Note: Descriptions are shown in the official language in which they were submitted.
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PRODUCTION OF BIOLOGICALLY ~CTIVE
FORMS OF CSF USING A BACULOVIRUS (AcNPV)-
INSE~l CELL EXPR~SSION SYSTEM
The present invention relates to the field of molecular
biology and proteins. In particular, the invention relates to the
achievement of high levels of expression of biologically active colony
stimulating factors using an insect cell/baculovirus expression
system.
Numerous different expression systems are now available to
; 10 the genetic engineer who wishes to express a cloned gene of
~; interest. Generally the goal is the production of high levels of
biologically active material. The requirements for the expression
system chosen to accomplish this goal are dependent upon the nature of
the protein to be expressed.
The value of utilizing prokaryotic host vector systems for
the synthesis of desirable eukaryotic proteins is diminished by
certain limitations inherent in such systems. For instance, the mRNA
transcript or protein product of such systems may be unstable in the
prokaryote. In addition, before a protein will be synthesized within
a prokaryotic cell, the DNA sequence introduced into the microorganism
must be free of intervening DNA sequences, nonsense sequences, and
initial or terminal sequences which encode for polypeptide sequences
which do not comprise the active eukaryotic protein. Further, some
eukaryotic proteins require modification after synthesis (e.g.,
glycosylation and all membrane associated processing) to becorne
biologically active, and prokaryotic cells are generally incapable of
such modifications.
Various nonviral eukaryotic host vector systems are also
available for the expression of heterologous proteins. Certain
limitations are inherent in each of these systems as well. For
example, high levels of expression are frequently difficult to obtain
in yeast systems where autonomously replicating vectors may be
unstable. Additionally, glycosylation patterns in yeast differ from
those in higher eukaryotes.
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Limitations encountered with mammalian host vector systems
include dif~iculties in host cell culturing and its scale-up. The
expense of mammalian cell culture media as well as a requirement ~or
serum often restricts its use on a large scale and complicates the use
of these systems for production of parenterally administered
pharmaceuticals. Furthermore, levels of expression in these systems
are generally substantially lower than that obtained in prokaryotic or
viral expression systems.
The use of viruses in eukaryotic host-vector systems has
been the subject of a considerable amount of recent investigation and
speculation. However, some viral vector systems also suffer from
significant disadvantages and limitations which diminish their
utility. For example, a number of eukaryotic viral vectors are either
tumorgenic or oncogenic in mammalian systems and create potentially
health and safety problems associated with resultant gene products and
accidental infection.
The baculovirus expression vector system involved in the
instant invention overcomes many of the above-mentioned limitations.
Baculoviruses are insect pathogenic viruses which, until recently9
were studied mostly for their potential use as viral insecticides for
control of agriculturally important insect pests. Because certain
baculoviruses are highly virulent for pest insects, some of the most
promising have been commercially developed and are used as bio10gical
pesticides (Miltenburger and Krieg 1984 Bioinsecticides:II:
Baculoviridae. Adv. Biotechnol~ Proces_es 3:291; Granados, R.R. and
Federici, B.A. eds. The Biol. o~ Baculoviruses Vol II, Boca Raton, FL:_
CRC Press, Inc. 1986). Baculoviruses are very stable and are able to
persist for longer times in the environment than other animal
viruses. This unusual biological stability is the result of a unique
association of the infectious virus particles and a viral occlusion
which is a crystalline assembly of a viral encoded structural protein
called polyhedrin. Late in viral replication, baculovirus particles
become embedded in a protein occlusion composed of the polyhedrin
protein. Insects acquire a baculovirus disease by ingesting these
occluded virus (0~) which contaminate their food supply. The
1~72~4
polyhedrin matrix protects ~he virus particles ;n the environment and
during their passage through the foregut of the insect. In the insect
midgut, the alkaline pH activates the dissolution of the polyhedrin
crystalline matrix resulting in the release of many viruses. The
virus become absorbed by the midgut epithelial cells and initiate the
infect;on process.
There is a second infectious form of nuclear polyhedrosis
viruses (NPVs), known as the extracellular or nonoccluded virus (NOV)
form, which is generated by the budding of viral nucleocapsids through
the plasma membrane of the infected cells. NOVs are responsible for
spreading a secondary infection via the hemolymph of the insect. It
is the NOU form of the virus which is infectious in insect cell
cultures; the occluded (OV) form is not infectious in cell cultures
since dissolution of the crystalline matrix occurs only at high
alkaline pH (i.e., pH 10~5) ~
The Formation of NOVs and OVs occurs in a biphasic manner
during the infection process. NOVs are abundantly produced before
occlusion is initiated. During a typical synchronous infection of
fully permissive cell lines, the majority of NOVs are produced between
12 and 24 hr post-infection (p.i.). The synthesis of polyhedrin is
initiated at 20 hr p.i. and does not reach maximal levels until 48 to
72 hr p.i. The significance of this temporal regulation with respect
to the expression vector system is that foreign gene products that may
have adverse effects on the cell should not diminish the production of
progeny NOVs to be used for further infection.
Of the 450~500 species of known baculoviruses, practically
all encode for a polyhedrin protein. As previously discussed, the
viral occlusion is a paracrystalline assembly of a polyhedrin monomer
which, for most viruses, has an average molecular weight of 28~000-
30,000 daltons (Summers, M.D. and Smith, G.E., 1978 Virolo~y
84 390)o Baculoviruses are unique among animal viruses, not only in
the protective function of the viral occlusion in the viral life cycle
but also because the polyhedrin gene is the most highly expressed
eucaryotic virus gene known. The polyhedrin protein can accumulate to
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greater than 1 mg/ml of infected cul tured insect cells (70-75% of the
total cellular protein) or can comprise up to 25% of the total protein
of an infected insect. Although very highly expressed, nei ther the
polyhedrin gene nor its protein is essential for viral infection or
replication in cultured insect cells or insects, thus making the
polyhedrin gene an ideal target for genetic manipulation.
The most extensively studied baculovirus is the Auto~raeha
californica nuclear polyhedrosis virus (AcNPV). The Autographa
californica host for AcNP\~ is a moth commonly referred to as the
alfalfa looper. Studies of the physical and functional organization
of the AcNPV genome have resulted in the mapping, cloning, and
sequencing of the AcNPV polyhedrin gene and its regulatory sequences
(Iddekinge et al. (1983) Virology 131:561; Smith et al. (1983) J.
Virol. 46:584). Not only does the polyhedrin gene exhibit a strong
promoter, but expression can continue late in infection well beyond
the point of repression of nearly all other baculovirus and host
genes. I
The genetic engineering of the bacul ovirus polyhedrin gene
for high level expression of a heterologous protein, in this case
recombinant human beta-interferon was first reported by Smith et al.
Mol. Cell Biol. 3(12):2156-2165 (1983)). Since then, human ,,
interleukin-2 has been expressed in insect cells by a baculovirus
expression vector as described by Smith et al. (Proc. Natl Acad. Sci.
USA 32:8404-8408 (1985)). Recently, the synthesis of functional human
T-cell leukemia virus Type I p40x protein using a baculovirus
expression vector has been reported (Jeang, K.T., et al., J. Virol.
61:708-713 (1987)). Other heterologous proteins that have been
expressed in this system are summari~ed in Summers et al. ("Genetic
Engineering of the Genome of the Auto~apha californica Nuclear
Polyhedrosis Uirus" Banbury Report: Genetically Altered _iruses in
the Environment, 22:319-329, Cold Spring Harbor Laboratory (19~5)).
The bacul ovirus expression system has several advantages for
the expression of foreign genes in comparison to other prokaryotic,
yeast or Inammalian cell expression vector systems. First, high levels
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of expressed proteins are possibl e. ~reater than 1.0 mg per ml of
polyhedrin protein is nonnally produced in infected cells. Another
non-essential occlusion-related viral protein, plO, is also abundantly
produced and its promoter has reportedly been used to drive forei gn
gene expression ~D.W. Miller et al. Genetic En~ineering Principles &
Methods 8:277-298, Setlow and Hollaender, eds. New York: Plenum Press,
. _
1986). Using the polyhedrin gene promoter, heterologous gene
expression levels never reach polyhedrin levels but are usually in the
range of tens to hundreds of micrograms per ml (Summers et al. ~198S),
p. 321, supra). Secondly, in contrast to those produced in bacterial
or yeast cells, recombinant proteins produced in insect cells may be
co- and post-translationally processed in a manner similar to what
occurs in mammalian cells. In at least one case, glycosylation of
IFN-beta in infected insec~ cells has been reported (G.E. Smith et al.
(1983) supra). Whereas about 40% of the natural iL-2 produced in
human Jurkat cells is not glycosylated, there was no evidence of any
glycosylation of the recombinant IL-2 produced in insect cells ~G.E~
Smith, et al., (1985), supra). In addition, correct cleavage of
mammalian secretory signal peptides has been obser~red (G.E. Smith et
al., (1983), supra; G.E. Smith et al., (1985), supra; D.W. Miller et
al., (1986), supra.
As described by M. B. Ladner et al., (EMBO J. (1987) 6:2683-
2698, - the production of
biologically active CSF-l is complicated by its high degree of
posttranslational processing which includes glycosylation and
dimerization. In addition there are a large number of cysteine
residues, in particular, in the N-terminal portion of the protein.
There are, in fact, a total of ten cysteine residues in the "long
form" of CSF and seven in the "short form". Both thus contain
cysteine residues at positions 7, 31, 48, 90, 102, 139, 146, the long
form has additional cysteines at 157, 159 and 225. It is belieYed
that processing to form dimer includes formation of multiple
intrachain and at least one interchain disulfide bond(s). Ilhile
prokaryotic systems may be employed for CSF-1, refolding protocols are
3s necessary to produce high yields of biologically active product. It
~',
1 3 ~ r~? 2 1 ~
would be desirable to have a high level expression system that
produces biologically active dimers of CSF-1.
As described in PCT Publication No. W088/01297 published
February 25, 1988, ~ - the native
DNA sequence encoding G-CSF has not proved to be effective for the
production of high levels of G-CSF in prokaryotic hosts. Not only
does the insect cell expression system provide an intriguing
alternative but the possibilities of achieving the appropriate post-
translational processing and recovery of G-CSF from the culture medium
would be advantageous in the production of pharmaceutically useful
CSFs.
The invention relates, in one respect, to methods for
producing by recombinant DNA technology biologically active CSFs and
in the case of CSF-l, a dimerized form of CSF-1~ using host insect
cells infected with a recombinant baculovirus expression vector.
Accordingly, one aspect of the invention relates to growing the
infected insect cells under suitable conditions to produce the desired
recombinant CSFs and recovering the biologically active polypeptide
or dimerized polypeptide from the culture medium.
In another aspect, the invention is directed to recombinant
baculovirus expression vectors which are capable of effecting the
expression of CSFs, to the host cells infected with such vectors, and
to cultures thereof.
One aspect of the invention concerns recombinant baculovirus
2s expression vectors in which CSFs are expressed under the
transcriptional control of a baculovirus promoter~ In one aspect of
the invention the baculovirus promoter is the polyhedrin gene
promoter.
Yet another aspect of the invention relates to recombinant
baculovirus expression vectors that are capable of expressing CSFs and
which may encode either the long or short forms of CSF-l, muteins
thereof, CSF-1 in which the amino terminal 4 codons are altered, or G-
CSF.
~ 3 ~
Preferably, the baculovirus genoma is derived
from Autographa ca]ieornica, Trichoplusia ni, Rachiplusia
ou or Galleria mellonella.
Also, aspects of the invention are the recombinant
baculovirus transfer vectors which are used to transfer the desired
recombinant gene into the baculovirus genome.
Figure 1 shows the DNA sequence of the CSF-1 gene from
pcCSF-17.
Figure 2 shows the DNA sequence of original cDNA clone
number 4. The sequence is missing a few nucleotides at the 5' end
compared to the LCSF sequence in pcDBhuCSF-4 which was completed by
substitution of upstream codons from pcCSF-17 (see Example 2, Sect;on
A.2.)
Figure 3 shows the DNA sequence of G-CSF from pP12.
Figure 4 shows the DNA sequence comparison between the
recombinant baculovirus transfer vectors pAcC1-CS. The carrots
indicate restriction endonuclease cleavage sites.
Figure S shows the DNA sequence comparison for the regions
around the CSF- 1 translational start codon between the recombinant
baculovirus transfer vectors pAcM1, pAcM2, pAcM3, pAcM4, pAcM6, pAcM10
and the polyhedrin gene.
Figure 6 shows SDS-PAGE analysis of immunoprecipitated
baculovirus expression system products.
Figure 7 shows expression levals of various recombinant
polyp~ptides in the. baculovirus expression vector system.
Modes for Carrying Out the Invention
A. Definitions
. .
"Colony stimulating factor-1 (CSF-1)" refers to a protein
which exhibits the spectrum of activity understood in the art for CSF-
1, i.e., when applied to the standard in vitro colony stimulating
assay of Metcalf, D., J. Cell Physiol. (1970) 76:89, it results in the
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formation of primarily macrophage colonies. Native CSF~l is a
glycosylated dimer; dimerization may be necessary for activity.
Contemplated within the scope of the invention and within the
definition of CSF-1 are both the dimeric and monomeric forms. The
monomeric form may be converted to the dimeric form by in vitro
provision of host intracellular conditions suitable for dimerization
of the monomer.
There appears to be some species speci ficity: Human CSF-1
is operative both on human and on murine bone marrow cells; murine
CSF-1 does not show activity with human cells. Therefore, "human"
CSF-1 should be positive in the specific murine radioreceptor assay of
Das, S. K., et al., Blood (1981) 58:630, although there is not
necessarily a complete correlation. The biological activity of the
protein will generally also be inhibited by neutralizing antiserum to
human urinary CSF-l (Das, S. K., et al., suera). However, in certain
special circumstances (such as, for example, where a particular
antibody preparation recognizes a CSF-1 epi tope not essential for
biological function, and which epitope is not present in the
particular CSF-1 mutein being tested) this criterion may not be met.
Certain other properties of CSF-1 have been recognized more
recently, incl uding the ability of this protein to stimul ate the
secretion of series E prostaglandins, interleukin-1, and interferon
from mature macrophages (Moore, R., et al., Science (1984) 223:178;
Ralph, P. et al. (1986) Immunobiol.s 172:194i Ralph~ P. et al. (1987)
Cell. Immunol., 105:270). The mechanism for these latter activities
is not at present understood, and for purposes of definition herein,
the criterion for fulfillment of the definition resides in the ability
to stimulate the formation of monocyte/macrophage colonies using bone
marrow cells from the appropriate species as starting materials, under
most circumstances (see above) the inhibition of this activity by
neutralizing antiserum against purified human urinary CSF-1, and,
where appropriate for species type, a positive response to the
radioreceptor assay. (It is known that the proliferative effect of
CSF-1 is restricted to cells of mononuclear phagocytic lineage
(Stanley~ E. R., The Lymphokines (19~1), Stewart, W. E., Il, et al.,
~3~7~4
;
9 l
ed., Humana Press, Clifton, JN), pp. 102-132) and that receptors for
CSF-1 are restricted to these cell types (Byrne, P. V., et al., Cel1
Biol. (1981) 91:848)).
Figure 1 shows the amino acid sequence for a particular form
of hunlan CSF-l encoded by the recombinant cDNA clone pc~SF-17
(Kawasaki, F.S., et al. (1985), Science 230:291). This protein
contains 224 amino acids in the mature sequence and a leader sequence
of 32 amino acids. The protein produced as the expression product of
this clone is active in assays specific for CSF-1, namely, the bone
marrow proliferation assay (wherein the activity is destroyed by
addition of anti-CSF-1 antibodies), colony stimulation assays, and a
radioreceptor assay. Further characterization of the biological and
molecular properties of human and murine csF-1 is disclosed by P.
Ralph et al. in Molecular Basis of Lymphokine Action tD. R. Webb et
al., eds., The HUMANA Press, Inc., 1987, pp. 295-311).
For convenience, the mature protein amino acid sequence of
the monomer portion of a dimeric protein shown in Figure 1, deduced
from the cDNA clone illustrated herein, is designated mCSF-1 (mature
CSF-1). Figure 1 shows the presence of a 32 residue putative signal
sequence, which is presumably cleaved upon secretion from mammalian
cells; mCSF-1 is represented by amino acids 1-224 shown in that
figure. Specifically included in the definition of human CSF~l are
muteins which monomers and dimers are mCSF-1 and related forms of
mCSF-1. CSF-1 derived from other species may fit the definition oF
"human" CSF-1 by virtue of its display of the requisite pattern of
activity as set forth above with regard to human substrate.
Also for convenience, the amino acid sequence of mCSF-1 will
be used as a reference and other sequences which are substantially
equivalent to this in terms of CSF-1 activity will be designated by
referring to the sequence shown in Figure 1. The substitution of a
particular amino acid will be noted by reference to the amino acid
residue which it replaces. Thus, for example, sergOCSF-1 refers to
the protein which has the sequence shown in Figure 1 except that the
amino acid at position 93 is serine rather than cysteine. Numerous
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muteins have been described in commonly owned PCT Publication Nos.
W086/04607 published August 14, 1986 and PCT Publication No.
W088/03173 published May S, 1988 ~
and are included within the definition of CSF-1 according to the
invention so long as the biological activity of CSF-1 is maintained.
It is expected, of course, that such modifications may quantitatively
or qualitatively affect the activityl either by enhancing or
diminishing the activity of the protein in various assays.
Additional CSF-1 proteins, which have the same colony
stimulating activity described in Metcalf supra, contain substantially
longer amino acid sequences deduced from the recovered cDNA, which
include a 298 amino acid "extra" segment at approximately residue 150
of the pcCSF-17 encoded protein and expression of this cDNA results in
CSF-1 activity in these specific CSF-1 assays, as well.
For convenience, the CSF-1 encoded by pcCSF-17 will be
referred to as the "short form" of the protei n, and for consistency
with previous designations, as "mature" or mCSF-1. The 522 amino acid
sequence encoded in, for example, pcDBhuCSF-4 and its corresponding
clones will be referred to as the "long form", or lCSF-1~ This
sequence is shown in Figure 2. LCSF-1 is further described in PCT
Publication No. W087/0695~ published November 19, 1987, and in
commonly owned PCT Publication No. W088/03173 published May 5, 1988.
The abbreviations used herein also include "huCSF-1" for all human
forms of the protein.
"G-CSF" as used herein means a protein having the effect of
stimulating the production of primarily granulocyte colonies or
granulocyte-macrophage colonies in a colony forming assay using bone
marrow cell progenitors of an appropriate species. A protein haYing
this activity has the deduced amino acid sequence shown in Figure 3 as
disclosed in commonly owned EP Publication No. 256,843 published
February 24, 1988, and in PCT Publication No. W088/01297 published
February 25, 1988.
G-CSF may be isolated from the MIA PaCa-2 cell line~ In
addition, G-CSF specific RNA sequences are clearly detectable in the
mRNA of LD-1 cells and 5637 cells. Thus, G-CSF obtained by cloning
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11 '
mRNA of LD-1 cells and 5637 cells. Thus, G-CSF obtained by cloning
from these cell lines as well as any other cell line which ;s G-CSF
probe positive, is considered within the scope of the invention.
Ma-ture G-CSF produced by the LD-1 cell line has been isolated and
amino-acid sequenced. 65% of the LD-1 produced protein has a Thr
residue at the NH2-terminus and 35% has a Pro residue at the NH2-
terminus. Further disclosure on the cloning and expression of G-CSF
are found in commonly owned PCT Publication No. ~088/01297 published
February 25, 1988 and EP Publication No. 256,~43 (supra3.
The mature G-CSF protein amino acid sequence shown in Figure
3, deduced from the cDNA clone, is designated mG-CSF (mature G-CSF)
beginning at the amino acid residue threonine designated ~1. There is
a 30 residue putative signal sequence, which is presumably cleaved
upon secretion from mammalian cells; mG-CSF is represented by amino
acids 1-174 shown in that figure. Specifically included in the
definition of human G-CSF are muteins which monomers and dimers, if
any, are G-CSF and related forms of G-CSF, designated by their
differences from mG-CSF. G-CSF derived from other species may fit the
definition of "human" G-CSF by virtue of its display of the requisite
pattern of activity as set forth above with regard to human substrate.
Also for convenience, the amino acid sequence of G-CSF will
be used as a reference and other sequences which are substantially
equivalent to this in terms of G-CSF activity will be designated by
referring to the sequence shown in Figure 3. The substitution of a
particular amino acid will be noted by reference to the number of the
amino acid residue which it rep1aced. Thus, for example, ser60 G-CSF
refers to the protein which has the sequence shown except that the
amino acid at position 60 is serine rather than proline.
As is the case for all proteins, the precise chemical
structure of CSF-1 or G-CSF depends on a number of factors. As
ionizable amino and carboxyl groups are present in the molecule, a
particular protein may be obtained as an acidic or basic salt9 or in
neutral form. All such preparations which retain their activity when
placed in suitable environmental conditions are included in the
definition. Further, the primary amino acid sequence may be augmented
1~72~
12
by derivatization using sugar moieties ~glycosylation) or by other
supplementary nlolecules such as lipids, phosphate, acetyl groups and
the like, more commonly by conjugation with saccharides. The primary
amino acid structure may also aggregate to form complexes. Certain
aspects of such augmentation are accomplished through post-
translational processing systems of the producing host; other such
modification may be introduced in vitro. ~n any event, such
modifications are included in the definition so long as the activity
of the protein, as defined above, is not destroyed. It is expected,
of course, that such modifications may quantitatively or qualitatively
affect the activity, ei-ther by enhancing or diminishing the activity
of the protein in the various assays. Further, individual amino acid
residues in the chain may be modified by oxidation, reduction, or
other derivatization, and the protein may be cleaved to obtain
fragments which retain activity. Such alterations which do not
destroy activity do not remove the protein sequence from the
definition.
Modifications to the primary structure itself by deletion,
addition, or alteration of the amino acids incorporated into the
sequence during translation can be made without destroying the
activity of the protein. Such substitutions or other alterations
result in proteins having an amino acid sequence which falls within
the definition of proteins "having an amino acid sequence
substantially equivalent to that of CSF-1 or G-CSF."
"Operably linked" refers to juxtaposition such that the
normal function of the components can be performed. Thus, a coding
sequence "operably linked" to control sequences refers to a
configuration wherein the coding sequence can be expressed under the
control of these sequences.
"Control sequences" refers to DNA sequences necessary for
the expression of an operably linked coding sequence in a particular
host organism. Eukaryotic cells including the insect cells o-f the
instant invention are known to utilize promoters and polyadenylation
signals.
~.172'~
13
"Expression system" refers to DNA sequences containing a
desired coding sequence and control sequences in operable linkage, so
that hosts transformed with these sequences are capable of producing
the encoded proteins. These DNA sequences may also direct the
synthesis of the encoded proteins in an ~n vitro cellular
enYironment. In order to effect transformation, the expression system
may be included on a transfer vector; however, the relevant DNA may
then also be integrated into the viral chromosome to result in a
recombinant viral genome.
As used herein "cell", "cell line"~ and "cell culture" are
used interchangeably and all such designations include progeny. Thus
"transformants" or "transformed cells" includes the primary subject
cell and cultures derived therefrom without regard for the number of
transfers. It is also understood that all progeny may not be
precisely identical in DNA content, due to deliberate or inadvertent
mutations. Mutant progeny which have the same functionality as
screened for in the originally transformed cell are included. Where
distinct designations are intended, it will be clear from the context.
"Infection" as used herein refers to the invasion of cells
by pathogenic viral agents where conditions are favorable for their
replication and growth. "Transfection" refers to a technique for
infecting cells with purified nucleic acids by adding calcium chloride
to solutions of DNA containing phosphate or other appropriate agents
such as dextran sulfate thereby causing the DNA to precipitate and be
taken up into the cells.
"Recombinant transfer vector" refers to a plasmid containing
a "heterologous" gene under the control of a functional promoter
(e.g., polyhedrin or plO promoter) and flanked by viral sequences.
The "recombinant expression vector" is formed after cotransfection of
the recombinant transfer vector and wild-type baculovirus DNA into
host insect cells whereupon homologous recombination occurs between
the viral sequences flanking the heterologous gene and the homologous
sequences in the wild-type viral DNA. This results in the replacement
of wild-type sequences in the virus with the transfer vector sequences
~3~ 72~
14
between the crossover points. The recombinant expression vector is
the recombinant viral DNA containing the desired heterologous gene.
B. General Description
Interest in the growth and differentiation oF granulocytes
and macrophages from bone marrow progenitors has prompted the study of
the colony-stimulating factors (CSFs) that regulate these processes.
The subject has been recently reviewed by D. Metcalf in Blood, 67:~57
(1985). One of these factors, termed macrophage-CSF ~M-CSF or CSF-1)
stimulates the in vitro growth of predominantly macrophage colonies
from bone marrow stem cells. Another of these factors, termed
granulocyte-CSF (G-CSF) stimulates the in vitro growth of
predominantly granulocyte colonies from bone marrow stem cells.
The CSF-l proteins of the invention are capable both of
stimulating monocyte-precursor-macrophage cell production from
progenitor marrow cells, thus enhancing the effectiveness of the
immune system, and of stimulating such functions of these
differentiated cells as the secretion of lymphokines in the mature
macrophages.
In one application, these proteins are useful as adjuncts to
chemotherapy. It is well understood that chemotherapeutic treatment
results in suppression of the immune system. Often, although
successful in destroying the tumor cells against which they are
directed~ chemotherapeutic treatments result in the death of the
subject due to this side effect of the chemotoxic agents on the cells
of the immune system. Administration of CSF-1 to such patients,
because of the ability of CSF-l to mediate and enchance the growth and
differentiation of bone marrow-derived precursors into macrophages,
results in a restimulation of the immune system to prevent this side
effect, and thus to prevent the propensity of the patient to succumb
to secondary infection. Other patients who would be helped by such
treatment include those being treated for leukemia through bone marrow
transplants; they are often in an immunosuppressed state to prevent
rejection. For these patients also, the immunosuppression could be
reversed by administration of ~SF-1~
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In addition to the cDNA encoding the 224 amino acid CSF-l
monomer described above, cDNAs from both human and murine systems have
been obtained which include DNA encoding an additiona1 298 amino acid
sequence. These cDNAs express to produce "long Form" CSF-l proteins
which fulfill the criteria for biological activity of this protein.
In general outline, ~he human cDNA encoding the 22~ amino
acid form of the protein was used as a probe to recover the sequences
encoding murine CSF-l from a cDNA bank prepared in lambda gtlO from L-
929 mRNA which had been enriched for CSF-l production capability. Two
Io clones were recovered which encode similar 520 amino acid proteins.
The clones diverge dramatically in the 3' untranslated region. One of
the 3' untranslated regions is more than 2 kb and bears little
resemblance to the corresponding human sequences, the other, shorter
clone contains approximately 530 bp in the untranslated region and
shows considerable homology to the corresponding human DNA.
The longer forms of CSF-l obtained from the murine library
were then used as a basis to prepare probes to retrieve longer human
sequences, if any. Based on comparison of the murine cDNAs to the
human genomic sequence, a region of the gene previously thought to lie
in an intron region putatively encodes an amino acid sequence showing
considerable homology to the "extra" 298 amino acid seqment contained
in the murine sequence. This permitted construction of an
oligonucleotide probe based on the "extra" DNA which had been, in the
murine system, translated to protein. However, since the human
genomic sequence was available, the probe was designed to accomodate
the precise human sequence.
While pcCSF-17 had been prepared from MIA PaCa mRNA enriched
for CSF-I-encoding materials, a subsequent cDNA library was prepared
from total mRNA extracted from MIA PaCa cells and cloned into gtlO.
The gtlO library was first screened using pcCSF-17 sequences as probe,
and selected probe-positive candidates were screened using an
oligonucleotide probe based on the "extra" translated sequence of the
murine cDNA, but modified to correspond to the related region in the
human genome. Several clones encoding a corresponding "long form" of
13~72~
16 J
a human protein were also obtained. Of course, the availability of
DNA encoding each of these sequences provides the opportunity to
modify the codon sequence so as to generate mutein forms also having
CSF 1 ac tivity.
S For example, glycosylation sites within the coding sequencemay be predicted by the presence of the sequence Asn. x. Ser or Thr
which is found at glycosylated Asn residues in all !~nown glycoproteins
(Hughes, R.C. (1983) Glyçoproteins New York: Chapman and Hall, p
42). The Asn residue within this sequence, for example, may be
substituted by site specific mutagenesis to remove the glycosylation
site. As disclosed herein, two such substitutions were carried out in `
the CSF-1 molecule.
C. Methods Emplo,yed
C.1. Transformations and Transfections
Transformation of E. coli cells was done according to
procedures set forth in T. Maniatis, E.F. Fritsch and J. Sambrook
Molecular Cloning: A Laboratory Manual (1982) Cold Spring Harbor
Press.
Transfections of Sf9 cells are accomplished using a
modi-fication of the calcium phosphate precipitation technique (Graham,
F.L. et al., 1973, Virology 52 :456) as modified for insect cells
(Burand, J.P. et al. (1980), Virol., 101:286; Carstens, E.B. et al.
(1980) Virol., 101:311) and further described by Summers, M.D. and
Smith, G.E. (A Manual of Methods for Baculovirus Vectors and Insect
Cell Culture Procedures, Texas A & M Press: 1986)
C.2. Probing mRNA by Northern Blot; Probe of cDNA or Genomlc
Libraries
RNA is fractionated for Northern blot by agarose slab gel
electrophoresis under fully denaturing conditions using formaldehyde
(Maniatis, T., et al., ~e~ PP 202-203) or 10 mM methyl mercury
(CH3HgOH) (Bailey, J. M., et al., Anal. Biochem. (1976) 70:75-85; and
Sehgal, P. B., et al. 9 Nature (1980) 288 :95-97 ) as the denaturant.
~ ~72~
17
For methyl mercury gels, 1.5~ ge'ls are prepared by me'lting agarose in
running buffer (100 mM boric acid, 6 mM sodium borate7 10 mM sDdiurn
sulfate, 1 mM EDTA, pH 8.2), cooling to 60 C and adding 1/100 vol~me
of 1 M CH3HgOH. The RNA is dissolved in 0.5 x running buf~er and
denatured by incubation in 10 mM methyl mercury for 10 minutes at room
temperature. Glycerol (20~) and bromophenol blue (0.05%) are added
for loading the samples. Samples are electrophoresed for 500-600
volt-hr with recirculation of the buffer. After electrophoresis, the
gel is washed ~or 40 minutes in 10 mM 2-mercaptoethanol to detoxify
the methyl mercury, and Northern blots prepared by transferring the
RNA from the gel to a membrane filter.
cDNA or genomic libraries are screened using the colony or
plaque hybridization procedure. Bacterial colonies9 or the plaques
for phage are lifted onto duplicate nitrocellulose filter papers (S &
S type BA-85). The plaques or colonies are lysed and DNA is fixed to
the filter by sequential treatment for five minutes with 500 mM NaO~I,
1.5 M NaCl. The filters are washed twice for five minutes each -time
with 5 x standard saline citrate ~SSC) and are air dried and baked at
80 C for two hours.
The gels for Northern blot or the duplicate filters for cDNA
or genomic screening are prehybridized at 25-42 C for 6-8 hours with
10 ml per filter of DNA hybridization buffer without probe (0-50%
formamide, 5-6 x SSC, pH 7.0, 5 x Denhardt's solution
(polyvinylpyrrolidine, plus Ficoll and bovine serum albumin; l x =
0.02% of each), 20-50 mM sodium phosphate buffer at pH 7.0, 0.2% SDS,
20 J~ g/ml poly U (when probing cDNA), and 50 ~ g/ml denatured salrnon
sperm DNA). The samples are then hybridized by incubation at the
appropriate temperature for about 24-36 hours using the hybridization
buffer containing kinased probe (for oligomers). Longer cDNA or
genomic fragment probes were labeled by nick translation or by primer
extension.
The conditions of both prehybridization and hybridization
depend or the stringency desired, and vary, ~or example~ with probe
length. For example, conditions for relatively long (e.g., more than
~L 3 :~ 7 ~
18
30-50 nucleotide) probes may employ a temperature of 42~C-S5C and
hybridization buffer containing about 20%-5~% formamide. For the
lower strin~encies needed for oligomeric probes of about 15
nucleotides, lower temperatures of about 25 C-4~ C, and lower
formamide concentrations (0%-~0%) are employed. For longer probes,
the filters may be washed, for example, four times ~or 30 minutes,
each time at 40 C-55 C with 2 x SSC, 0.2% SDS and 50 mM sodium
phosphate buffer at pH 7, then washed twice with 0.2 x SSC and 0.2%
SDS, air dried, and are autoradiographed at -70 C for 2 to 3 days.
Washing conditions are somewhat less harsh for shorter probes. Other
conditions are known in the art that may provide the results disclosed
herein.
C.3. Vector Construction
Construction of suitable vectors containing the desired
coding and control sequences employs standard ligation and restriction
techniques which are well understood in the art and are described in
Maniatis, T. et al., supra. Isolated plasmids, DNA sequences, or
synthesized oligonucleotides are cleaved, tailored, and religated in
the form desired.
Site specific DNA cleavage is performed by treating with the
suitable restriction enzyme (or enzymes) under conditions which are
generally understood in the art, and the particulars of which are
specified by the manufacturer of these commercially available
restriction enzymes. See, e.g., New England Biolabs, Product
2s Catalog. In general, about 1 ~ 9 of plasmid or DNA sequence is
cleaved by one unit of enzyme in about 20 ~A 1 of buffer solution, in
the examples herein, typically, an excess of restriction enzyme ~is
used to insure complete digestion of the DNA substrate. Incubation
times of about one hour to two hours at about 37~C are workable,
although variations can be tolerated. After each incubation; protein
is removed by extraction with phenol/chloroform, and may be followed
by ether extraction, and the nucleic acid recovered from aqueous
fractions by precipitation with ethanol. If desired9 size separation
of the cleaved fragments may be performed by polyacrylamide gel or
~1 12~
19
agarose gel electrophoresis using standard techniques. A general
description of size separations is found in Methods in Enzymology
(1980) 65;49g-560.
Restriction cleaved fragments may be blunt ended by treating
S with the large fragment o~ E. coli DNA polymerase I (Klenow) in the
presence of the four deoxynucleotide triphosphates (dNTPs~ using
incubation times of about 15 to 25 minutes at 20 to 25 C in S0 mM Tris
pH 7.6, 50 mM NaCl, 5 mM MgCl2, 6 mM dTT and S-10 r.M dNTPs. The
Klenow fragment fills in at 5' sticky ends but chews back protruding
3' single strands, even though the four dNTPs are present. If
desired, selective repair can be performed by supplying only one of
the, or selected, dNTPs within the limitations dictated by the nature
of the sticky ends. After treabment with Kleno ~ the mixture is
extracted with phenol/chloroform and ethanol precipitated. Treatment
under appropriate conditions with Sl nuclease results in hydrolysis of
any single-stranded portion.
Synthetic oligonucleotides rnay be prepared by the triester
method of Matteucci, et al., J Am. Chem. Soc. (1981) 103:3185-3191 or
_ . _ _ _ _ _ _
using automated synthesis methods. Kinasing of single strands prior
to annealing or for labeling is achieved using an excess1 eOg.,
approximately 10 units of polynucleotide kinase to 1 nM substrate in
the presence of 50 mM Tris, pH 7.6, 10 mM MgC12, 5 mM dithiothreitol,
1-2 mM ATP. If kinasing is for labeling or probe, the ATP will
contain high specific activity gamma32P.
Z5 Ligations are performed in 15-30 ~l volumes under the
following standard conditions and temperature: 20 mM Tris-Cl pH 7.5,
10 mM MgC12, 10 mM dTT, 33 ~Lg/ml BSA, 10 mM-50 mM NaCl, and either
40 ~M ATP, 0.01-0.02 (Weiss~ units T4 DNA ligase at 0 C (for "sticky
end" ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14
C (for "blunt end" ligation~. Intenmolecular "sticky end" ligations
are usually performed at 33-100 ~glml total DNA concentrations (5-100
nM total end concentration). Intermolecular blunt end ligations
(usually employing a 10-30 fold molar excess of linkers) are performed
at 1 ~M total ends concentration.
:~3~72~
In vector construction employing "vector fragments", the
vector fragment is csmmonly treated with bacterial alkaline
phosphatase (BAP) in order to remove the 5' phosphate and prevent
religation of the vector. BAP digestions are conducted at pH 8 in
approximately 150 mM Tris, in the presence of Na+ and Mg~2 using about
1 unit of BAP per ~9 of vector at 60 C ~or about one hour. In order
to recover the nucleic acid fragments, the preparation is extracted
with phenol/chloroform and ethanol precipitated. Alternatively,
religation can be prevented in vectors which have been double digested
by additional restriction enzyme digestion of the unwanted fragments.
C.4. Modification of DNA Sequences
I
- For portions of vectors derived from cDNA or genomic DNA
which require sequence modifications, site specific primer directed
mutagenesis is used. This technique is now standard in the art, and
is conducted using a primer synthetic oligonucleotide complementary to
a single stranded phage DWA to be mutagenized except for limited
mismatching, representing the desired mutation. Briefly, the
synthetic oligonucleotide is used as a primer to direct synthesis of a
strand complementary to the phage, and the resulting double-stranded
DNA is transformed into a phage-supporting host bacterium. Oultures
of the transformed bacteria are plated in top agar, permitting plaque
formation from single cells which harbor the phage.
Theoretically, 50% of the new plaques will contain the phage
having, as a single strand, the mutated form; 50% will have the
original sequence. The plaques are hybridized with kinased synthetic
primer at a temperature which permits hybridization of an exact match,
but at which the mismatches with the orignal strand are sufficient to
prevent hybridization. Plaques which hybridize with the probe are
then picked, cultured, and the DNA recovered. Details o~ site
specific mutation procedures are described below in specific examples.
1 3 ~ 7 2 ~ ~ A
Z1
C.5. Verification of Construction
In the construct;ons set forth below, correct li~ations for
pl asmid construction are confi rmed by fi rst transforming E. coli
strain MM294, or other suitable host with the ligation mixture.
Miniprep DNA was prepared according to Ish-Horowicz, D. et al. (Nucl.
Acids Res. (19i31) 9:2989) and screened by restriction analysis. DNA
may be further analyzed by restriction and/or sequenced by the dideoxy
method of Sanger, F., et al., Proc. Natl. Acad. Sci. (USA) (1977)
74:5463 as further described by Messing, et al., Nucleic Acids Res.
(1981) 9:309, or by the method of Maxam, et al., Methods in Enzymology
(1 g80) 65 :499.
C.6. Transfer Vector Construction
Because the genome of AcNPV is so large (125kb), there are
too many restriction sites to allow site-specific insertion of
heterologous genes. Therefore, it is necessary to derive recombinant
virus, containing the gene to be expressed, through homologous
recombination between viral DNA and genetically engineered chirrleric
plasmids called transfer vectors.
The transfer vectors which have been described by Smith,
G.E., et al., 1983, uera, were originally constructed by cloning the
AcNPV EcoRI-1 fragment containing the polyhedrin gene into the EcoRI
site of E. coli plasmid pUC8 (Vieira, J.9 et al., Gene 19:259-268
(1982)). A series of plasmids or transfer vectors having single BamHI
cloning sites at various locations in the polyhedrin gene were created
as described (Smith et al., 1983, supra). One of these, pAc373, has a
single BamHI site 50 bp downstream from the polyhedrin cap site i.e.,
8 bp before the polyhedrin ATG translation initiation codon (Smith et '3
al., (1985), supra and in U.S. Patent No. 4,745,051). The transfer
vectors, pAc610 and pAc611 have the polylinker from M13mplO and
M1~mpll, respectively, inserted at this BamHI si te of pAc373 (Luckow,
V.A. et al. (1988) Biotechnol. 6:47-55. Partial nucleotide sequence
of pAc401 and pAc436 transfer vectors for the production of
polyhedrin/foreign gene fusion proteins has been reported ~Summers,
M.D. et al., 19~7, p. 53, supra).
i
22
C.7. Preparation and Isolation of_Recombinant AcNPV Virus
Detailed methods for the generation of recombinant virus can
be found in European Patent Application No. 0127839 to G.E. Smith and
M.D. Summers of the Texas A & M University System. In general, 2~g
of genetically engineered transfer vector DNA and 1 ~9 of AcNPV viral
DNA are cotransfected onto monolayer culture cells of Spodop-tera
frugiperda. The infected cells usually show viral occlusions by day 3
or 4, with 10-50% of the cells being infected. The virus titer of the
medium is expected to be 107 pfu/ml and 0.1%~0.5% are expected to be
recombinant virus.
Several methods -for the detection of recombinant virus are
known in the art. Visual detection of the plaques is best achieved
using a low power dissecting microscope and observing the plaques on
inverted plates with a black background and illumination from the
side. More unequivocal methods for detecting recombinants are plaque
hybridization using DNA probes to the cloned gene. Antibody probes to
the product of the cloned gene may also be employed.
Isolation of the recombinant virus is achieved through
plaque purification of serially infected monolayer cells overlayed
with soft agar. After two or three cycles the recombinant virus would
be seen as separate plaques showing the characteristic occlusion-
negative morphology. The plaques, containing about 10,000 pfu of
virus, are picked using a sterile Pasteur pipet and transferred to 2
ml of medium.
C.8. Electrophoretic Analysis of Expression Products
In order to concentrate expression products, culture
supernatants were incubated with Protein A-Sepharose CL-4B beads
cross-linked to antibody to either CSF~1 or G-CSF for 1 hour. The
beads were then pelleted and the supernatants reserved. The beads
were washed once in .5 ml buffer containing lM LiCl9 20 mM Tris-HCl pH
8.0 and O.S~ NP40, three times in .5 ml buffer containing 150 mM NaCl,
20 mM Tris-HCl pH8 and 0.5% NP40 and then suspended in sample loading
huffer lacking reducing agent. The samples were then heated to 37 C
'2`~
~1
~L 3 ~ '7 ~
23
for 5 minutes, pell eted and the supernatant removed to new tubes
These supernatants were adjusted to contain 0.14 M beta-
mercaptoethanol or 20 mM DTT, heated to 100 C ~or 4 min. and run on
SDS-PAGE. SDS-PAGE was performed essentially according to the
procedure of Laemmli (Nature (1970) 227:680-685).
Immunoblotting procedures have been described in commonly
owned EP Publication No. 219,286 published April 22, 1987 essentially
as described below. Immunoblotting of the gel onto nitrocellulose
(Schleicher and Schuell, 0.45 ~meter) was performed in a Bio-rad
Trans-blot cell at 35 V for one hour at room temperature essentially
according to published methods (Towbin et al. (1979) Proc. NatlO Acad!
of Sci , USA, 76:4350-4354; Bittner et al. (1980) Anal. Bi_chem.,
102:459-0471; Burnette et al. (19~1) Anal. Biochem., 112:195 203).
Following transfer, nonspecific antibody bind;ng si tes on the
nitrocellulose were blocked by incubation for 30 minutes at room
temperature with gentle agitation in 250 ml of 0.1% Tween 20 in
phosphate buffered saline (PBS). Then the blot was washed three times
with gentle agitation at room temperature for five minutes each in 250
ml volumes of 0.1~ nonfat dry milk, 0.1% ovalbumin in PBS, and
incubated with gentle agitation for three hours at room temperature in
5 ml of a 1/500 to 1/1000 dilution in the preceding buffer of rabbit
antiserum against G-CSF (supplied by BABC0). After washing three
times as described above, the blot was incubated for one hour at room
temperature with 5 ml of a 1/2000 dilution of goat anti-rabbit IgG
conjugated to horseradish peroxidase (this conjugate supplied by
Zymed ) and washed again three times as above.
In order to concentrate expression products, culture
supernatants were incuba-ted with Protein A-Sepharose CL-4B beads
cross-linked to antibody to a CSF protein for 1 hour. The beads were
then pelleted and the supernatants reserved. The beads were washed
once in .5 ml buffer containing lM LiCl, 20 mM Tris-HCl pH 8~0 and
0.5% NP40, three times in .5 ml buffer containing 150 mM NaCl, 20 mM
Tris-HCl pH8 and 0.5% NP40 and then suspended in sample loading buffer
lacking reducing agent. The samples were then heated to 37 C for 5
minutes, pelleted and the supernatant removed to new tubes These
13~72~l~
24
supernatants were adiusted to contain 0.14 M beta-mercaptoethano1 or
20 mM DTT, heated to 100 C For 4 min. and run on SDS-PAGE.
C.9. Insect Cell Culture
Methods for insect cell cultures are well known in the art
and detailed procedures for the-,r cultivation can be found in Summers,
M.D. et al. (1987, ~ ), EP Publication No. 127,839 to G.E. Smith et
al. or in U.S. Patent No. 4,745,051 (supra). The insect expression
host of the current invention, Spodoptera frugiperda (Sf9) is well
suited to the production of heterologous proteins because of its
ability to grow in either monolayer or suspension culture.
As monolayer cultures, Spodoptera frugiperda cells will
divide every 18-24 hours depending on the culture media. The cells do
not require carbon dioxide to maintain the pH of the medium and they
will grow well at temperatures between 25-30 C. Subculturing is done
2 or 3 times a week when the cells are confluent. Because insect
cells are loosely adherent they are easily resuspended without the
need of proteases.
Suspension culture conditions will vary depending on the
medium and culture volume and should be determined empirically.
Subculturing is required when the cell density reaches 2X106 cells/ml
by replacing 80% or more of the culture with an equal volume of fresh
medium. With suspension cultures larger than 500 ml it becomes
necessary to aerate by either bubbling or diffusion.
Pre~erred media and culture conditions have been described
by Inlow, D. et al. (1987) "Large-scale insect culture for recombinant
protein production, "Presented at S~nposium on Strategies in Cell-
Culture Scale-Up at the American Chemical Society National Meeting in
New Orleans, Louisiana. Appropriate serum-free media include:
(a) a basal medium;
(b) a lipid component; and
(c) a peptone component.
~L3~72~
A "basal medium" is herein defined as a nutrient mixture o~
inorganic sal tS9 sugars, amino acids, optionally also containing
vitamins, organic acids and/or buffers. Basal media to~ether with
supplements provide the nutrients necdessary to support cell life,
growt~ and reproduction. The preferred basal media used as the
starting point for the preparation of the serum free media of the
present invention contain neither serum, nor proteins, nor preferably
any peptones. The choice of basal medium for the preparation of the
media of this invention is not critical. The basal medium can also be
considered optinal in the sense that appropriate peptone and lipid
components can be selected which provide such necessary nutrients as
amino acids and vitamins required to support cell life, growth and
reproduction.
As indicated, IPL-41 is a prererred basal medium for the
preparation of the media used in this invention. IPL-41 basal medium
is commercially avai'lable from a number of vendors and is described in
Weiss, et al. In Vitro, 17(6):495-502 (June 1981~ and in Weiss et al.,
CRC Press, supra, pp. 70-72 (1986). Table 1 of Weiss et al. (In Vitro
at page 4g6, and Table 3 of Weiss et al. CRC Press, at pages 71-72
outline all the components of IPL-41 and provide their proportions in
mg/l. - At page 497
of Weiss et al. (In Yitro), the preparation of the complete medium
IPL-41 is described wherein tryptose phosphate broth (TPB) and fetal
bovine serum (FBS~ are added. The lPL~41 basal medium employed in
preparing the serum-free media of this invention does not contain
tryptose phosphate broth (TPB) or fetal bovin serum (FBS).
The serum-free media of this invention can further comprise
a protective agent. Such a protective agent is a preferred component
of the media of this invention~ especially under well-aerated culture
conditions. Therefore, the serum-free media of this invention
include:
(a) a basal medium;
(b) a lipid component;
B~
~ 3 ~- r~
26
(c) a peptone component; and
(d) a protective agent.
A protective agent is necessary to prevent cell damage and
death under well-aerated conditions as found in well agitated and
sparged cultures. The protective agent prevents a
disintegration/clumpding phenomenon of insect cells growth in shake
flasks and adherence of the cells to the vessel walls. Further, the
protective agent reduces the amont of cellular debris in shake flask
cultures indicating that cell lysis is reduced by the presence oF the
lo protectant. The protective agent further preferably acts as an anti-
foaming agent preventing the loss of cells from the free suspension
into a foam layer, and can act as a bubble surface tension reducing
agent and/or as a cell surface stabilizing agent and/or as a
vciscosifying agent.
Protective agents are herein defined as non-toxic, water
soluble compounds that functionally act to protect insect cells from
damage and death in agitated and sparged insect cell culture. The
protective agents oF this invention are preferably non toxic, water
soluble polymers. A protective agent candidate can be selected by
first confirming that it is not toxic to the insect cells to be
cultured by methods known to those skilled in the art of insect cell
culture, for example, by adding it to a suspension or monolayer of the
insect cells of choice for cultivation and comparing the growth oF the
culture to a control. Then, the non-toxic protective agent candidates
can be tested for protective ability by adding the candidate agent to
an agiated or sparged culture of the insect cells of choice at small
scale and observing viability and growth rate of the cells of said
culture to the viability and growth rate of the cells in a control
culture.
The protective agents in the media of this invention are
preferably cell surface stabilizing agents and/or viscosiFying agents
and/or bubble surface tension reducing agents. Examples of preferred
protective agents are hdydroxyethyl starch, methyl cellulose,
carboxymethyl cellulosed (as~ sodium carboxydmethyl cellulose),
~72l~
27
dextrant sulfate, piolyvinylpyrrolidone, ficoll, alginic acid,
polypropyleneglycol, and non-toxic polymeric detergents. Preferred
non-toxic, non-ionic polymeric detergents are block polymers of
propylene oxide and ethylene oxide ~polyoxypropylene polyoxyethylene
condensates), preferably Pluronic polyols, such as, Pluronic F68, F77,
F88 and F108, preferably F68 and F88, more preferably F680 The
Pluronic polyols are commercially available from BASF Wyandotte Corp.
(101 Cherry Hill Road, P.O. Box 181, Parsippany, NJ 07054, U.S.A~).
If the protective agent is not also functioning as an
emulsifier, the media of this invention further comprise another
emulsifier that acts to emulsify the lipid component in conjunction
with another emulsi~ier present at a small concentration in the lipid
component itself as described below. Such an emulsifier alternative
to a protective agent/emulsifier is preferably a detergent, preferably
non-ionic which is non-toxic to the insect cell culture at the
concentrations required for emulsification of the lipid component.
The introduction of the lipid component in the form of a
microemulsion enhances the availability of lipids in the media to the
cells. One option for emulsifying the lipld component is a dual
emulsifier system wherein the protective agent is an emulsifier as
well as a protective agent and can act in conjunctlon with an
emulsifier or combination of emulsifiers present in the lipid/organic
solvent solution making up the lipld component of the media of this
invention. Another option for emulslfying the lipid component of the
media of this invention is a system wherein the protective agent is
not significantly emulsifying but wherein one or more additional
emulsifiersd are present in an aqueous solution which is added to the
lipid component organic solution and act in conjuction with the
emulsifiers present therein to form a microdemulsion.
Preferred emulsifiers added to the lipid component of the
media of this invention include phospholipids, preferably lecithin and
non-toxi non-ionic polymeric detergents, preferably a polysorbate
compound having the formula:
2 ~ ~
28
H2 H-CH2-COOR
H(C2H4)tHOC f H~(C2H4)UH
CHo(c2H4)uH
wherein R is a saturated or unsaturated fatty acid having
from 16 to 20 carbons, inclusively
wherein t is an integer between 10 and 30, inclusively; and
wherein u is an integer between 10 and 20, inclusively~
Most preferably the non-toxic, non-ionic, polymeric
detergent/emulsifier is polyoxyethylene ~20) sorbitan monooleate,
otherwise known as polysorbate 80. Such a non-toxic, non-ionic
polymeric detergent is commercially available as Tween 80 from ICI
Americas Inc. (New Murphy Road & Concord Pike, Wilmington, DE 19897,
USA). Another polysorbate 80 is commercially available as Durfax 80
from Durkee Industrial Foods Group/SCM Corp. (900 Union Commerce
Bldg., Cleveland, Ohio 44115, USA)~ Other non-toxic9 non-ionic,
polymeric detergent candidate emulsifiers can be found in editions of
McCutcheon's Emulsifiers and Detergents, ~
Said non-ionic~ non-toxic, polymeric detergent/emulsifier,
such as, polysorbate 80. is present in the media used in this
invention at a concentration from about 5 mg/l to about 75 mg/l, more
preferably from about 20 mg/l to about 30 mg/l, and most preferably
about 25 mg/l.
A preferred example of a dual emulsifier system of the media
of this invention is the combination of a protective agent/emulsifier,
preferably a Pluroniic polyol, more preferably Pluronic F68 or Pluronc
F88s and still more preferably Pluronic F58s and a non-toxic, non-
ionic polymeric detergent, preferably a polysorbate compound, and more
~`25 preferably polysorbate 80.
.~
~ 3 ~ r~
29
The lipid component is preferably added to the bulk of the
media of the invention as a microemulsiQn. An advantage of the lipid
component being in the Form of a microemulsion, in addition to
enhancing the availability of the lipids to the insect cells, is in
providing the option of not having to filter skerilize the lipid
component and the rest of the media components separately. The lipid
component in the form of a microemulsion can be added to the media
without being filter sterilized, and the entire media can then be
filter sterilized without the concern that lipids, for example, in
lo globular form, could be lost during the filter sterilization
process. For large scale production, such an advantage is
significant.
An exemplary lipid component of the media used in this
invention comprises per liter of media:
10 mg cod liver oil
25 mg Tween 80
41.5 mg cholesterol
2 mg alpha-tocopherol
1 ml ethanol.
To the optionally filtered/sterilzied lipid component
solution (1 ml), then, in this exemplification, 10 ml of 10~ Pluronic
F68 in water (optionally filtered/sterilizsed~ is slowly added with
agitation as by vortexing.
The peptone component of the serum-free media o~ this
invention can be selected from a wide variety of hydrolyzed protein
products, either alone or in combination~ including, without
limitation, ox liver digest, such as Panmede (commercially available
from Paines & Byrnes Ltd., Greenford, Eryland), yeast extract~ such as
Yeastolate (preferably TC Yeastolate from Difco, VSA), caseine digest,
such as Bactocasitoner~ (commercially avai1able from Difco, USh~
tryptose phosphate broth (TPR~ wherein tryptose is the peptone,
Lactalbumin Hydrolyzate (LH) (commercially available from Difco, USA)
gelatin peptone, glycerin-gelatine peptone, and beef peptone among
many other proteolytic di~est products of proteins.
B~
~ 3 ~
~'l
Pre~erably the peptone component is composed of peptone
fractions either alone or in combination selected from the group
comprising TPB, caseine digest preferably Bactocasitone, ox liver
digest preferably Panmede, yeast extract, preferably Yeastolate, and
Lactalbumin Hydrolyzate (LH). More preferably, the peptone component
comprises either LH or Yeastolate, alone or in combination. Still
more preferably, the peptone component comprises either a combination
of LH and Yeastolate or Yeastolate alone.
The total peptone concentration present in the media of this
invention can be as high as the sum of the highest concentrations of
the individual peptone fractions wherein said highest concentration
for each peptone fraction is that which is non-toxic and non-
inhibitory to cell growth and wherein the total peptone concentration
of said highest, non-toxic, non-inhibitory concentrations of the
peptone fractions is similary non-toxic and non inhibitory to cell
growth. The highest peptone concentrations can vary not only with the
particular peptone fractions used but also with the particular insect
cell line that is selected. In general, preferred total peptone
concentration present in the media of this invention can range From
2Q about 1 g/l to about 12 g/l, more preferably from about 2 g/l to about
g/l, and still more preferably from about 3 g/l to about 5 g/l.
Example I
A. Construction of New Baculovirus Transfer Vectors
A.1. Construction of pAcC1
pAcC1 is similar to pAc401 (described previously in
Section C.5. except that the recognition site for EcoRI endonuclease
has been removed. To accomplish this, pAc401 was digested to
completion with EcoRI and the ends were made blunt using Klenow
fragment. After ligation and transformation, candidates were screened
for the absence of an EcoRI site~
~3:~72~'~
31 ;,
A.2. Construction of pAcC2
pAcC2 is similar to pAc436 (described previously in 3
Section C.5. except that the recognition site for EcoRI endonuclease
has been removed. To accomplish this, pAc436 was digested to 3~
completion with EcoRI and the ends were made blunt using Klenow
fragment. After ligation and transformation, candidates were screened 3
for the absence of an EcoRI site.
A.3. Construction of pAcC3
pAcC3 differs from pAcC2 in that an NcoI restriction
site has been introduced at the ATG translational start of the 3
polyhedrin gene. To accomplish this the new transfer vector, pAcC2,
was digested to completion with SmaI endonuclease. Following phenol
extracton and ethanol precipitation, SmaI digested pAcC2 was dissolved
in TE buffer (10 mM Tris HCl pH 7.4; 1 mM EDTA). In a 50 ~l volume
of ExoIII buffer ~50 mM Tris-HCl pH 8.0; 5 mM MgCl2; 10 mM beta-
mercaptoethanol), 10 ~ 9 of SmaI digested pAcC2 was treated with 50
units of E. coli ~onuclease III (ExoIII) at 30 C for 5 minutes. The
sample was phenol extracted and ethanol precipitated twice. Then 50
pmoles of a primer EK85, 5'AACCTATAAACCATGGCGGCCCGG3', was kinased
with cold ATP in a 20 ~l reaction volume (50 mM Tris-HCl pH 7.8; 10
mM MgCl2; 10 mM beta-mercaptoethanol). To 5 ~ g of ExoIII treated
pAcC2 was added 10 p~oles of kinased EK85 in a final volume of 20 ml
NET (100 mM NaCl; 10 mM Tris-HCl pH 7.5; 1 mM EDTA) buffer To anneal
the plasmid and primer, the reaction was heated to 65 C for 10
minutes, incubated at 37 C for 10 minutes and placed on ice. The
extension reaction was performed by adding 20 ~l 2 x Klenow buffer
(40 mM Tris-HCl pH 7.5; 20 mM MgCl2; 2 mM beta-mercaptoethanol)
containing 1 ~l 10 mM dNTPs, 1 ~ 1 10 mMATP, 1 l~l (about 2 units)
Klenow fragment and 1 ~l (about 1-2 units) T4 DNA ligase. The
reaction was incubated at 16C for about 4 hours and then transformed
into MM294. Minilysates were screened by analyzing for the presence
of an NcoI site. Miniprep DNA was then used to retransform and obtain
the desired pure clone.
~3~7~
32
A.4 Construction of pAcC4 and eAcC5
pAcC4 and pAcC5 are derivatives of pAcC3 containing a
polylinker sequence at the SmaI site. The poly1inker contains
recognition sites for restrictionendonucleases SmaI, KpnI, PstI,
BglII, Xbaf ~cleavable when DNA is ur,methylated), EcoRI, BamHI and
BclI. pAcC4 contains the sequence in one orientation whlle pAcC5
contains the polylinker in the opposite orientation (see Figure 4). To
construct these vectors pAcC3 was digested to completion with XmaI
endonuclease and ligated with two complementary self-annealed
oligomers having the sequence
5'-CCGGGTACCTGCAGATCTAGAATTCGGATCCTGATCA-3'
3'- CATGGACGTCTAGATCTTAAGCCTAGGACTAGTGGCC-5'
After transformation of MM294, miniprep DNAs of transformants were
analyzed for the presence of restriction sites in the polylinker
sequence.
Example II
A. Isolation of cDNA Encoding Human CSF-1
A.l pcCSF-17
The human derived pancreatic carcinoma cell line MIA
PaCa-2 was used as a source of mRNA to validate probes and for the
formation of a cDNA library containing an intronless form of the human
CSF-1 coding sequence. The MIA PaCa cell line produces CSF-1 at a
level approximately 10 fold below that of the murine L 929 cells.
Negative control mRNA was prepared from MIA PaCa cells
maintained in serum-free medium, i.e. under conditions wherein they do
not produce CSF-l. Cells producing CSF-I were obtained by reinducing
CSF-1 production after removal of the serum.
Cells were grown to confluence in roller bottles using
Dulbecco's Modified Eagles' Medium ~DMEM) containing 10% fetal calf
serum, and produced CSF-I at 2000-6000 units/ml. The cell cultures
were washed, and reincubated in serum-free DMEM to suppress CSF-l
formation for negative controls. No detectable CSF-l was produced
Z
33
after a day or two. Reinduced cel1s were obtained by addition of
phorbol myristic acetate (100 ng/ml) to obtain production after
several days of 1000-2000 units/ml.
The mRNA was isolated by lysis of the cell in isotonic
buffer with 0.5% NP-40 in the presence of ribonucleoside vanadyl
complex ( ~rger, S. L., et al. Biochemistry (1979) 18:51~3) followed
by phenol chloroform extraction, ethanol precipitation, and oligo dT
chromatography, and an enriched mRNA preparation obtained. In more
detail, cells were washed twice in P~S ~phosphate bu,fered saline) and
are resuspended in IHB (140 mM NaCl, 10 mM Tris, 1.5 mM MgCl2, pH 8)
containing 10 mM vanadyl adenosine complex (Berger, S. L. et al.
supra).
A non-ionic detergent of the ethylene oxide polymer type
(NP-40) was added to 0.5% to lyse the cellular, but not nuclear
membranes. Nuclei were removed by centrifugation at l,OO0 x 9 for 10
minutes. The postnuclear supernatant was added to two volumes of TE
(10 mM Tris, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.5)
saturated phenol chloroform (1:1) and adjusted to 0.5% sodium dodecyl
sulfate (SDS) and 10 mM EDTA. The supernatant was reextracted 4 times
and phase separated by centrifugati~n at 2,000 x 9 for 10 minutes.
The RNA was precipitated by adjusting the sample to 0.25 M NaCl,
adding 2 volumes of 100% ethanol and storing at -20 C. The RNA was
pelleted at 5,000 x 9 for 30 minutes, washed with 70% and 100% Zi
ethanol, and was then dried. Polyadenylated (poly A+) messenger RNA
(mRNA) was obtained from the total cytoplasmic RNA by chromatography
on oligo dT cellulose (Aviv, J.7 et al., Proc. Natl. Acad. Sci. (1972)
69:1408-l412). The RNA was dissolved In ETS (lO mM Tris, l mM EDTA,
0.5% SDS, pH 7.5) at a concentration of 2 mg/ml. This solution was
heated to 65 C for 5 minutes, then quickly chilled to 4~C. After
bringing the RNA solution to room temperature, it was adjusted to 0.4
M NaCl and was slowly passed through an oligo dT cellulose column
previously equilibrated with binding buffer (S00 mM NaCl9 10 mM Tris,
l mM EDTA, pH 7.5 0.05% SDS). The flowthrough was passed over the
column twice more. The column was then washed with 10 volumes of
binding buffer. Poly A+ m~NA was eluted with aliquots of ETS,
~3~ 72~
3~
extracted once with TE-saturated phenol chloroform and was
precipitated by the addit;on of NaCl to 0.2 M and 2 volumes of 100%
ethanol. The RNA was reprecipitated t~ice, was washed once in 70% and
then in 100 X ethanol prior to drying~
Total mRNA was sub~ected to 5-20% by weight sucrose gradient
centrifugation in 10 mM Tris HCl, pH 7.4, 1 mM EDTA, and 0.5~ SDS
using a 3echman S~40 rotor at 20 C and 27,00C rpm for 17 hours. The
mRNA fractions were then recovered from the gradient by ethanol
precipitation, and injected into Xenopus oocytes in the standard
translation assay. The oocyte products of the RNA fractions were
assayed in the bone marrow proliferation assay ~or in the bone marrow
proliferation assays of Moore, R. N., et al., J. Immunol. (1983)
131:2374, and of Prystowsky, M. B., et al.~ Am. J. Pathol. (1984)
114:149) and the fractions themselves were assayed by dot blot
hybridization to a 32-mer probe corresponding to the DNA in the second
exon of the genomic sequence (exon II probe). The overlining in
Figure 1 shows the exon II probe.
The bone marrow proliferation assay results of the
supernatants from the Xenopus oocytes did not correlate exactly with
the dot-blot results. The most strongly hybridizing fraction, 11,
corresponds to 18S, while the most active fractions 8 and 9 correspond
to 14-16S. Fraction 8, 9 and 11 were used to form an enriched cDNA
library as described below.
The mRNA was also fractionated on a denaturing formaldehyde
gel, transferred to nîtrocellulose, and probed with exon II probe.
Several distinct species ranging in size from 1.5 kb to 4~5 kb were
found, even under stringent hybridization conditions. To eliminate
the possibility of multiple genes encoding CSF-1, digests of genomic
DNA with various restriction enzymes were subJect to Southern blot and
probed using pcCSF-17 DNA. The restriction pattern was consistent
with the presence on only one gene encoding CSF-1.)
The enriched mRNA pool was prepared by combining the mRNA
from the gradient fractions having the highest bone marrow
proliferative activity, although their ability to hybridize to probe
~L3~72~
is relatively low (14S-16S) with the ~ractions hybridizing most
intensely to probe (18S). Higher molecular weight factions which also
hybridized to exon II probe were not ;ncluded because corresponding
mRNA from uninduced MIAPaCa cells also hybridized to exon II probe.
cDNA libraries were prepared from total or enriched human
mRNA in two ways. One ~ethod uses gt1C phage vectors and is described
by Huynh, T. V., et al., in DNA Clonin~_ Techniques: A Practical
Approach IRL Press, Oxford 1984, D. Glover, ed.
A preferred method uses oligo dT priming of the poly A tails
and AMV reverse transcriptase employing the method of Ckayama, H., et
al., Mol Cell Biol. 1(1983) 3:280-289
This method results in a higher proyortion of full length
clones than does poly dG tailing and effectively uses as host vector
portions of two vectors therein described, and readily obtainable from
the authors, pcDV1 and pLl . The resulting ~ectors contain the insert
between vector fragments containing proximal BamHI and XhoI
restriction sites; the vector contains the pBR322 origin of
replication, and Amp resistance gene and SV40 control elements which
result in the ability of the vector to effect expression of the
inserted sequences in COS-7 cells.
A 300,000 clone library obtained from above enriched MIAPaCa
mRNA by the Okayama and Berg method was then probed under conditions
of high stringency, using the exon II probe. Ten colonies hybridizing
to the probe were picked and colony purified. These clones were
2s assayed for the presence of CSF-1 encoding sequences by transient
expression in COS-7 cells. The cloning vector, which contains the
SV40 promoter was used per se in the transformation of COS-7 cells.
Plasmid DNA was purified from the 10 positive clones usin~ a
CsCl gradient, and the COS-7 cells transfected using a modification
tWang, A. M., et al., Science (1985) 228:149) of the calcium phosphate
coprecipitation technique. After incubation for three days, CSF-1
production was assayed by subjecting the culture supernatants to the
radioreceptor assay performed substantially as disclosed by Das, S.
K.9 et al., Blood (1981) 58:630, and to a colony stimulation ~bone
~7
13~72~
36
marrow proliferation) assay performed substantially as disclosed by
Prystowsky, M. B., et al., Am. J. Pathol. (1984) 114:149. Nine o-f the
ten clones picked failed to show transient CSF-l production in COS-7
cells. One clone, which did show expression, was cultured, the
plasmid DNA isolated, and the insert was sequenced. The DNA sequence,
along with the deduced amino acid sequence, are shown in Figure 1.
The full length cDNA is 1.64 kb and encodes a mature CSF-1 protein of
224 amino acids. The clone was designated CSF-17 with Cetus
depository number OMCC 2347 and was deposited with the American Type
Culture Collection on June 14, 1985, as accession No. 531~9. The
plasmid bearing the CSF-l encoding DNA was designated pcCSF-17.
A~2. Additional Clones
pcCSF-17, prepared as described above, was used as a
probe to obtain additional CSF-l encoding clones from a human cDNA
library. Total mRNA was prepared from MIAPaCa cells exactly as
described above and used to obtain a cDNA library in gtlO phage
vectors by ligating the reverse transcripts to EcoRI linkers and
inserting the EcoRI digest of the cDNA thus provided into the EcoRI
site of ggtlO, as described by Huynh, T. V., et al., in DNA Clonin~
~0 Techniques: A Practical Approach, IRL Press, Oxford, 19~, D. Glover,
ed.
Over one million phage were screened using a single stranded
highly labeled probe derived from CSF-17 using standard procedures,
which are briefly summarized as follows.
A BglI-Ava II fragment of pcCSF-17 DNA9 which includes the
entire coding sequence for CSF-1 plus about 600 bp of 3' untranslated
region was inserted into M13, and a labeled second strand synthesized
as follows: Approximately 1 pmol of M13 containing the single-
stranded BglI-AvaII-digested pcCSF-17 was annealed with 10 pmol of
3~ sequencing primer in 20 mM Tris, pH 7.9, 10 mM MgCl2, 100 mM NaCl, and
20 mM beta-mercaptoethanol at 67 ~ for 5 minutes and then transferred
to 42 C for 45 minutes. The annealed preparation was supplied with
100 ~ mol each of dATP, dCTP, and dTTP, and 2.5 /~ mol P32-labeled
(alpha)GTP, along with 5 U Klenow fragment. The reaction was
37
incubated at 37C for 20 minutes, and the DNA recovered on a P-6 dG
(Bio-Rad) spin column and boiled for 10 minutes to separate the
strands.
The probes, thus prepared, were used to screen the plaques
s (which had been transferred to nitrocetlulose) by hybridization under
stringent conditions (50~ formamide, 5 x SSC, 5 x Denhardt's) at 42C
for 18 hours.
Approximately 150 phage plaques were positive, and 20 which
were particularly strongly hybridized were selected for plaque
purification to obtain individual clones.
The 20 clones were then subiected to hybridization under the
same conditions to the oligonucleotide GM11, which has the sequence
complementary to nucleotides 506-545 in Figure 2. As described above,
this sequence is an exact match to that portion of the human genomic
sequence which corresponds to the "extra" portion of the murine cDNA,
described below, that encodes the "extra" 295 amino acid segment in
the "long form" of the murine CSF-1 protein.
The complete DNA sequence for the pertinent coding regions
of the cDNA insert in clone 4 along with the deduced amino acid
sequence, are shown in Figure 2. The sequence was derived by
integrating the known sequence of the genomic clone9 phCSF-la~
described above, using the 295 amino acid "extra" insert of the murine
sequences described below as a guide to deduce the complete sequence
shown. The sequence depicted shows the splicing of the "extra"
segment, sufficient to encode 2g8 "extra" amino acids contained in khe
gene at the 5' side of exon 6, into the sequence of pcCSF 17 between
the first nucleotide of the codon for the Gly residue at amino acid
position 150 into reading frame with the remaining CSF-17 sequence.
The insert changes the codon at 150 to a codon for aspartic acid, the
subsequent codon at the end of the insert is reconstituted to a Gly,
and the remaining sequence of residues continuing with His-Glu-Arg
etc. down to the C-terminal Val residue remain the same as in pcCSF-
17.
38 ~3~72~
Two of the clones, 4 and 25, were cloned into M13mpl8 or
M13mpl9 for sequencing to confirm the results sho~n in Figure 2 (only
clone 4 sequence shown) using the EcoRI restriction s;tes. These two
clones appeared identical from restriction enzyme analysis. They were
then subclones into the modified Okayama~Berg vector pcDB, which was
prepared from the published Okayama-Berg vectors pcDV1 and pLl
(Okayama, Ho~ et al., Mol. Cell Biol. (19B3) 3:280-289) as follows:
pcDV1 was disgested with HlndIII and Kp_I and the large
fragment, providing Ampr, the pBR322 origin of replication, and a
polyadenylation site, was isolated. pLl was digested with HindIII and
PstI and the small fragment, providing the SV40 promoter and origin of
replication, was isolated. These fragments were religated in a three-
way ligation with the synthetic, KpnI/~stI-digested oligonucleotide
fragment
CTGCAGGAGCTCAGATCTTCTAGAGAATTCTCGAGCGG~CGCATCGATGGTACC
GACGTCCTCGAGTCTAGAAGATCTCTTAAGAGCTCGCCGGCGTAGCTACCATG6
to obtain pcDB, a plasmid corresponding to pcD-x shown in the
reference, wherein "x" is replaced by a polylinker. Thus, pcDB
contains the SV40 early promoter and an immediately downstream linker
followed by a polyadenylation site. Sequences ligated into the
polylinker region should be expressed under the control of the SV~O
early promoter.
Before testing expression, because clones 4 and 25 appeared
to be missing some 5' end sequences as compared to CSF-17~ the
upstream portions from pcÇSF-17 were substituted for those of clones
and 25.
The protocol for this substitution was as follows: pcCSF-17
was digested with SmaI, which cuts at the extreme 5' end of the
untranslated sequence (see Figure 1) and the linearized plasmid was
ligated to EcoRI linkers and resealed. The religated plasmid was then
digested with EcoRI, which removes the entire coding region from just
upstream of the SmaI site at the extreme 5' end to the EcoRI site
immediately downstream of the stop codon. This was ligated into the
polylinker of pc~B a~ the EcoRI site. The coding sequences downstream
~L3~72~ ,
39
of the BstXI site, which site is located at approximately the codon
for amino acid 8 of the CSF-17 mature protein sequence (see Figure 1)
was removed by digestion with BstXI and ~e~ e~I cuts into the
linker slightly downstream of the EcoRI site past the stop codon.)
This deleted downstream sequence was replaced by the analogous
BstI/KpnI fragment from mpl3-4 and mpl3-25. The resulting clones,
pcDBhuCSF 4 and pcDBhuCSF-25 thus contained the coding sequences
downstream of the codon for approximately amino acid 8 from clones 4
and 25, respectively, and the upstream sequences from pCSF-17. The
ligated sequences are in reading frame and under control of the SV40
early promoter.
B. Construction of Recombinant Baculovirus Transfer Vectors
Containing the CSF-l Gene
B.1. Construction of pAcM1 and pAcM2
pAcM1 was constructed by taking advantage of the SmaI
and EcoRI sites located outside the CSF-1 coding sequence but within
the insert containing the CSF-1 gene in pcCSF-17 (see Kawasaki, E. S.
et al., (1985) supra; SmaI recognition site begins at bp 13 and EcoRI
begins at bp 953) . The pAc611 transfer vector was doubly digested
with EcoRI and SmaI endonucleases. The 937 bp SmaI-EcoRI fragment
from pcCSF-17 containing the CSF-l coding sequence was isolated by gel
electrophoresis and ligated to Smal-EcoRI digested pAc611 using T4 DNA
ligase. E. coli MM2 ~ was transformed with the ligation mixture and
transformants were screened for the presence of the 937 bp SmaI-EcoRI
CSF-l fragment.
pAcM2 was constructed in a manner analogous to that
described for pAcMl except that the SmaI-EcoRI fra~ment from pCSF-
BamBcl described in commomly owned PCT Publication No. W086/046079
supra was inserted into pAc611 rather than the SmaI-EcoRI fragment
from pcCSF-17. pCSF-BamBcl differs from pcCSF-17 in that a mutation
has been introduced to change the serine at position 159 in the CSF-l
coding sequence to a stop codon. This mutation was accomplished by
excising the coding sequence from pcCSF-17 and ligating into M13 for
site-specific mutag~nesis using the primer,
13~7f~
4~
5'-GAGGGATCCTGATCACCGCAGCTCC-3'.
This results in a ne~ BclI site at codons 159-160. The
mutated DNA was excised with BstXI and EcoRI and ligated into BstXI-
EcoRI digested pcCSF-17. The ligation mixture was transformed into E.
coli DG105, a dam~host, and the plasmid DNA isolated. This host was
used because BclI is sensitive to dam methylation. The resulting
plasmid, pCSF-BamBcl, was the source of the CSF-1 sequence present in
pAcM2.
B.2. Construction of pAcM3
pAcM3 was constructed by using M13 site-specific
oligonucleotide directed mutagenesis of the pAcM2 vector to delete the
CSF-1 5' untranslated leader sequence and to reconstruct the AcNPV
polyhedrin gene 5' untranslated leader sequence. The approximately
900 bp EcoRV-EcoRI fragment from pAcM2 was ligated into SmaI-EcoRI
digested M13RF. The EcoRV site is located at -96 bp in the polyhedrin
S' leader (see Smith, G. E. et al., Mol. Cell. Biol. 3:2158) and the
EcoRI site begins at bp953 following the CSF-1 coding sequence as
previously described in Section B1. Using the primer,
5'-GTTTTGTAATAAAAAAACCTATAAATAATGACCGCGCCGGGC-3'
the polyhedrin 5' untranslated leader is juxtaposed with the ATG
translational start of CSF-1. The CSF-1 5' untranslated leader
sequence as well as the polylinker sequence of the transfer vector are
deleted. The mutated DNA sequence (EK82RF) was restricted with XbaI,
the ends were made blunt by Klenow repair and a second endonuclease
digestion with EcoRI resulted in a XbaI(blunt)-EcoRI fragment which
was inserted into EcoRV-EcoRI digested pAc611. After ligation and
transformation into DH5, transformants were screened by restriction
analysis. The desired construct designated pAcM3 contains a small
insertion in the polyhedrin promoter region at the EcoRV site as a
result of subcloning the M13 fragment back into pAc611.
B.3. Construction of pAcM4
pAcM4 is an in frame translational fusion vector constructed
by cloning a mutated CSF-1 cDNA fragment into pAcC1. The source of
, r~ 2
41
the CSF-1 cDNA fragment was pCSF-glyl50 described in PCT Publication
No. W086/04607, supra which contains a TGA stop codon instead of
histidine codon at position 151. It was prepared from the pcCSF-17
insert by site-specific mutagenesis using the appropriate primer,
5'-AGCCAAGGCTGATCAAGGCAGTCC-3'.
Thus, the downstream portion of the coding se4uence was excised from
pcCSF-17 with BstXI^EcoRI, put into an M13 vector for mutagenesis and
returned after plaque purification as a BstXI-EcoRI insert. The
resulting plasmid is designated pCSF-glyl50.
The SmaI-EcoRI fragment (bp 13-953) in pCSF-glyl50 was then
inserted into an M13 vector for a second round of mutagenesis. The
objective this time was to introduce a SmaI recognition site starting
3 bp after the ATG translational start codon of the CSF-glyl50 coding
sequence. This was accomplished by site-specific mutagenesis using
the appropriate primer 5'-GCCCGTATGTCCCCGGGGGGCGCCG-3'. The
mutagenized DNA in M13 is designated EK83RF.
EK83RF was used as the source of the CSF mutein used in the
construction of pAcM4. EK83RF was digested with EcoRI endonuclease,
repaired with Klenow fragment and digested with SmaI endonuclease.
2~ The 770 bp fragment containing the CSF sequence was gel purified and
ligated into SmaI digested pAcC1, using T4 DNA ligase. The ligation
mixture was used to transform MMZ94 and transformants were screened by
minilysates or colony hybridization.
The desired construct pAcM4 thus contains the intact
polyhedrin gene promoter and 5' untranslated leader but three altered
codons in the CSF-1 signal peptide. Instead of Met-Thr-Ala-Pro-
Gly...the sequence in this construct encodes Met-Arg-Pro-Gly Gly...
The CSF-1 5' untranslated leader sequence has been removed so that all
of the sequence 5' to the ATG translational start is derived from the
AcNPV polyhedrin gene (see Figure 5).
B.4 Construction of pAcM5 and pAcM6 Containing "Long Form"
pAcM5 was prepared by placing the coding region of CSF-
4 from pcDBhuCSF-4 into pAc610. pcDBhuCSF-4 was digested with EcoRI
~3~72~
42
and the 1826 bp fragment containing the 522 amino acid coding sequence
of the "long -form" of CSF-1 was isolated by gel electrophoresis. The
pAc610 transfer vector was also digested with EcoRI endonuclease and
ligated with the E RI fragment containing the CSE-1 gene. E. coli
MM294 were transformed with the ligation mixture and transformants
were screened for hybridization to the oligonucleotide. GM11
(described in Section A.2) which hybridizes to the extra amino acid
sequence not found in pcCSF-17. The resul ting construct was
desi gnated pAcM5.
pAcM6 was constructed by excising the 250 bp StuI (positions
511-821 bp on the pcCSF-17 sequence) fragment from pAcM4 (described in
Section B.3) and replacing it with the 1141 bp StuI (positions 343-
1484 bp on the CSF-4 sequence) fragment from pcDBhuCSF-4. The 1141 bp
StuI fragment from pcDBhuCSF-4 contains the sequence from within amino
acid 101 to within amino acid 184 of the shorter pcCSF-17 CSF
sequence, with an additional coding sequence for 298 amino acids
inserted after amino acid 149. Said in another way, the resulting
construction, pAcM6, has the same 5' and 3' sequence as found in pAcM4
with the additional 894 bp of additional codin~ sequence from pcDB
huCSF-4 inserted within the CSF-1 coding sequence.
B.5. Construction of pAcM9
pAcM9 was constructed by replacing a fragment of pAcM4
with a mutated fragment of CSF-1 cDNA derived fro~ M13 clone
EK90/91. The mutations in this fragment alter the coding sequence of
pAcM4 such that amino acids Asnl22 and Asnl40 are replaced with Gln.
EK90/91 was constructed as follows. The 9~3 bp SmaI-
EcoRI fragment from pCSF-17-asp59-glyl50 was cloned into M13mpl8 and
subjected to oligonucleotide directed mutagenesis using the primer 5'-
GMTGTCTTCCAAGAMCAAAGA-3' to produce M13 clone EK90 (codon 122
altered to Gln). EK90 was then mutated with the primer 5'-
CMGMCTGTCAAAACAGCTTTG-3' to produce the double mutant EK90/91 ~codon
122 and 140 altered to Gln). The muta-ted 940 bp SmaI-EcoRI fragment
from EK90/91 was then subcloned into pUC18 to product pUC90/91.
13~72~ ~
43
The construction pAcM9 was co~pleted by excising the
250bp StuI fragment (511-821bp in pCSF17) from pACM4 and ligating it
to the 250bp from pUC90/91. The ligated DNA was transformed into
MM294 and the pAcM9 clone was identified by colony hybridization.
5B.6. Construction of pAcM10
pAcM10 was constructed by inserting a CSF-1 sequence
modified by the introduction of an NcoI site at the ATG translational
start into the pAcC4 transfer vector. The modified CSF-1 sequence was
derived as follows. The double mutein, pCSF-17-asp59-glyl50,
described in PCT Publication No. W086/04607, supra was constructed by
a first site specific mutagenesis to change amino acid 59 to aspartic
acid and create a new EcoRV site at codons 59-60 using the primer 5'-
GGTACAAGATATCATGGAG-3'. The resulting plasmid, pCSF-17-asp59, was
digested with BstXI and EcoRI to p7ace the C-terminal fragment into
M13 for a second site-specific mutagenesis with the same primer
described in Section B.3 for the generation of pCSF-17-glyl5Q. The
mutated fragment was then returned to the vector to obtain pCSF-17-
asp59-glyl50.
pCSF-17-asp59-glyl50 was digested with XmaI (bp 13-18 in
pcCSF-17) and NarI (bp 47-52, bp 99-104, bp 191-196 in pcCSF-17) to
completion. NarI cut twice within the 5' untranslated sequence of
CSF-1 and the large NarI-XmaI fragment containing the CSF-1-asp59-
glyl50 sequence from amino acid 6 on through the vector was puriFied
by gel electrophoresis. Complementary primers having the sequence
5'-CCGGGACCAT&GCCGCGCCGGG-3'
3'-CTGGTACCGGCGCGGCCCGC-5'
were self annealed and llgated to the NarI-XmaI fragment containing
CSF-1 sequence. Transformants of E. coli MM294 were screened For the
presence of the NcoI site introduced in the primer sequence.
pAcM10 was constructed by digesting pCSF-17-NcoI with NcoI
and BamHI. pAcC4 was also doubly digested with NcoI and BamHI and
after fragment isolation, ligation and transformation, minilysates of
E. coli DH5 condidates were screened for the appropriate NcoI-BamHI
fragment sizes.
~72~ ~
44
B.7. Construction of æAcM11
pAcM11 was produced by replacing a fragment of pAcM6
with a mutated fragment of CSF~1 cDNA derived from pUC90/91 (described
above). The mutations in this fragment alter the 522 amino acid
coding sequence of pAcM6 such that amio acids Asnl22 and Asnl40 are
replaced with Gln. pUC90/91 and pcD8huCSF4 (described in Section A.2)
were cleaved with both EcoRI and BsmI. The 1137 bp BsmI (position
477) to EcoRI (position 1614) fragment from pcDBhuCSF~ was agarose-gel
purified and ligated to the large fragment of pUC90/91. The ligation
reaction was transformed into MM294 cells and colonies were screened
by hybridization to the oligomer GMll (described in Section A.2). The
resultant plasmid, pUC90/9lEB, contains the entire coding region of
CSF4 ~ith the changes previously mentioned. The 1138bp StuI fragment
from pUC90/9lEB (343-1481bp) was exchanged with the corresponding
fragment in pAcM6 to produce pAcM11. pAcM11 was transforrned into
MM294 and isolated by colony hybridization.
B.8. Construction of pAcM12 and pAcM13
pAcM12 and pAcM13 ~ere derived from pAcM6 and pAcM11
respectively by replacing a 721 bp BamHI fragment (between positions
683 and 1404) with the corresponding fragment derived from M13 clone
4/29/33, an m13mp18 mutant of CSF4. The 4/29/33 fragment has a stop
codon immediately following the codon for the proline at amino acid
position 221. Therefore, pAcM12 encodes a "long form" CSF 1 protein
that is truncated after proline 221 "long form" CSF-l protein that is
truncated after proline 221 that may be glycosylated at Asn 122 and
Asn 140. pAcM13 encodes a truncated "long form" CSF~1 protein that
cannot be glycosylated at those positions. Clone 4/29/33 was produced
by oligonucleotide directed mutagenesis of 4/29 with the primer GM33
which changed the Asnl22 and Asn140 to Gln residues.
C. Expression of CSF-1 in Insect Cells
Transfection of insect cell s, pl aque purification of
recombinant virus and infection of insect cells (105cells/~l) with the
transfer vectors containing CSF-1 described hereinabove, yielded
~3~72~
between 7500 and 500,000 U/ml of CSF-1 activity in small scale
infections of 2-25 ml. Expression levels for each of the vectors are
compared in Figure 8.
Protein products obtained from the Baculovirus expression
system were analyzed by immunoprecipitation of supernatants from
infected Sf9 cells. Samples were eluted from rabbit anti-CVl-CSF-1
covalently coupled to Sepharose beads, run on SDS-PAGE and stained
wi-th Commasie blue. Figure 6 shows CSF-l produced by cells
cotransfected with pAcM4 (lane 3), pAcM3~1ane 4 and pAcM6 (lane 5).
lo Since the expression products in most cases were
precipitated from cell supernatants it is clear that the products were
secreted. Molecular weights of the CSF proteins produced indicated
that the si3nal peptide was cleaved though the precise site of
cleavage has not yet been determined. The products also appear to be
glycosylated as indicated by their increased molecular weight as
compared to the CSF produced intracellularly (in the absence of a
signal peptide) in E. coli.
Example III
Isolation and Expression of cDNA Fncoding Human G-CSF
A cDNA clone encoding human granulocyte stimulating factor
was isolated from the MIA PaCa-2 cell line, and was expressed using a
recombinant Baculovirus vector in insect cells. The MIA PaCa-2 cell
line described in Yunis, A. A. et al., Experimental Hematol., 12:838
843 (1984), is an established cell line publicly available from the
Cell Repository Lines (CRL) collection of the American Type Culture
Collection, 12301 Parklawn Avenue, Bethesda, MD 20895 under accession
number ATCC CRL 142~. The cDNA clone was sequenced and the
corresponding amino acid sequence was deducedO
A. Isolation of G-CSF mRNA From MIA PaCa-2 Cells
Confluent MIA PaCa-2 cells were stimulated in serum free
Dulbecco's minimum essential medium (DMEM) for 4 days with phorbol
13~72~
46
myristate acetate (50 ng/ml) and retinoic acid (10~ M). The RNA ~as
prepared as described by Chirguin et al.; briefly, the cells were
lysed in 5 M guanidine isothiocyanate Followed by centrifugation
through a 5.7 M cesium chloride (CsCl) cushion; poly A+ RNA was
prepared from the lysed cells by one selection cycle on oligo (dT)
cellulose as described in Maniatis et al., supra.
In more detail, cells were lysed in a solution which
contained 5 M guanidine isothiocyanate, 0.025 M Na-citrate, pH 7, 0.5%
sarcosine and 8~ beta-mercaptoethanol. Molecular biology grade CsCl
was made up to 5.7 M or 40% w/v and buffered with 0.02 M Tris pH 7.5
and 0.002 M Na-EDTA. All solutions were prepared under RNase free
conditions and passed through 0.45 Millipore filters before use.
The lysed cells were then layered onto SW28 ultracentrifuge tubes
which contained layers of 10 ml 5.7 M CsCl and 6 ml 40~ CsCl.
After centrifugation at 26,000 rpm for 18 hours, the RNA
pellet was dissolved in H20 and ethanol precipi-tated twice.
Polyadenylated (Poly A~) messenger RNA (mRNA) was obtained by
chromatography of the total RNA on oligo (dT) cellulose as described
in Maniatis, supra (at page 197).
The RNA was dissolved in sterile H20 and heated to 65CC for
five minutes. An equal volume of a solution containing 0.040 M Tris
Cl pH 7.6, 1.0 M NaCl, 0.002 M EDTA and 0.2% SDS was quickly added and
the sample was cooled to room temperature. The sample was then loaded
on an oligo (dT) column that had been equilibrated with a buFfer
containing 0.020 M Tris pH 7.6, 0.5 M NaCl, 0.001 M EDTA and 0.1%
SDS. The flow through was collected, heated to 65 C, cooled to room
temperature and passed over the column once more. The column was then
washed with 10 volumes of wash buffer (0.02 Tris, 0.1 M NaCl, 0.001 M
FDTA~ 0.1% SDS). Poly A+ mRNA was eluted with aliquots of 0.01 M Tris
pH 7.5, 0.001 M EDTA and ethanol-precipitated twice.
334~9 of MIA PaCa-2 Poly A~ mRNA was fractionated on 5-25%
by weight sucrose gradient centrifugation in 20 mM Tris HCl, pH 7.57 1
mM EDTA, and 0.5% sarcosine using a Beckman SW40 rotor at 20CC and
27,800 rpm for 16 ho~rs. The mRNA fractions were collected in 400~-1
~3~2~
47
fractions and ethanol precipitated twice. Fractions were pooled and
resuspended in 15~A1 H20.
Northern blots were prepared by the electrophoresis of S ~9
of poly A~ RNA per lane or 1 ~ l of each pooled RNA fraction through
1% agarose gels containing 0.5 M formaldehyde followed by blotting
onto nitrocellulose filters. After baking the filters for 1.5 hours
at 80CC, they were prehybridized (5 x SSC, 10 x Denhardt's solution
(0.2% polyvinylpyrrolidone, 0.2~ Ficoll, 0.2% BSA), 0.1% SDS, 50 mM
sodium phosphate pH 7.09 and 100 ~ g/ml tRNA) for one hour at 55 C.
The blots were hybridized for 16 hours at 55 C in a similar solution
that also contained 10% dextran sulfate and 106 cpm per ml of an
oligonucleotide probe labeled with gamma32P-ATP and polynucleotide
kinase. The blot shown in Figure 38 was washed at 55 C in 3 x SSC,
0;1% SDS. The oligonucleotide probe had the sequence 5 ' -
GTAG6TGGCACACAGCTTCTCCTG-3 ' and was designed based on the sequence of
the CHU-2 cDNA clone described by Nagata et al. 9 Nature, 319:415-418
(1986).
The RNA pools that were probe-positive in the above assay
were translated in Xenopus laevis oocytes by the injection of 50 nl of
RNA into each oocyte as described in Gurdon et al., Nature, 233:177-
180 (1971). Supernatants of 10 ~l per oocyte were collected after 40
hours and assayed for CSF activity.
The Xenopus laevis supernatants from each hybridization
fraction were assayed for CSF activity in a murine bone marrow cell
proliferation assay as described in Moore, et al., J. Immunol.,
131:2374-2378 (1983). Briefly, in this assay, 5 x 104 murine bone
marrow cells per/well were incubated in ~-well plates (12 x 8) with
serially diluted Xenopus laevis oocyte supernatants made from
positively hybridizing mRNA fractions. After three days, 3H
thymidine (0.5 ~ Ci/well) was added, and after six hours the cells
were harvested and counted in a liquid scintillation counter.
Peak bone marrow proliferation was found in Xenopus laevis
oocyte supernatants made with mRNA fractions that were most strongly
positive in Northern blots with the above-desoribed oligonucleotide
probe.
48
B. Identification of G-CSF Clones in a MIA PaCa~2 cDNA Library
A cDNA library was prepared from the enriched MIA PaCa-2
mRNA as described in Kawasaki, et al., Science, 30:291-296 (1985).
Briefly, the method used oligo (dT~ priming of the poly A~ tails and
AMV reverse transcriptase employing the method of Okayama, H. et al.,
Mol. Cell Biol., 3:280-289 (19B3). ---~
This method results in a higher proportion of full length
clones than does poly (dG) tailing and effectively uses as host vector
portions of two vectors therein described, and readily obtainable from
lo the authors, pcDV1 and pL1. The resulting vectors contain the insert
between vector fragments containing proximal BamHI and XhoI
restriction sites; the vector contains the pBR322 origin of
replication. and ampicillin resistance ~ene and SV40 control elements
which result in the ability of the vector to effect expression of the
inserted sequences in COS-7 cells.
A 1.2 x 106 clone library in _ coli obtained from the above
enriched MIA PaCa-2 mRNA by the Okayama and Berg method was then
probed using the same oligonucleotide probe that yielded a positive
signal on the most active pooled MIA PaCa-2 mRNA fractions. To probe
the library, E. coli containing the Okayama-Berg vectors were grown up
on nutrient medium. Colonies were lifted onto nitrocellulose filter
papers and were lysed. DNA was fixed to the filter by treatment for
five minutes with 0.5 mM NaOH, 1.5 M NaCl. Filters were then washed
twice for five minutes each time with 1.5 M Tris pH 8, 3 M NaCl and
were air dried and baked at 80 C for two hours.
The filters for the screening were prehybridized and
hybridized to the gamma32P labeled probe as described above in Section
B of the example, but both prehybridization and hybridization were
carried out at 50 C. Plasmids pP12 and pP28 were determined to be
probe positive and were further characterized.
C~ Sequencing of G-CSF MIA PaCa-2 Plasmids cDNA
pP12 was digested with B HI and subcloned into a M13mp19
vector and sequenced using the dideoxy chain-termination method.
`B~
49 ~3~7~
The DNA sequence and predicted protein sequence of pP12 are
shown in Figure 4. The cDNA insert in pP12 is 1510 base pairs long
excluding the poly (dG) and poly (dA) tails; it contains 11 more bases
of 5' untranslated sequence than the CHU-2 G-CSF clone. The major
difference between this clone derived frorn MIA PaCa-2 and CHU-2 cDNA
clones of Nagata et al. is a 9 base pair insertion (GTGATGGAG) in the
CHU-2 clone that would encode the amino acid residues Val-Ser-Glu just
prior to cys-36 in the MIA PaCa-2 G-CSF as indicated by the arrow in
Figure 4. There are two other differences; an A at position 588 in
the MIA PaCa-2 clone (G in the CHU-2 clone) is a silent third base
change, and a T at position 1237 in the MIA PaCa-2 clone (C in the
CHU-2 clone) is in the 3' untranslated region.
2. Construction of Recombinant BaculoYirus Transfer Vectors
Containing the G-CSF Gene
i
D.1. Construction of pAcG1 (also known as pJD2)
The Baculovirus G-CSF expression ~ector9 pAcG1, was
constructed using the pAc610 transfer vector and the G-CSF coding
sequence from pP12. pAc610 was digested with EcoRI endonuclease and
the sticky ends were made blunt using the Klenow fragment as
described. A second digestion with BamHI endonuclease prepared the
transfer vector for receipt of the G-CSF coding sequence immediately
downstream from the polyhedrin gene 5' leader sequence. pP12
containing the G-CSF coding sequence ~as digested with NcoI
endonuclease which contains within its recognition sequence the ATG
translational initiation codon of the G-CSF gene. Following Klenow
repair to generate blunt ends, pP12 was digested with BamHI
endonuclease. The NcoI (blunt)-BamHI fragment containing the G-CSF
gene was purified by gel electrophresis. After ligation wi~h T4
ligase and transformation into c. coli MM294 candidates were screened
by restriction analysis. Double digestion with EcoRV and BamHI
endonucleases resulted in two fragments 1.5 and 8 kb in length.
2 ~ ~
so
D.2 Construction of pAcG2
The pAcG1 plasmid lacks the native G-CSF leader sequence but
does contain a G-C rich segment from the pAc610 polylinker adjacent to
the first ATG of the coding sequence. In addition~ this construction
has a T at the ~3 position from the ATG translational start codon
which has been reported to result in low translational efficiency
(Kozak, M. Cell, (1986) 44:283). The new transfer vector pAcC3 was
designed to eliminate the G-C rich region, restore the complete
polyhedrin leader and place an A residue at the -3 position to the
0 translational start.
pAcG2 was constructed by introducing an NcoI-~I fragment
from pP12 containing the G-CSF coding sequence into pAcC3. pP12 was
digested with SspI endonuclease which cuts some 4~ bp 3' to the
translational termination codon of G-CSF to produce blunt ends. A
second digestion with NcoI endonuclease results in a 654 bp fragment
containing the G-CSF coding sequence (minus leader) which was gel
purified. pAcC3 was doubly digested with NcoI and SmaI endonucleases
and purified by gel electrophoresis for ligation to the G-CSF
fragment. After ligation with T4 ligase and transformation into
MM294(?), transformants were screened for presence of insert by
digesting with restriction endonucleases. The presence of an NcoI
restriction endonuclease recognition site ensured that the
! translational initiation codon had been regenerated.
E. Ex~ression of G-CSF i n Insect Cells
Transfection of insect cells, plaque purification of
recombinant virus and infection of insect cells (105 cells/ml) with
pAcG1 yielded about 90,000 units/ml of G-CSF. Expression levels
obtained with pAcG2 after similar procedures were about 2.4 x 106
units/ml of G-CSF. These levels are compared to levels of CSF-l
produced in at least two different constructions in Figure 8.
Protein products obtained from the ~culovirus expression
system were analyzed by immunoblotting of supernatants from the
infected Sf9 cells.
~3~7~
51
Other modifications of the above described embodiments of
the invention which are obvious to those of skill in the area of
molecular biology and related disciplines are intended to be within
the scope of the claims that follow.
Deposits
The materials listed below were deposited with the American
Type Culture Collection, Rockville, MD, USA (ATCC). The deposits were
made under the provisions of the Budapest Treaty on the International
Recognition of the Deposit of Microorganisms for the ~rposes of
10Patent Procedure and the Regulations thereunder ~Budapest Treaty)~
Maintenance of a viable culture is assured for 30 years from date of
deposit. The organism will be made available by ATCC under the terms
of the Budapest Treaty, and subject to an agreement between Applicants
and ATCC which assures unrestricted availability upon issuance of the
15pertinent U.S. patent. Availability of the deposited strain is not to
be construed as a license to practice the invention in contravention
of the rights granted under the authority of any government in
accordance with its patent laws.
~,
Recombinant Deposit
20Transfer Vector CMCC# ATCC#Date
pAcM3 6/12/87
pAcM~ 6/12/87
pAcM6 6/12/87
pAcM10 6/12187
pAcG2
