence (I),
wherein the nucleotide sequence is slightly modified by
artificial mutation, insertion, deletion and the like, on
condition that the function of these variants is the same as
that of SV40 replication origin or the DNA of the promoter
region of human EF-la gene.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a graph showing a process for the
construction of an expression plasmid pEF-SV-CSF.
Fig. 2 is a graph showing a process for the
construction of an expression plasmid pEF-BOS.
Fig. 3 is a graph showing the structure of pEF-BOS.
Figs. 4-7 show the construction of several plasmids
described in Figs. 4-7 of Laid-open Canadian Patent
Application No. 2,005,016.
DETAILED DESCRIPTION OF THE INVENTION
Expression plasmids of the present invention can be
prepared in the following manner.
Human EF-la chromosomal gene having promoter region
may be obtained by Southern hybridization method from a human
gene library using appropriate probes. The gene library
constructed from human fetal liver (R. M. Lawn et al.; Cell,
vol. 15, p. 1157, 1978), human placenta (H. Matsushime et al.;
Mol. Cell. Biol., vol. 6, p. 3000, 1986) and the like can be
72736-61
CA 02051085 1999-02-15
8
used as the human gene library.
The human EF-la promoter region may also be obtained
in accordance with the method of T. Uetsuki et al. (J. Biol.
Chem., vol. 264, p. 5791, 1989) or by chemically synthesizing
it based on the report of Uetsuki et al.
According to the method of T. Uetsuki et al. (J.
Biol. Chem., vol. 264, p. 5791, 1989), human EF-la cDNA is
isolated from cDNA library constructed with mRNA from human
fibroblast GM637 cells by using yeast EF-la cDNA as a probe.
To isolate human EF-1a gene, above human gene libraries are
screened using the obtained human EF-la cDNA as a probe. It
is known that human chromosomes are possessed of pseudogenes
each of which having the same DNA sequence of a certain gene
but showing no function. For the purpose of avoiding cloning
of the pseudogene, sequence of 3'-non-coding region of the
human EF-la cDNA can be used as the probe. Then Southern
hybridization analysis and nucleotide sequencing are carried
out to confirm the isolated gene is the active gene of human
EF-la but not a pseudogene.
The transcription initiation site is determined by
primer extension method using an mRNA extracted from a human
cell line HL-60 cells and a synthetic oligonucleotide which is
complementary to 5' region of the obtained human EF-la cDNA.
The promoter region of human EF-1a contains typical TATA box
located at about 30 nucleotides upstream of the transcription
initiation site.
Preferably, however, the DNA fragment of interest
may be isolated from some kinds of plasmids in which human
72736-61
CA 02051085 1999-02-15
8a
EF-la promoter region has already been incorporated, such as
plasmid pEF-321, pEF-204, pEF-223 or pEF-220 prepared by D. W.
Kim et al. (Gene, vol. 91, p. 217, 1990; see Figs. 4-7 of this
application which are described in Figs. 4-7 of Laid-open
Canadian Patent Application No. 2,005,016). Of these, plasmid
pEF-321 may be most convenient from high expression efficiency
point of view.
Four transformants were obtained by transforming an
E. coli strain with each of these four plasmids introduced
chloramphenicol acetyl transferase (CAT) gene into the
downstream of the promoter region. These transformants, named
E. coli DH5 (pEF220-CAT), E. Coli DH5 (pEF223-CAT), E. coli
DH5 (pEF204-CAT) and E. coli DH5 (pEF321-CAT), have been
deposited on March 2, 1989, in Fermentation Research
Institute, Agency of Industrial Science and Technology, and
have been assigned the designations as FERM P-10595, FERM
P-10596, FERM P-10594 and FERM P-10597, respectively.
Any DNA fragment may be useful as the starting
material for the construction of expression plasmids of the
present
72736-61
'91-~l9-86 ,:59 ION PATENT ~~,W3-864-628lJ T-156 P. 11
9
invention, provided that it. is possessed of the same
~itxr_leotide sequence as that of the human EF-1CC promoter
region contained in the plaemid pEF~321. Tha DNA fragment
containing human EF-loc promoter region comprises 2.5 kilo
base paixs and contains a DNA fragment represented by the
fpllowing nucleotide sequence (I).
CCCGGGCTGGGCTGAGACCCGCAGAGGAAGACGCTCTAGG 90
GArl"T".~G~'CCCGGACTAGCGAGATGGCAAGGCTGAGGACGG 80
GAGGCTGATTrAGAGGCGAAGGTACACCCTAATCTCAATA 120
CAACCTTTGGAGCTAAGCCAGCAATGGTAGAGGGAAGATT 160
C'fGCACGTCCCTTCCAGGCGGCCTCCCCGTCACCACCCCC 200
CCCAACCCGCCCCGACCGGAGCTGAGAGTAATTCATACAA 240
AAGGACTCGCCCCTGCCTTGGGGAATCCCAGGGACCGTCG 280
T'rAAACTCCCACTAACGTAGAACCCAGAGATCGCTGCGTT 320
CCCGCCCCCTCACCCGCCGGCTCTCGTCATCACTGAGGTG 360
GAGAAGAGCATGCGTGAGGCTCCGGTGCCCGTCAGTGGGC 900
AGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGA 990
C-:GGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCG 9$0
GGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCT 520
TTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTA 560
GTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCA 600
GAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGG 690
CCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACT 680
TCCACGCCCCTGGCTGCAGTACGTG~1TTCTTGATCCCGAG 720
'91-09-05 .~:51 ION PATENT ~ , 03-854-6280 T-156 P.12
Zo
CTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCG 760
CTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTG 800
GCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGG'1'GGCAC 890
CTTt:GC'GCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCA 880
TTTAAAATTTTTGATGACCTGCTGCGACGCT'PTTTT1'CTG 920
GCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACT 960
GGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCC1000
GTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCG1090
AGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCT1080
GGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTA1120
TCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACC1160
AGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCT1200
GCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGC1240
GGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCC1280
GTCCTCAGCCG'1'CGCTTCATGTGACTCCACGGAGTACCGGi320
C,CGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGA1360
GTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGAT1900
GGAGTTTCCCCACACTGAGTGGGTGGAGAGTGAAGTTAGG1440
CCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCC1480
TTTTTG,AGT.TTGGA~'CTTGGTTCATTCTCAAGCCTCAGAC1520
AGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA1560
A 15 61 --- ( z )
' 91-09-~, '.7: 52 lOtJ FF1TENT ' '«.' 03-854-6280 T-156 P. 13
,.
When plasmi.d pEF-321-CAT is used as the starting
material, expression plasmids which show high expression
eff3.cienr_y in transient expression systems can be obtained by
inserting a DNA fragment containing SV90 replication origin
into an appropriate site in each of the expression plasmids.
'lhe SV90 replication origin can be obtained by digesting
any plasmids containing the replication origin with,
appropriate restriction enzymes. For example, an SV40 DNA
fragment (311 bp) containing SV40 replication origin can be
obtained by digesting plasmid pMLSV.
According to some of the expression plasmids of the
present invention, a portion of the upstream Side of the DNA
fragment containing human EF-1a, promoter region may be
removed and replaced by a DNA fragmen~ containing SV40
replication origin, provided that function of the human EF"-1d
promoter is net altered by the removal of the upstream
portion. For example, an upstream portion of the DNA
fragment containing human EF-lOC promoter region, namely a
SphI-Sphl DNA fragment of about 1.3 kilo base pairs, can be
replaced by a DNA fragment containing SV90 replication
origin. In this case of thA expression plasmid, the fragment
containing human EF-1a, promoter. region comprises a nucleotide
sequence represented by the following sequence (zz).
373 CGTGAGGCTCCGGTGCCCGTCAGTGGGC 900
AGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGA 990
GC~GGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCG 480
'91-0q-06 .:53 IOI-1 FATENT ,: J3-864-6280 T-156 F.14
,.. .. . ... .' ,~_,;:
12
GGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCT 520
TT'I'TCCCGRGGGTGGGGGAGAACCGTATATAAGTGCAGTA 560
GTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCA 600
GAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGG 690
CCTCTTTACGGGTTATGGCCCTTGCG'I'GCCTTGAATTACT 680
TCCACGCCCCTCYGCTGCAGTACGTGATTCTTGATCCCGAG 720
CTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCG 760
G'PTAAGGAGCCCC'TTCGCCTCGTGCTTGAGTTGAGGCCTG 800
GCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCAC 890
CTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCA 880
TTTAAAAT'TTTTGATGACCTGCTGCGACGCTTTTTTTCTG 920
GCAAGATAGTC:TTGTAAATGCGGGCCAAGATCTGCACACT 960
GGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCC1000
GTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCG1090
AGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCT1080
GGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTA1120
TCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGG'PCGGCACC1160
AGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCT1200
GCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGC1240
GGGCGGGTGAGTCAC:CC:ACACAAAGGAAAAGGGCCTTTCC1280
GTR'.CTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGG1320
GCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGA1360
GTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGAT1900
'91-09-~_ 17:54 ION PRTENT 03-8b4-6c80 T-156 P.15
.. ... ..
13
GGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGG1440
CCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCC7.480
TTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGAC1520
AGTGGTTCAAAGTTTTTTTCTTCCATTTCAGG'.CGTCGTGA1560
A 1561 ---(II)
For the purpose of exhibiting function of the expression
plasmid o:~ the present z.nvention in mammalian cells, it is
necessary to add a polyadenyl.ation signal to the plasmi.d.
For this purpose, the SV40 polyadenylation signal contaic~ed
in plasmid pEF-321--CAT may be used conveniently, as well as
those in other genes, such as the polyadenylation signal in
human granulocyte-COloriy stimulating factor (G-CSF).
In addition, the expression plasmid of the present
invention may preferably have appropriate restriction enzyme
recognition sates downstream of its promoter region, so that
a gene to be expressed or cloned can be inserted easily into
downstream of the promoter region. For this purpose, various
cloning site-containing DNA fragments can be used, such as a
stuffer frsgment of plasmid CDMB. This stuffer fragment has
an advantage in that self-ligation does not occur when BstXT
reCOgnit i.On SINS COIItalIled in the fragment are used in
cloning. The stuf~fer fragment can be isolated from plasmid
Ct7hi8 as a ~Ybal-XbaI DNA fragment (451 bp) .
when the expression plasmid contains a recognition site
;identical to its cloning Site, the object of the present
.,_..,
'91-09-06 :55 ION PRTENT 03-864-6280 T-156 P.16
14
i.nv~ention can be attained by removing the recognition site.
Even when such a recognition site is found in a DNA fragment
containing human E>~-1a promoter region, a DNA fragment
containing SV90 replicat.~.on origin or a DNA fragment
containing a polyadenylation signal, the recognition site can
be removed with no inconvenience as long as basic functions
o~ these pNA fragmonts do not change by the removal. For
example, removal of Bst.XI recognition site from a DNA
fragment containing human GF--la promoter region does not
alter Function of the promoter region, in this instance, the
fragment conte~i.ning human EE-1pC promoter region comprises a
nucleotide sequence represented by the following sequence
(III) .
373 CGTGAGGCTCCGGTGCCCGTCAGTGGGC 400
AGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGA 490
GGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCG 980
GGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCT 520
TTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTA 560
GTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCA 600
GAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGG 690
CC'TCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACT 680
TCCAC CTGGCTGCAGTACGTGATTCTTGATCCCGAG 720
CTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCG 760
C'TTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTG a00
~
'91-09-t. 17:56 ION FHTENT 03-864-6280 T-156 F.:17
GCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCAC 840
CTTCGCGCCTGTCxCGCTGCTTTCGATAAGTCTCTAGCCA 880
TTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTG 920
GCAAGATAGTCTTG'.L'AAATGCGGGCCAAGATCTGCACACT 960
GGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCC1000
GTGCGTGCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCG1040
AGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCT1080
GGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTA1120
TCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACC1160
AGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCT1200
GCAGGGAGCTCAAAATGGAGGACGCGGCGCTGGGGAGAGC1240
GGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCC1280
GTCCTCAGCCGTCGCTTCATGTGACTCCACGGAG'IACCGG1320
GCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGA1360
GTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGAT1400
GGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGG1990
CCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCC1480
TTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGAC1520
AGTGGTTCAAAGTTTTTT'1'CTTCCATTTCAGGTGTCGTGA1560
A 1561 ---(III)
A trr~nsformant was obtained by transforming an E. cold
strain with one of the obtained plasmids of the present
'\
' gi-99-96 : 5'? I Oh! PRTENT 93-86d-6289 T-156 P. 18
16
invention. The transformant plasmid pEF-sOS, named E. coli
DH,S (pEF-BOS), has been deposited by one of the present
inventors on September 6, 1991, in Fermentation Research
Institute, Agency of 2ndustrial Science and Technology, and
has been assigned the designation as FERM BF-359.
Llsdf_ulness of the expression plasmfd of the present
invention can be confirmed by inserting an appropriate gene
into dow.nsta=eattt of the human EF-1a, promoter region making use
of appropriate restriction enzyme recognition sites or the
like and by subsequently checking expression efficiency of
the inserted gene. As a gene to be expressed for this
purpose, CAT gene may be used preferably because its
expressed amount can be judged easily, though human G--CSF
cPNA is also useful. When CAT gene is used, the amount of
formed CAT can be measured as a formation ratio of acetyiated
chloraznpheriicol using thin layer chromatography. In the case
of G-CSF, the amount of formed product can be measured by a
biological assay technique using an appropriate cell line.
EXAMPLES
Examples of the present invention are given below by way
of .~.llustrtltion, and not by way of limitation.
Unless otherwise stated, commonly used abbreviations in
this field of studies are used in the following descriptions.
Each experiment in the following examples is based on the
common gene manipulation tachniques which can be performed in
accordance with any commonly used manual, such as r7oleculax~
u:~
' 91-09-E~~. 1~r : 58 I OtJ PRTENT 03-864-6280 T-156 P. 19
.., .~ ._ ._ . :.e ~:
17
C,lon,iug, A Laboratory Manual Second Edition (Maniatis et al.,
Cold Spring Harbar Laboratory, 1989) and Labomanual Gene
Technology (written in Japaneset M. Muramatsu, Maruzen Go.,
Ltd., 1988).
Ex~m1?~
'"Construction of expression plasmids"
(1) Q7.asm~.d pEF-SV-CSF
This plasmid was constructed in the following manner.
Hasic process Eor its construction is shown in Fig. 1.
hay EcoRI-ECORI cDNA fragment containing CAT gene was
removed from an expression plasmid pEF-321-CAT whickx has been
constructed by D.W.Yim et a1. (Gene, vo1.91, p.217, 1990).
An EcoRT~-EcoRT DNA fragment containing G-CSF cDNA was cut out
from plasmid pBR-VII and ligated with the above EcoRI-EcoRI
large fragment to obtain plasmid pEF-321-CSF, Thereafter, an
EcoRI-EcoRI DDIA fragment containing a 311 by DNA fragment in
which 5V90 replication origin is contained was inserted into
the H.indIII recognition site located upstream of EF-lcx
promoter region in the thus prepared plasmid pEF-321-CSF to
yield the titla expression plasmid pEF-SV-CSF.
(2) Plasmid pEF-BOS
A gist of the process for the construction of this
plasmid is illustrated iu Fig. 2. From the G-CSF -containing
cDNA moiety in the plasmid pEF-321-CSF prepared in Example 1-
(1), a region encoding G-CSF was removed by cutting out its
upstream portion starting from the Eco8lI recognition site,
°
91-09-06 :59 InIJ FRTENT 03-864-6280 T-156 P.20
1 J3
At the same time, a XbaT linker was inserted into the EcoRI
recognition site located between the EF-loc promoter region
and the G-CSF polyadenylation signal. In this way, a plasmid
rontaininy a XbaI recognition site was obtained. Into the
XbaI recognition site of the thus constructed new plasmid was
inserted t~ 951 by stuffer gene of plasmid CDM8 (Seed, B:
Nature, ~ro1.329, p.840, 1987).
Next, a SphT-.Sphl DNf, fragment which is located upstream
portion of the DNA fragment including the EF-loc promoter
region in the thus obtained plasmid was removed and replaced
>7y EcoRI-ECORI ONA fragment containing a 311 by DNA fragment
including SV40 replication origin in the same manner as in
Example 1-(1). Thereafter, the BstXI recognition site in the
nucleotide sequence containing EF-la, promoter region was
removed by deleting a nucleotide sequence GCCC to yield the
title e~cpxession plasmid pEF-BOS.
"Production of human GTCSF in COS cells"
Human G-CSF cDNA was inserted into BstXI site of pl;F-BOS
or CDMB, or into BamHI site of pKCR vectox containing SV40
early promoter (0'Hare,K., Benoist,C, and Breathnach,R.
(1981) P.roc. Natl. Aead. Sci. USA 78, 1527-1531). As shown
in Table A, when COS cells were transfected with these
plasmids by DEAF-dextran/chloroquine method, the construct in
pEF-BOS has directed the synthes~.s of human G-CSF alaout 20
I'91-09-c 18:00 IOh! PRTENT 03-8E4-6280 T-156 P.21
a.. ....1 :.,. .., Xt.:l.i
:'c ,'~ r.
f~,; ;r ."x .:
19
times more efficiently than the construct in CLIMB, and 50 -
200 t~.rnes more ef~icientJ.y than the construct in pKCR.
G-CSF activities were titrated by a biological assay
with mouse interleukin-3 (IL-3)-dependant cell, line NFS-60,
which can grow in the presence of G-CSF. Since the number of
surviving NFS-60 cells after addition of a sample that
contains human G-CSF correlates to the sum of DNA synthesis
in the cells. a half-maximal value of [3H]thymidine
incarporation into the cells indicates the G-CSF activity of
the sample.
Table A.
Vector G-CSF activity in mediums
(unitsb/ml)
pEF-HOS 33,000 12,000
CDMB 1, 600 1, 200
pKCR160 160
gAt 72 hrs post transfection, the G-CSF activity in the
medium was assayed.
b 1 unit of the activity corresponds to about 62 pg of human
G-CSF.
AS Shown in Fig. 3, pEF-BOS Carries the SV40 replication
origin (311 by of EcoRZI fragment), the promoter region of
human IaF-lOt c:hromosamal gene (1.2 kb), the stuffer fragment
(451 bp) from CDMB vector and polyadenylation signal from
human G-CSF cDNA (about 700 by ECO81I - ECORI DNA fragment)
(NagatR,S., Tsuchiya,M., Asano,5., Kaziro,Y., Yamazaki,Y.,
~' 91-09-OE ~ ~~: c~1 I ON FRTENT 03-864-6280 T-156 P. 22
"' ~::, .. , a ~ ,' ~ -;
~.: ~,,..s . _ _ .. ..
Yarnamoto,0., Hirata.Y., Kubota,N., Oheda,M., Npmura,H, and
Ono,lvl. (1986) Nature 319, 415-918) in HindITT-EcoRT site of
pUC119. The promoter region of EF-1a gene is from nucleotide
position 373 to 1561, as shown in the sequence (ITI), which
includes 203 by S' flanking region, 33 by the first axon, 939
by the first intron and l0bp of the part of the second axon
located at 20 by upstream of the ATG initiation cpdon. The
size of pf,f-BOS is 5.8 kb, and the cDNA to be expressed can
be inserted at bstXT site using BstXI adapter, or XbaT site
using XbaI linker.
°'Effect of SV40 replication origin on the expression in
COS cells"
Plasmids pEF-BOS, pEF-Sv and pEF-321 were compared in
terms ofi their efficiencies to express human G-CSF cDNA in
COS cells in the same manner as in Example 2. Of these
plasmids, plasmid pEF-SV contains EF-la promoter region of
2.5 kilo base paa.xs and SV40 replication origin, while
plasmid pEF-321 Contains EF-1CG promoter region of 2.5 kilo
base pairs but with no SV40 replication origin. As
summarized in Table B, plasmids pEF-SV and pEF-BOS both
containing the SV40 replication origin showed more than ten
times higher leveJ.s of G--CSF gene expression in comparison
with plasmid pEF-321 whioh contained no SV40 replication
Oric~izl.
' ~ .~w~
'A1-09-~ 18:02 I0N PRTENT 03-864-6280 T-156 P.23
r:, ',: :_? _ _ ~,; ,. , ,
21
~abl~ 8
hF-la Sv40
Vector promoter replicationG-CSF activity in mediums
region origin unitsb/ml
pEF-3212.S Kbp - 700
pEF-SV 2.5 Kbp + 10,000
pF.~_-13051 . 2 ICbp+ 12, 000
.
~: At 72 hrs post tranefection, the G-CSF activity in the
medium was assayed.
f: One unit of the activity carresponds to about 62 pg of
human G-CSF.
Fxam~nle 9
"Effect of the inventive plasmids in various cell lines"
when a CAT gene was inserted into pEF-BOS, the CAT
activit9.es observed with pEF-BOS-CAT were 1.5 -- 50 times
higher than that of pSV2-CAT or pRSV-CAT after introduction
into various cell lines including murine L929, human HeLa,
CHU-2 and simian COS cells (Table C). The pEF-BOS vector,
therefore, will be used to produce a large amount of growth
factors and proteins in mammalian cells, to express a high
level of anti-sense RNA. Furthermore, the pEE'-BOS-CAT will
be an ideal positive control gor CAT assay in various cell
types.
F
'' 91-09-06 ~ : 03 I ON PATF_I~lT 03-864-6280 T-156 P. 24
22
Table G. Promoter activitieain various cells
Relative activities
CAT
Vector L929 ~TeLa CHU-2 COS
pSV2 2.0 74.3 82.7 10.2
pRSV $,Q 8.5 19.2 22.5
p~~'-gOS 100 100 100 100
CDMB n,t,t n.t. n.t. 25.4_
~CA'r activities are presented as a percentage of that o~ thp
pEF-BOS
bn.t., nct tested.
