DNA encoding for CSF-1 and accompanying recombinant systems

A colony stimulating factor, CSF-1, is a lymphokine useful in overcoming the immunosuppression induced by chemotherapy or resulting from other causes. CSF-1 is obtained in usable amounts by recombinant methods, including cloning and expression of the murine and human DNA sequences encoding this protein.

TECHNICAL FIELD 
The present invention relates to the use of recombinant technology for 
production of lymphokines ordinarily produced in low concentration. More 
specifically, the invention relates to the cloning and expression of a DNA 
sequence encoding human colony stimulating factor-1 (CSF-1). 
BACKGROUND ART 
The ability of certain factors produced in very low concentration in a 
variety of tissues to stimulate the growth and development of bone marrow 
progenitor cells into granulocytes and/or macrophages has been known for 
nearly 15 years. The presence of such factors in sera, urine samples, and 
tissue extracts from a number of species is demonstrable using an in vitro 
assay which measures the stimulation of colony formation by bone marrow 
cells plated in semi-solid culture medium. There is no known in vivo 
assay. Because these factors induce the formation of such colonies, the 
factors collectively have been called Colony Stimulating Factors (CSF). 
More recently, it has been shown that there are at least four subclasses of 
human CSF proteins which can be defined according to the types of cells 
found in the resultant colonies. One subclass, CSF-1, results in colonies 
containing macrophages predominantly. Other subclasses produce colonies 
which contain both neutrophilic granulocytes and macrophages; which 
contain predominantly neutrophilic granulocytes; and which contain 
neutrophilic and eosinophilic granulocytes and macrophages. 
There are murine factors analogous to the first three of the above human 
CSFs. In addition, a murine factor called IL-3 induces colonies from 
murine bone marrow cells which contain all these cell types plus 
megakaryocytes, erythrocytes, and mast cells, in various combinations. 
These CSFs have been reviewed by Dexter, T. M., Nature (1984) 309: 746, 
and Vadas, M. A., et al, J Immunol (1983) 130: 793. 
The invention herein is concerned with the recombinant production of 
proteins which are members of the first of these subclasses, CSF-1. This 
subclass has been further characterized and delineated by specific 
radioimmunoassays and radioreceptor assays--e.g., antibodies raised 
against purified CSF-1 are able to suppress specifically CSF-1 activity, 
without affecting the biological activities of the other subclasses, and 
macrophage cell line J774 contains receptors which bind CSF-1 
specifically. A description of these assays was published by Das, S. K., 
et al, Blood (1981) 58:630. 
Purification methods for various CSF proteins have been published and are 
described in the following paragraphs. 
Stanley, E. R., et al, J Biol Chem (1977) 252: 4305 reported purification 
of a CSF protein from murine L929 cells to a specific activity of about 
1.times.10.sup.8 units/mg, which also stimulated mainly macrophage 
production. Waheed, A., et al, Blood (1982) 60: 238, described the 
purification of mouse L-cell CSF-1 to apparent homogeneity using a rabbit 
antibody column and reported the first 25 amino acids of the murine 
sequence (Ben-Avram, C. M., et al, Proc Natl Acad Sci (USA) (1985) 882: 
4486). 
Stanley, E. R., et al, J Biol Chem (1977) 252: 4305-4312 disclosed a 
purification procedure for CSF-1 from human urine and Das, S. K., et al, 
Blood (1981) 58: 630; J Biol Chem (1982) 257: 13679 obtained a human 
urinary CSF-1 at a specific activity of 5.times.10.sup.7 units/mg which 
produced only macrophage colonies, and outlined the relationship of 
glycosylation of the CSF-1 proteins prepared from cultured mouse L-cells 
and from human urine to their activities. Wang, F. F., et al, J Cell 
Biochem (1983) 21: 263, isolated human urinary CSF-1 to specific activity 
of 10.sup.8 U/mg. Waheed, A., et al, disclosed purification of human 
urinary CSF-1 to a specific activity of 0.7-2.3.times.10.sup.7 U/mg on a 
rabbit antibody column (Exp Hemat (1984) 12: 434). 
Wu, M., et al, J Biol Chem (1979) 254: 6226 reported the preparation of a 
CSF protein from cultured human pancreatic carcinoma (MIAPaCa) cells which 
resulted in the growth of murine granulocytic and macrophagic colonies. 
The resulting protein had a specific activity of approximately 
7.times.10.sup.7 units/mg. 
Partially purified preparations of various CSFs have also been reported 
from human and mouse lung-cell conditioned media (Fojo, S. S., et al, 
Biochemistry (1978) 17: 3109; Burgess, A. W., et al, J Biol Chem (1977) 
252: 1998); from human T-lymphoblast cells (Lusis, A. J., et al, Blood 
(1981) 57: 13; U.S. Pat. No. 4,438,032); from human placental conditioned 
medium to apparent homogeneity and specific activity of 7.times.10.sup.7 
U/mg (Wu, M., et al, Biochemistry (1980) 19: 3846). 
A significant difficulty in putting CSF proteins in general, and CSF-1 in 
particular, to any useful function has been their unavailability in 
distinct and characterizable form in sufficient amounts to make their 
employment in therapeutic use practical or even possible. The present 
invention remedies these deficiencies by providing purified human and 
murine CSF-1 in useful amounts through recombinant techniques. 
A CSF protein of a different subclass, murine and human GM-CSF has been 
purified and the cDNAs cloned. This protein was shown to be distinct from 
other CSFs, e.g., CSF-1, by Gough, et al, Nature (1984) 309: 763-767. 
Murien IL-3 has been cloned by Fung, M. C., et al, Nature (1984) 307: 233. 
See also Yokota, T., et al, PNAS (1984) 81: 1070-1074; Wong, G. G., et al, 
Science (1985) 228: 810-815; Lee, F., et al, PNAS (1985) 82: 4360-4364; 
and Cantrell, M. A., et al, PNAS (1985) 82: 6250-6254. 
DISCLOSURE OF THE INVENTION 
In one aspect, the present invention relates to recombinant CSF-1 protein, 
including the biologically active proteins containing modifications of 
primary amino acid sequence of the native protein. CSF-1 protein in 
recombinanat form can be obtained in quantity, can be modified 
advantageously through regulation of the post-translational processing 
provided by the host, and can be intentionally modified at the genetic or 
protein level to enhance its desirable properties. For example, muteins 
having deletions of substantial portions of the carboxy terminal one-third 
of the polypeptide are thus active. Thus, the availability of CSF-1 in 
recombinant form provides both flexibility and certain quantitative 
advantages which make possible applications for use of the protein 
therapeutically, that are unavailable with respect to the native protein. 
In other aspects, the invention relates to an isolated DNA sequence 
encoding recombinant CSF-1, to recombinant expression systems for this 
sequence and to vectors containing them, to recombinant hosts which are 
transformed with these vectors, and to cultures producing the recombinant 
protein. The invention further relates to methods for producing the 
recombinant protein and to the materials significant in its production. 
In addition, the invention relates to compositions containing CSF-1 which 
are useful in pharmaceutical and therapeutic applications, and to methods 
of use for such compositions.

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 formation of primarily 
macrophage colonies. Native CSF-1 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 dimer by in 
vitro provision of intracellular conditions, and the monomer is per se 
useful as an antigen to produce anti-CSF-1 antibodies. 
There appears to be some species specificity: 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-1 (Das, S. K., et al, supra). 
However, in certain special circumstances (such as, for example, where a 
particular antibody preparation recognizes a CSF-1 epitope 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, 
including the ability of this protein to stimulate the secretion of series 
E prostaglandins, interleukin-1, and interferon from mature macrophages 
(Moore, R., et al, Science (1984) 223: 178). 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 (Stanely, E. R., 
The Lymphokines (1981), Stewart, W. E., II, et al, ed, Humana Press, 
Clifton, NJ), pp. 102-132) and that receptors for CSF-1 are restricted to 
these cell lines (Byrne, P. V., et al, Cell Biol (1981) 91: 848)). 
As is the case for all proteins, the precise chemical structure 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 
salt, 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 
by derivatization using sugar moieties (glycosylation) or by other 
supplementary molecules 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, most frequently 
dimers. Indeed, native human urinary CSF-1 is isolated as a highly 
glycosylated dimer. Certain aspects of such augmentation are accomplished 
through post-translational processing systems of the producing host; other 
such modifications may be introduced in vitro. In 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, either 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". Indeed, 
human and murine derived CSF-1 proteins have non-identical but similar 
primary amino acid sequences which display a high homology. 
For convenience, the mature protein amino acid sequence of the monomeric 
portion of a dimeric protein shown in FIG. 5, deduced from the cDNA clone 
illustrated herein, is designated mCSF-1 (mature CSF-1). FIG. 5 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-1 are muteins which monomers and dimers are mCSF-1 
and related forms of mCSF-1, designated by their differences from 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 FIG. 5. The substitution of a particular amino acid will be noted 
by reference to the amino acid residue which it replaces. Thus, for 
example, ser.sub.90 CSF-1 refers to the protein which has the sequence 
shown in FIG. 5 except that the amino acid at position 90 is serine rather 
than cysteine. Deletions are noted by a .gradient. followed by the number 
of amino acids deleted from the N-terminal sequence, or by the number of 
amino acids remaining when residues are deleted from the C-terminal 
sequence, when the number is followed by a minus sign. Thus, 
.gradient..sub.4 CSF-1 refers to CSF-1 of FIG. 5 wherein the first 4 amino 
acids from the N-terminus have been deleted; .gradient..sub.130- refers to 
CSF-1 wherein the last 94 amino acids following amino acid 130 have been 
deleted. Illustrated below are, for example, asp.sub.59 CSF-1, which 
contains an aspartic residue encoded by the gene (FIG. 5) at position 59 
rather than the tyrosine residue encoded by the cDNA, and 
.gradient..sub.158- CSF-1, which comprises only amino acids 1-158 of 
mCSF-1. 
"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. The 
control sequences which are suitable for procaryotes, for example, include 
a promoter, optionally an operator sequence, a ribosome binding site, and 
possibly, other as yet poorly understood, sequences. Eucaryotic cells are 
known to utilize promoters, polyadenylation signals, and enhancers. 
"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. In order to effect transformation, the expression system may be 
included on a vector; however, the relevant DNA may then also be 
integrated into the host chromosome. 
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. 
B. General Description 
The CSF-1 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 appplication, 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-1 to mediate and 
enhance the growth and differentiation of bone marrow-derived precursors 
into macrophages and monocytes and to stimulate some of the functions of 
these mature cells, 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 CSF-1. 
In general, any subject suffering from immunosuppression whether due to 
chemotherapy, bone marrow transplantation, or other, accidental forms of 
immunosuppression such as disease (e.g., acquired immune deficiecny 
syndrome) would benefit from the availability of CSF-1 for pharmacological 
use. In addition, subjects could be supplied enhanced amounts of 
previously differentiated macrophages to supplement those of the 
indigenous system, which macrophages are produced by in vitro culture of 
bone marrow or other suitable preparations treated with CSF-1. These 
preparations include those of the patient's own blood monocytes, which can 
be so cultured and returned for local or systemic therapy. 
The ability of CSF-1 to stimulate production of lymphokines by macrophages 
and to enhance their ability to kill target cells also makes CSF-1 
directly useful in treatment of neoplasms and infections. 
CSF-1 stimulates the production of interferons by murine-derived macrophage 
(Fleit, H. B., et al, J Cell Physiol (1981) 108: 347), and human, 
partially purified, CSF-1 from MIAPaCa cells stimulates the poly(I):poly 
(C)-induced production of interferon and TNF from human monocytes as 
illustrated below. In addition, CSF-1 stimulates the production of myeloid 
CSF by human blood monocytes. 
Also illustrated below is a demonstration of the ability of murine CSF-1 
(from L-cell-conditioned medium) to stimulate normal C3H/HeN mouse 
peritoneal macrophages to kill murine sarcoma TU5 targets. This activity 
is most effective when the CSF-1 is used as pretreatment and during the 
effector phase. The ability of CSF-1 to do so is much greater than that 
exhibited by other colony stimulating factors, as shown in FIG. 6 
hereinbelow. In addition, the ability of murine cells to attack viruses is 
enhanced by CSF-1. 
Murine CSF-1 is inconsistently reported to stimulate murine macrophage to 
be cytostatic to P815 tumor cells (Wing, E. J., et al, J Clin Invest 
(1982) 69: 270) or not to kill other leukemia targets (Ralph, P. et al, 
Cell Immunol (1983) 76: 10). Nogawa, R. T., et al, Cell Immunol (1980) 53: 
116, report that CSF-1 may stimulate macrophages to ingest and kill yeast. 
Thus, in addition to overcoming immunosuppression per se, CSF-1 can be used 
to destroy the invading organisms or malignant cells indirectly by 
stimulation of macrophage secretions and activity. 
The CSF-1 of the invention may be formulated in conventional ways standard 
in the art for the administration of protein substances. Administration by 
injection is preferred; formulations include solutions or suspensions, 
emulsions, or solid composition for reconstitution into injectables. 
Suitable excipients include, for example, Ringer's solution, Hank's 
solution, water, saline, glycerol, dextrose solutions, and the like. In 
addition, the CSF-1 of the invention may be preincubated with preparations 
of cells in order to stimulate appropriate responses, and either the 
entire preparation or the supernatant therefrom introduced into the 
subject. As shown hereinbelow, the materials produced in response to CSF-1 
stimulation by various types of blood cells are effective against desired 
targets, and the properties of these blood cells themselves to attack 
invading viruses or neoplasms may be enhanced. The subject's own cells may 
be withdrawn and used in this way, or, for example, monocytes or 
lymphocytes from another compatible individual employed in the incubation. 
Although the existence of a pattern of activity designated CSF-1 has been 
known for some time, the protein responsible has never been obtained in 
both sufficient purity and in sufficient amounts to permit sequence 
determination, nor in sufficient purity and quantity to provide a useful 
therapeutic function. Because neither completely pure practical amounts of 
the protein nor its encoding DNA have been available, it has not been 
possible to optimize modifications to structure by providing such 
alternatives as those set forth in A above, nor has it been possible to 
utilize this protein in a therapeutic context. 
The present invention remedies these defects. Through a variety of 
additional purification procedures, sufficient pure CSF-1 has been 
obtained from human urine to provide some amino acid sequence, thus 
permitting the construction of DNA oligomeric probes. The probes are 
useful in obtaining the coding sequence for the entire protein. One 
approach, illustrated below, employs probes designed with respect to the 
human N-terminal sequence to probe the human genomic library to obtain the 
appropriate coding sequence portion. The human genomic cloned sequence can 
be expressed directly using its own control sequences, or in constructions 
appropriate to mammalian systems capable of processing introns. The 
genomic sequences are also used as probes for a human cDNA library 
obtained from a cell line which produces CSF-1 to obtain cDNA encoding 
this protein. The cDNA, when suitably prepared, can be expressed directly 
in COS or CV-1 cells and can be constructed into vectors suitable for 
expression in a wide range of hosts. 
Thus these tools can provide the complete coding sequence for human CSF-1 
from which expression vectors applicable to a variety of host systems can 
be constructed and the coding sequence expressed. The variety of hosts 
available along with expression vectors suitable for such hosts permits a 
choice among post-translational processing systems, and of environmental 
factors providing conformational regulation of the protein thus produced. 
C. Suitable Hosts, Control Systems and Methods 
In general terms, the production of a recombinant form of CSF-1 typically 
involves the following: 
First a DNA encoding the mature (used here to include all muteins) protein, 
the preprotein, or a fusion of the CSF-1 protein to an additional sequence 
which does not destroy its activity or to additional sequence cleavable 
under controlled conditions (such as treatment with peptidase) to give an 
active protein, is obtained. If the sequence is uninterrupted by introns 
it is suitable for expression in any host. If there are introns, 
expression is obtainable in mammalian or other eucaryotic systems capable 
of processing them. This sequence should be in excisable and recoverable 
form. The excised or recovered coding sequence is then placed in operable 
linkage with suitable control sequences in a replicable expression vector. 
The vector is used to transform a suitable host and the transformed host 
cultured under favorable conditions to effect the production of the 
recombinant CSF-1. Optionally the CSF-1 is isolated from the medium or 
from the cells; recovery and purification of the protein may not be 
necessary in some instances, where some impurities may be tolerated. For 
example, for in vitro cultivation of cells from which a lymphokine factor 
will be isolated for administration to a subject, complete purity is not 
required. However, direct use in therapy by administration to a subject 
would, of course, require purification of the CSF-1 produced. 
Each of the foregoing steps can be done in a variety of ways. For example, 
the desired coding sequences can be obtained by preparing suitable cDNA 
from cellular messenger and manipulating the cDNA to obtain the complete 
sequence. Alternatively, genomic fragments may be obtained and used 
directly in appropriate hosts. The constructions for expression vectors 
operable in a variety of hosts are made using appropriate replicons and 
control sequences, as set forth below. Suitable restriction sites can, if 
not normally available, be added to the ends of the coding sequence so as 
to provide an excisable gene to insert into these vectors. 
The control sequences, expression vectors, and transformation methods are 
dependent on the type of host cell used to express the gene. Generally, 
procaryotic, yeast, or mammalian cells are presently useful as hosts. 
Since native CSF-1 is secreted as a glycosylated dimer, host systems which 
are capable of proper post-translational processing are preferred. 
Accordingly, although procaryotic hosts are in general the most efficient 
and convenient for the production of recombinant proteins, eucaryotic 
cells, and, in particular, mammalian cells are preferred for their 
processing capacity. Recombinant CSF-1 produced by bacteria would require 
in vitro dimerization. In addition, there is more assurance that the 
native signal sequence will be recognized by mammalian cell hosts making 
secretion possible, and purification therefore easier. 
C.1. Control Sequences And Corresponding Hosts 
Procaryotes most frequently are represented by various strains of E. coli. 
However, other microbial strains may also be used, such as bacilli, for 
example Bacillus subtilis, various species of Pseudomonas, or other 
bacterial strains. In such procaryotic systems, plasmid vectors which 
contain replication sites and control sequences derived from a species 
compatible with the host are used. For example, E. coli is typically 
transformed using derivatives of pBR322, a plasmid derived from an E. coli 
species by Bolivar, et al, Gene (1977) 2: 95. pBR322 contains genes for 
ampicillin and tetracycline resistance, and thus provides additional 
markers which can be either retained or destroyed in constructing the 
desired vector. Commonly used procaryotic control sequences which are 
defined herein to include promoters for transcription initiation, 
optionally with an operator, along with ribosome binding site sequences, 
include such commonly used promoters as the beta-lactamase (penicillinase) 
and lactose (lac) promoter systems (Chang, et al, Nature (1977) 198: 1056) 
and the tryptophan (trp) promoter system (Goeddel, et al Nucleic Acids Res 
(1980) 8: 4057) and the lambda derived P.sub.L promoter and N-gene 
ribosome binding site (Shimatake, et al, Nature (1981) 292: 128), which 
has been made useful as a portable control cassette, as set forth in U.S. 
Ser. No. 685,312 now U.S. Pat. No. 4,711,845, issued Dec. 8, 1987 which is 
a CIP of U.S. Ser. No. 646,693 now abandoned which was a CIP of abandoned 
application Ser. No. 578,133, filed Feb. 8, 1984, and assigned to the same 
assignee. However, any available promoter system compatible with 
procaryotes can be used. 
In addition to bacteria, eucaryotic microbes, such as yeast, may also be 
used as hosts. Laboratory strains of Saccharomyces cerevisiae, Baker's 
yeast, are most used although a number of other strains are commonly 
available. While vectors employing the 2 micron origin of replication are 
illustrated, Broach, J. R., Meth Enz (1983) 101: 307, other plasmid 
vectors suitable for yeast expression are known (see, for example, 
Stinchcomb, et al, Nature (1979) 282: 39, Tschempe, et al, Gene (1980) 10: 
157 and Clarke, L, et al, Meth Enz (1983) 101: 300). Control sequences for 
yeast vectors include promoters for the synthesis of glycolytic enzymes 
(Hess, et al, J Adv Enzyme Reg (1968) 7: 149; Holland, et al, Biochemistry 
(1978) 17: 4900). Additional promoters known in the art include the 
promoter for 3-phosphoglycerate kinase (Hitzeman, et al, J Biol Chem 
(1980) 255: 2073), and those for other glycolytic enzymes, such as 
glyceraldehyde- 3-phosphate dehydrogenase, hexokinase, pyruvate 
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, 
phosphoglucose isomerase, and glucokinase. Other promoters, which have the 
additional advantage of transcription controlled by growth conditions, are 
the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid 
phosphatase, degradative enzymes associated with nitrogen metabolism, and 
enzymes responsible for maltose and galactose utilization (Holland, ibid). 
It is also believed terminator sequences are desirable at the 3' end of 
the coding sequences. Such terminators are found in the 3' untranslated 
region following the coding sequences in yeast-derived genes. Many of the 
vectors illustrated contain control sequences derived from the enolase 
gene containing plasmid peno46 (Holland, M. J., et al, J Biol Chem (1981) 
256: 1385) or the LEU2 gene obtained from YEp13 (Broach, J., et al, Gene 
(1978) 8: 121), however, any vector containing a yeast compatible 
promoter, origin of replication and other control sequences is suitable. 
It is also, of course, possible to express genes encoding polypeptides in 
eucaryotic host cell cultures derived from multicellular organisms. See, 
for example, Tissue Culture, Academic Press, Cruz and Patterson, editors 
(1973). Useful host cell lines include murine myelomas N51, VERO and HeLa 
cells, and Chinese hamster ovary (CHO) cells. Expression vectors for such 
cells ordinarily include promoters and control sequences compatible with 
mammalian cells such as, for example, the commonly used early and later 
promoters from Simian Virus 40 (SV 40) (Fiers, et al, Nature (1978) 273: 
113), or other viral promoters such as those derived from polyoma, 
Adenovirus 2, bovine papilloma virus, or avian sarcoma viruses, or 
immunoglobulin promoters and heat shock promoters. General aspects of 
mammalian cell host system transformations have been described by Axel; 
U.S. Pat. No. 4,399,216 issued Aug. 16, 1983. It now appears also that 
"enhancer" regions are important in optimizing expression; these are, 
generally, sequences found upstream of the promoter region. Origins of 
replication may be obtained, if needed, from viral sources. However, 
integration into the chromosome is a common mechanism for DNA replication 
in eucaryotes. Plant cells are also now available as hosts, and control 
sequences compatible with plant cells such as the nopaline synthase 
promoter and polyadenylation signal sequences (Depicker, A., et al, J Mol 
Appl Gen (1982) 1: 561) are available. 
C.2. Transformations 
Depending on the host cell used, transformation is done using standard 
techniques appropriate to such cells. The calcium treatment employing 
calcium chloride, as described by Cohen, S. N., Proc Natl Acad Sci (USA) 
(1972) 69: 2110, is used for procaryotes or other cells which contain 
substantial cell wall barriers. Infection with Agrobacterium tumefaciens 
(Shaw, C. H., et al, Gene (1983) 23: 315) is used for certain plant cells. 
For mammalian cells without such cell walls, the calcium phosphate 
precipitation method of Graham and van der Eb, Virology (1978) 52: 546 is 
preferred. Transformations into yeast are carried out according to the 
method of Van Solingen, P., et al, J Bact (1977) 130: 946 and Hsiao, C. 
L., et al, Proc Natl Acad Sci (USA) (1979) 76: 3829. 
C.3. Probing mRNA by Northern Blot; Probe of cDNA or Genomic Libraries 
RNA is fractionated for Northern blot by agarose slab gel electrophoresis 
under fully denaturing conditions using formaldehyde (Maniatis, T., et al, 
Molecular Cloning (1982) Cold Spring Harbor Press, pp 202-203) or 10 mM 
methyl mercury (CH.sub.3 HgOH) (Bailey, J. M., et al, Anal Biochem (1976) 
70: 75-85; and Sehgal, P. B., et al, Nature (1980) 288: 95-97) as the 
denaturant. For methyl mercury gels, 1.5% gels are prepared by melting 
agarose in running buffer (100 mM boric acid, 6 mM sodium borate, 10 mM 
sodium sulfate, 1 mM EDTA, pH 8.2), cooling to 60.degree. C. and adding 
1/100 volume of 1M CH.sub.3 HgOH. The RNA is dissolved in 0.5.times. 
running buffer and denatured by incubation in 10 mM methyl mercury for 10 
min 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 for 40 min 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 colonies, 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 5 min with 500 mM NaOH, 1.5M NaCl. The filters are washed 
twice for 5 min each time with 5.times.standard saline citrate (SSC) and 
are air dried and baked at 80.degree. C. for 2 hr. 
The gels for Northern blot or the duplicate filters for cDNA or genomic 
screening are prehybridized at 25.degree.-42.degree. C. for 6-8 hr with 10 
ml per filter of DNA hybridization buffer without probe (0-50% formamide, 
5-6.times.SSC, pH 7.0, 5.times.Denhardt's solution (polyvinylpyrrolidine, 
plus Ficoll and bovine serum albumin; 1.times.=0.02% of each), 20-50 mM 
sodium phosphate buffer at pH 7.0, 0.2% SDS, 20 .mu.g/ml poly U (when 
probing cDNA), and 50 .mu.g/ml denatured salmon 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 on the 
stringency desired, and vary, for example, with probe length. Typical 
conditions for relatively long (e.g., more than 30-50 nucleotide) probes 
employ a temperature of 42.degree.-55.degree. C. and hybridization buffer 
containing about 20%-50% formamide. For the lower stringencies needed for 
oligomeric probes of about 15 nucleotides, lower temperatures of about 
25.degree.-42.degree. C., and lower formamide concentrations (0%-20%) are 
employed. For longer probes, the filters may be washed, for example, four 
times for 30 minutes, each time at 40.degree.-55.degree. C. with 
2.times.SSC, 0.2% SDS and 50 mM sodium phosphate buffer at pH 7, then 
washed twice with 0.2.times.SSC and 0.2% SDS, air dried, and are 
autoradiographed at -70.degree. C. for 2 to 3 days. Washing conditions are 
somewhat less harsh for shorter probes. 
C.4. 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. 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 Catalog. In general, about 1 .mu.g of 
plasmid or DNA sequence is cleaved by one unit of enzyme in about 20 .mu.l 
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.degree. 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 desired, size 
separation of the cleaved fragments may be performed by polyacrylamide gel 
or agarose gel electrophoresis using standard techniques. A general 
description of size separations is found in Methods in Enzymology (1980) 
65: 499-560. 
Restriction cleaved fragments may be blunt ended by treating with the large 
fragment of E. coli DNA polymerase I (Klenow) in the presence of the four 
deoxynucleotide triphosphates (dNTPs) using incubation times of about 15 
to 25 min at 20.degree. to 25.degree. C. in 50 mM Tris pH 7.6, 50 mM NaCl, 
6 mM MgCl.sub.2, 6 mM DTT and 5-10 .mu.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 desried, 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 treatment 
with Klenow, the mixture is extracted with phenol/chloroform and ethanol 
precipitated. Treatment under appropriate conditions with S1 nuclease 
results in hydrolysis of any single-stranded portion. 
Synthetic oligonucleotides may 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 excess, e.g., approximately 10 units of 
polynucleotide kinase to 1 nmole substrate in the presence of 50 mM Tris, 
pH 7.6, 10 mM MgCl.sub.2, 5 mM dithiothreitol, 1-2 mM ATP. If kinasing is 
for labeling of probe, the ATP will contain high specific activity 
32.UPSILON.P. 
Ligations are performed in 15-30 .mu.l volumes under the following standard 
conditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl.sub.2, 10 mM 
DTT, 33 .mu.g/ml BSA, 10 mM-50 mM NaCl, and either 40 .mu.M ATP, 0.01-0.02 
(Weiss) units T4 DNA ligase at 0.degree. C. (for "sticky end" ligation) or 
1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14.degree. C. (for "blunt 
end" ligation). Intermolecular "sticky end" ligations are usually 
performed at 33-100 .mu.g/ml 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 .mu.M total ends 
concentration. 
In the vector construction employing "vector fragments", the vector 
fragment is commonly 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.sup.+ and Mg.sup.+2 using about 1 unit of BAP per .mu.g of 
vector at 60.degree. for 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.5. Modification of DNA Sequences 
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 DNA 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. 
Cultures 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 original strand are sufficient to prevent hybridization. Plaques 
which hybridize with the probe are then picked, cultured, and the DNA 
recovered. Details of site specific mutation procedures are described 
below in specific examples. 
C.6. Verification of Construction 
In the constructions set forth below, correct ligations for plasmid 
construction are confirmed by first transforming E. coli strain MM294, or 
other suitable host, with the ligation mixture. Successful transformants 
are selected by ampicillin, tetracycline or other antibiotic resistance or 
using other markers depending on the mode of plasmid construction, as is 
understood in the art. Plasmids from the transformants are then prepared 
according to the method of Clewell, D. B., et al, Proc Natl Acad Sci (USA) 
(1969) 62: 1159, optionally following chloramphenicol amplification 
(Clewell, D. B., J Bacteriol (1972) 110: 667). The isolated DNA is 
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 (1980) 65: 499. 
C.7. Hosts Exemplified 
Host strains used in cloning and expression herein are as follows: 
For cloning and sequencing, and for expression of construction under 
control of most bacterial promoters, E. coli strain MM294 obtained from E. 
coli Genetic Stock Center GCSC #6135, was used as the host. For expression 
under control of the P.sub.L N.sub.RBS promoter, E. coli strain K12 MC1000 
lambda lysogen, N.sub.7 N.sub.53 cI857 SusP80, ATCC 39531 is used. 
For M13 phage recombinants, E. coli strains susceptible to phage infection, 
such as E. coli K12 strain DG98, are employed. The DG98 strain has been 
deposited with ATCC July 13, 1984 and has accession number 39768. 
Mammalian expression has been accomplished in COS-7 and CV-1 cells. 
D. Preferred Embodiments 
The recombinant CSF-1 of the invention can be considered a set of muteins 
which have similar but not necessarily identical primary amino acid 
sequences, all of which exhibit, or are specifically cleavable to a mutein 
which exhibits, the activity pattern characteristic of CSF-1--i.e. they 
are capable of stimulating bone marrow cells to differentiate into 
monocytes, preponderantly, and, within the limitations set forth in the 
Definitions section above, are immunoreactive with antibodies raised 
against native CSF-1 and with the receptors associated with CSF-1 
activity. Certain embodiments of these muteins are, however, preferred. 
The primary sequence shown in FIG. 5 for mCSF-1 has the required activity, 
and it is, of course, among the preferred embodiments. Also preferred are 
muteins wherein certain portions of the sequence have been altered by 
either deletion of, or conservative substitution of, one or more amino 
acids in mCSF-1. By a "conservative" amino acid substitution is meant one 
which does not change the activity characteristics of the protein, and in 
general is characterized by chemical similarity of the side chains of the 
two residues interchanged. For example, acidic residues are conservatively 
replaced by other acidic residues, basic by basic, hydrophobic by 
hydrophobic, bulky by bulky, and so forth. The degree of similarity 
required depends, of course, on the criticality of the amino acid for 
which substitution is made, and its nature. Thus, in general, preferred 
substitutions for cysteine residues are serine and alanine; for aspartic 
acid residues, glutamic acid; for lysine or arginine residues, histidine; 
for leucine residues, isoleucine, or valine; for trytophan residues, 
phenylalanine or tyrosine; and so forth. 
Regions of the CSF-1 protein which are most tolerant of alteration include 
those regions of known low homology between human and mouse species 
(residues 15-20 and 75-84); regions which confer susceptibility to 
proteolytic cleavage (residues 51 and 52 and residues 191-193); cysteine 
residues not participating in disulfide linkages, or residues which are 
not absolutely essential for activity (residues 159-224). It also appears 
residues 151-224 are not essential. 
Therefore, particularly preferred are those CSF-1 muteins characterized by 
the deletion or conservative substitution of one or more amino acids 
and/or one or more sequences of amino acids between positions 159 and 224 
inclusive of mCSF-1 or positions 151-224 inclusive. In particular, 
.gradient..sub.158- CSF-1 has CSF activity comparable to that of the 
native protein, even is a limited number of additional amino acid residues 
not related to CSF-1 are included at the truncated C-terminus; 
.gradient..sub.150- CSF-1 has similar activity. Indeed, the native protein 
is reported to have a molecular weight of 14-15 kd (as opposed to the 26 
kd predicted from the cDNA sequence) and the hydrophobicity deduced from 
the recombinant (predicted) amino acid sequence corresponds to a 
transmembrane region normally susceptible to cleavage. It may, therefore, 
be that the truncated version corresponds in a rough way to the CSF-1 as 
isolated. 
Also preferred are muteins characterized by the deletion or conservative 
substitution of one or more of the amino acids at postitions 51 and 52 
and/or positions 191, 192 and 193 of mCSF-1. Especially preferred is 
gln.sub.52 CSF-1; a corresponding proline substitution is not 
conservative, and does not yield an active CSF. Since they represent 
regions of apparently low homology, another preferred set of embodiments 
is that characterized by the deletion or conservative substitution of one 
or more of the amino acids at positions 15-20 and/or positions 75-84 of 
mCSF-1. Also preferred are those muteins characterized by the deletion or 
conservative substitution of the cysteine residue at any position not 
essential for disulfide bond formation. Also preferred are those muteins 
characterized by the deletion or substitution of the tyrosine residue at 
position 59 of mCSF-1; particularly substitution by an aspartic acid 
residue. 
E. Cloning and Expression of Human CSF-1 
The following illustrates the methods used in obtaining the coding sequence 
for human CSF-1, for disposing this sequence in expression vectors, and 
for obtaining expression of the desired protein. 
E.1. Purification of Native Human CSF-1 and Probe Design 
Human urinary CSF-1 was partially purified by standard methods as described 
by Das, S. K., et al, Blood (1981) 58: 630, followed by an affinity 
purification step using a rat monoclonal antibody to murine CSF-1, 
designated YYG106, attached to a Sepharose B column (Stanley, E. R., 
Methods Enzymol (1985) 116: 564). The final step in purification was 
reverse phase HPLC in a 0.1% TFA/30% acetonitrile--0.1% TFA/60% 
acetonitrile buffer system. 
For MIAPaCa CSF-1, which was produced serum-free by induction with phorbol 
myristic acetate, the cell supernatant was subjected to calcium phosphate 
gel chromatography (according to Das (supra)), followed by affinity 
chromatography using lentil lectin (in place of the ConA affinity step of 
Das), and then to the immunoaffinity step employing the YYG106 monoclonal 
antibody conjugated to Sepharose B and to the reverse phase HPLC, both as 
above described. 
The urinary and MIAPaCa proteins, having been purified to homogeneity, were 
subjected to amino acid sequencing using Edman degradation on an automated 
sequencer. Sufficient N-terminal sequence of human CSF was determined to 
permit construction of probes shown in FIG. 3. 
E.2. Preparation of the Human Genomic Sequence 
A human genomic sequence encoding CSF-1 was obtained from the Maniatis 
human genomic library in .lambda. phage Charon 4 using probes designed to 
encode the N-terminal sequence of human protein. The library was 
constructed using partial HaeIII/AluI digestion of the human genome, 
ligatiion to EcoRI linkers, and insertion of the fragments into EcoRI 
digested Charon 4 phage. A Charon 4A phage containing the CSF-1 sequence 
as judged by hybridization to probe as described below, and designated 
pHCSF-1, was deposited with the American Type Culture Collection (ATCC) on 
Apr. 2, 1985 and has accession no. 40177. Upon later study of this phage, 
it was found that rearrangements and/or deletions had occurred and the 
correct sequences were not maintained. Therefore, an alternative colony 
obtained from the genomic library in identical fashion, and propogated to 
confirm stability through replication, was designated pHCSF-1a and was 
deposited with ATCC on May 21 1985, and given accession number 40185. 
pHCSF-1a contained an 18 kb insert and was capable of generating 
restriction enzyme digests which also hybridized to probe, and was used 
for sequence determination and additional probe construction as outlined 
below. 
If the CSF-1 encoding sequence is present in its entirety its presence can 
be demonstrated by expression in COS-7 cells, as described by Gluzman, Y., 
Cell (1981) 23: 175. The test fragment is cloned into a plasmid derived 
from pBR322 which has been modified to contain the SV40 origin of 
replication (pGRI Ringold, G., J Mol Appl Genet (1982) 1: 165-175). The 
resulting high copy number vectors are transformed into COS-7 cells and 
expression of the CSF-1 gene assayed after 24, 48, and 72 hours by the 
radioreceptor assay method described by Das (supra). Expression is under 
control of the native CSF-1 control sequences. The HindIII digests of the 
approximately 18 kb insert of pHCSF-1a tested in this manner failed to 
express, thus indicating that HindIII digests into the gene. This was 
confirmed by subsequent mapping. 
However, for initial sequencing, a 3.9 kb HindIII fragment was obtained 
from the pHCSF-1a phage and cloned into M13 cloning vectors. 
The HindIII fragment has been partially sequenced, and the results are 
shown in FIG. 4, along with a deduced peptide sequence. It contains the 
correct codons for the portion of the human CSF-1 protein for which the 
amino acid sequence had been determined, as set forth in FIG. 1. The 
presence of an intron of approximately 1400 bp was deduced from the 
available amino acid sequence. In addition, based on the genomic sequence 
encoding amino acids 24-34 (see overlined portion of FIGS. 4 and 5), a 
32-mer probe for the cDNA library was constructed and employed as 
described below. 
In more detail, to obtain the genomic clone, pHCSF-1a, the Maniatis library 
was probed using two mixtures of oligomers shown in FIG. 3. EK14 and EK15 
were selected, although the other oligomers shown are useful as well. A 
"full length" probe for the N-terminal sequence, EK14, was used as a 
mixture of sixteen 35-mers. A shorter oligomer, EK15, was employed as a 
mixture of sixty-four 18-mers. Phage hybridizing to both kinased probes 
were picked and cultured by infection of E. coli DG98 or other competent 
strain. 
Specific conditions for probing with EK14 and EK15 are as follows; for 
EK14, the buffer contained 15% formamide, 6.times.SSC, pH 7.0, 5.times. 
Denhardt's, 20 mM sodium phosphate, 0.2% SDS and 50 .mu.g/ml denatured 
salmon sperm DNA. Prehybridization and hybridization were conducted at 
42.degree. C. and the filters were washed in 2.times.SSC at 52.degree. C. 
For EK15, similar conditins were used for hybridization and 
prehybridization except for the formamide concentration, which was 0%; 
washing was at a slightly lower temperature, 42.degree. C. 
The approximately 18 kb DNA insert isolated from the positively hybridizing 
phage pHCSF-1a was treated with HindIII and the fragments were subjected 
to electrophoresis on agarose gel according to the method of Southern. The 
gels were replicated onto nitrocellulose filters and the filters were 
probed again with EK14 and EK15. Both probes hybridized to a 3.9 kb 
fragment. 
The positive fragment was excised from the gel, eluted, and subcloned into 
HindIII-treated M13mp19 for dideoxy sequencing. A partial sequence is 
shown in FIG. 4. The underlining corresponds precisely to the previously 
determined N-terminal sequence of human CSF-1; the residues with dot 
subscripts are homologous to the murine sequence. 
In FIG. 4, the 1.4 kb intron region between the codons for amino acids 22 
and 23, as deduced from the human sequence determined from the purified 
protein, is shown untranslated. The sequence upstream of the N-terminal 
residues contains the putative leader; the translation of the portion of 
this leader immediately adjacent to the mature protein, which was 
tentatively verified by the preliminary results of sequencing of the cDNA 
clone (see below) is shown. The upstream portions are, however, not shown 
translated; these portions are confirmed by comparison to the cDNA to 
comprise an intron. 
Further sequencing to obtain about 13 kb of the entire 18 kb gene shows 
that the gene contains 9 exons separated by 8 introns. The regions of the 
mature protein cDNA correspond exactly to the genomic exon codons except 
for codon 59, as further described below. 
An additional M13 subclone was obtained by digestion of the HindIII 3.9 kb 
fragment with PstI to generate a 1 kb PstI/PstI fragment which includes 
the known N-terminal sequence and about 1 kb of additional upstream 
sequence. 
E.3. cDNA Encoding Human CSF-1 
psCSF-17 
The human derived pancreatic carcinoma cell line MIAPaCa-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 
MIApaCa 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 MIAPaCa cells maintained in 
serum-free medium, i.e. under conditions wherein they do not produce 
CSF-1. Cells producing CSF-1 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 produce CSF-1 
at 2000-6000 units/ml. The cell cultures were washed, and reincubated 
serum-free to suppress CSF-1 formation. For negative controls, no 
detectable CSF-1 was produced after a day or two. Reinduced cells 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 (Berger, S. L., et 
al, Biochemistry (1979) 18: 5143) followed by phenol chloroform 
extraction, ethanol precipitation, and oligo dT chromatography, and an 
enriched mRNA preparation obtained. In more detail, cells are washed twice 
in PBS (phosphate buffered saline) and are resusupended in IHB (140 mM 
NaCl, 10 mM Tris, 1.5 mM MgCl.sub.2, 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) is added 
to 0.5% to lyse the cellular, but not nuclear membranes. Nuclei are 
removed by centrifugation at 1,000.times.g for 10 min. The post-nuclear 
supernatant is 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 is re-extracted 4 times and phase separated by 
centrifugation at 2,000.times.g for 10 min. The RNA is precipitated by 
adjusting the sample to 0.25M NaCl, adding 2 volumes of 100% ethanol and 
storing at -20.degree. C. The RNA is pelleted at 5,000.times.g for 30 min, 
is washed with 70% and 100% ethanol, and is then dried. Polyadenylated 
(poly A.sup.+) messenger RNA (mRNA) is obtained from the total cytoplasmic 
RNA by chromatography on oligo dT cellulose (Aviv, J., et al, Proc Natl 
Acad Sci (1972) 69: 1408-1412). The RNA is dissolved in ETS (10 mM Tris, 1 
mM EDTA, 0.5% SDS, pH 7.5) at a concentration of 2 mg/ml. This solution is 
heated to 65.degree. C. for 5 min, then quickly chilled to 4.degree. C. 
After bringing the RNA solution to room temperature, it is adjusted to 0.4 
M NaCl and is slowly passed through an oligo dT cellulose column 
previously equilibrated with binding buffer (500 mM NaCl, 10 mM Tris, 1 mM 
EDTA, pH 7.5 0.05% SDS). The flow-through is passed over the column twice 
more. The column is then washed with 10 volumes of binding buffer. Poly 
A.sup.+ mRNA is eluted with aliquots of ETS, extracted once with 
TE-saturated phenol chloroform and is precipitated by the addition of NaCl 
to 0.2M and 2 volumes of 100% ethanol. The RNA is reprecipitated twice, is 
washed once in 70% and then in 100% ethanol prior to drying. 
Total mRNA was subjected 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 Beckman SW40 
rotor at 20.degree. C. and 27,000 rpm for 17 hr. 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 (as described by 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 FIGS. 4 and 5 shows the exon 
II probe.) These results are summarized in FIG. 7. 
The broken line in FIG. 7A shows the respose in the bone marrow 
proliferation assay of the supernatants from the Xenopus oocytes; FIG. 7B 
shows the dot-blot results. The most strongly hybridizing fraction, 11, 
corresponds to a size slightly larger than the 18S marker, while the most 
active fractions 8 and 9 correspond to 14-16S. Fractions 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 nitrocellulose, 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 subjected to Southern blot and probed using 
pcCSF-17 DNA. The restriction pattern was consistent with the presence of 
only one gene encoding CSF-1.) 
The enriched mRNA pool was prepared by combining the mRNA from the gradient 
fractions (8 and 9) having the highest bone marrow proliferative activity, 
although their ability to hybridize to probe is relatively low (14S-16S) 
with the fractions (11) hybridizing most intensely to probe (slightly 
larger than 18S). Higher molecular weight fractions which also hybridized 
to exon II probe were not included because corresponding mRNA from 
uninduced MIAPaCa Cells also hybridized to exonII probe. 
cDNA libraries were prepared from total or enriched human mRNA in two ways. 
One method uses .lambda.gt10 phage vectors and is described by Huynh, T. 
V., et al, in DNA Cloning 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 Okayama, H., et al, Mol Cell 
Biol (1983) 3: 280-289, incorporated herein by reference. 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 the authors, pcDVl and pLl. 
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 Amp resistance gene and SV40 control 
elemetns 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 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 using a CsCl gradient, 
and the COS-7 cells transfected using a modification (Wang, 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., et al, Blood (1981) 
58: 630, and to a colony stimulation (bone marrow proliferation) assay 
performed substantially as disclosed by Prystowsky, M. B., et al, Am J 
Pathol (1984) 114: 149. Nine of the ten clones picked failed to show 
transient CSF-1 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 FIG. 5. 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 CMCC 2347 and was deposited with the American Type 
Culture Collection on June 14 1985, as accession no. 53149. The plasmid 
bearing the CSF-1 encoding DNA was designated pcCSF-17. 
Mutein-Encoding Sequences 
Modifications were made of the pcCSF-17 inserts to provide corresponding 
plasmids encoding muteins of the mCSF-1 protein. For site-specific 
mutagenesis, pcCSF-17 and M13mp18 were digested with the same restriction 
enzyme excising the appropriate region of the CSF-1 coding sequence, and 
the excised sequence ligated into the M13 vector. Second strand synthesis 
and recovery of the desired mutated DNA used the following oligonucleotide 
primers: 
for pro.sub.52 CSF-1, 5'-TACCTTAAACCGGCATTTCTC-3', which creates a new 
HpaII site at codons 52-53; 
for gln.sub.52 CSF-1, 5'-TACCTTAAACAGGCCTTTCTC-3', which creates a new StuI 
site at codons 52-53; 
for asp.sub.59 CSF-1, 5'-GGTACAAGATATCATGGAG-3', which creates a new EcoRV 
site at codons 59-60. 
After second strand extension using Klenow, the phage were transformed into 
E coli DG98 and the resulting plaques screened with kinased labeled probe. 
After plaque purification, the desired mutated inserts were returned to 
replace the unmutated inserts in pcCSF-1, yielding pCSF-pro52, pCSF-gln52, 
and pCSF-asp59, respectively. 
Plasmids containing three deletion mutants which encode .gradient..sub.158- 
CSF-1 were also prepared: pCSF-Bam, pCSF-BamBcl, and pCSF-BamTGA. For 
pCSF-Bam, pcCSF-17 was digested with BamHi, and the upstream BamHI/BamHI 
fragment of the coding region was isolated and religated to the vector 
fragment. The ligation mixture was transformed into E coli MM294 and 
plasmids with the correct orientation isolated. The resulting pCSF-Bam 
encodes 158 amino acids of the CSF-1 protein fused to six residues derived 
from the vector at the C-terminus: arg-his-asp-lys-ile-his. 
For pCSF-BamBcl, which contains the entire CSF-1 encoding sequence, except 
that the serine at position 159 is mutated to a stop codon, the coding 
sequence was excised from pcCSF-17 and ligated into M13 for site-specific 
mutagenesis using the primer: 5'-GAGGGATCCTGATCACCGCAGCTCC-3'. This 
results in a new BclI site at codons 159-160. The mutated DNA was excised 
with BstXI/EcoRI and ligated into the BstXI/EcoRI digested pcCSF-17, the 
ligation mixture was transformed into E coli DG105, a dam host, and the 
plasmid DNA isolated. 
For pCSF-BamTGA, in which the codons downstream of the 159-stop are 
deleted, pCSF-BamBcl was digested with XhoI and BcII, and the insert 
ligated into XhoI/BamHI digested pcCSF-17. 
In addition, pCSF-Gly150, which contains a TGA stop codon instead of 
histidine at position 151, was prepared from the pcCSF-17 insert by 
site-specific mutagenesis using the appropriate primer, as described 
above. 
E.4. Transient Expression of CSF-1 
Expression of pcCSF-17 
The expression of plasmid DNA from CSF-17 (pcCSF-17) in COS-7 cells was 
confirmed and quantitated using the bone marrow proliferation assay, the 
colony stimulation assay and the radioreceptor assay. It will be recalled 
that the specificity of the bone marrow proliferation assay for CSF-1 
resides only in the ability of CSF-1 antiserum to diminish activity; that 
for the colony stimulation assay, in the nature of the colonies obtained. 
Both assays showed CSF-1 production to be of the order of several thousand 
units per ml. 
Bone Marrow Proliferation 
For the bone marrow stimulation assay, which measures biological activity 
of the protein, bone marrow cells from Balb/C mice were treated with 
serial dilutions of the 72 hour supernatants and proliferation of the 
cells was measured by uptake of labeled thymidine, essentially as 
described by Moore, R. N., et al, J Immunol (1983) 131: 2374; Prystowsky, 
M. B., et al, Am J Pathol (1984) 114: 149. The medium from induced MIAPaCa 
cells was used as control. Specificity for CSF-1 was confirmed by the 
ability of rabbit antisera raised against human urinary CSF-1 to suppress 
thymidine uptake. The results for COS-7 cell supernatants transfected with 
pcCSF-17 (CSF-17 supernatant) at a 1:16 dilution are shown in Table 1. 
TABLE 1 
______________________________________ 
.sup.3 H-thymidine incorporation (cpm) 
no normal antihuman 
add'ns serum CSF-1 serum 
______________________________________ 
medium 861 786 2682 
MIAPaCa supernate 
12255 16498 3302 
CSF-17 supernate 
16685 21996 2324 
______________________________________ 
(The antihuman CSF-1 serum was prepared as described by Das, et al, supra.) 
The MIAPaCa supernatant (at the 1:16 dilution used above) contained 125 
U/ml CSF activity corresponding to 2000 U/ml in the undiluted supernatant, 
where 1 unit of colony stimulating activity is defined as the amount of 
CSF needed to produce one colony from 10.sup.5 bone marrow cells/ml in the 
assay of Stanley, E. R., et al, J Lab Clin Med (1972) 79: 657. 
These data show that the bone marrow stimulating activity is associated 
with CSF-1, since thymidine uptake is inhibited by anti-CSF-1 serum. 
Regression of results in this bone marrow proliferation assay obtained at 
four dilutions ranging from 1:8 to 1:64 gave an estimated activity for 
CSF-1 in CSF-17 supernatants of 2358 U/ml, which was diminished to 424 
U/ml in the presence of antiserum, but showed an apparent increase to 3693 
U/ml in the presence of non-immune serum. This was comparable to the 
levels shown in the radioreceptor assay below. 
Colony Stimulation 
Direct assay of the CSF-17 supernatants for colony stimulation (Stanley, E. 
R., et al, J Lab Clin Med (supra)) showed 4287 U/ml, which was 
substantially unaffected by the presence of non-immune serum but reduced 
to 0 U/ml in the presence of rabbit antihuman CSF-1. This compares to 2562 
U/ml in the MIAPaCa supernatants. Eighty-five percent of the pcCSF-17 
transformed COS-7 supernatant induced colonies had mononuclear morphology; 
MIAPaCa supernatant induced colonies showed a 94% macrophage-6% 
granulocyte ratio. 
Radioreceptor Assay 
The radioreceptor assay measures competition between .sup.125 I-labeled 
CSF-1 and the test compound for specific receptors on J774.2 mouse 
macrophage cells. MIAPaCa supernatant, assayed for colony stimulating 
activity as above, was used as a standard (2000 U/ml). The CSF-1 
concentration of the pcCSF-17 transformed COS-7 supernatant was found to 
be 2470 U/ml based on a 1:10 dilution and 3239 U/ml based on a 1:5 
dilution. 
Thus, comparable values for CSF-1 concentration in the media of COS-7 cells 
transformed with pcCSF-17 were found in all assays. 
Expression of Muteins 
In a similar manner to that described above for pcCSF-17, the 
mutein-encoding plasmids were transfected into COS-A2 cells and transient 
expression of CSF-1 activity assayed by the bone marrow proliferation 
assay and by radioimmunoassay using anti-CSF antibodies. The expression 
product of pCSF-pro52 was inactive, indicating that, as expected, 
substitution by proline is not conservative. All other muteins showed 
activity in both assays as shown by the results below: 
______________________________________ 
Expression of CSF-1 Constructs in COS Cells 
Radio- Bone Marrow Assay 
CSF-1 immunoassay Proliferation 
Colony 
Plasmid (units/ml) (units/ml) (units/ml) 
______________________________________ 
pcCSF-17 3130 2798 11,100 
3080 3487 9750 
3540 3334 11,500 
pCSF-pro52 54.8 &lt;25 &lt;100 
51.9 &lt;25 &lt;100 
45.3 &lt;25 &lt;100 
pCSF-gln52 1890 2969 6200 
2250 2308 5500 
1910 2229 4400 
pCSF-asp59 3210 3381 9000 
4680 3417 6800 
3470 2812 10,600 
pCSF-Bam 9600 8048 22,600 
8750 8441 21,900 
8400 10,995 21,700 
pCSF-BamBcl 
8800 26,000 
10,700 21,600 
15,450 24,200 
pCSF-BamTGA 
8450 22,600 
7550 23,200 
9700 20,000 
pCSF-Gly150 
26,850 55,710 
______________________________________ 
E.5. Stable Expression of CSF-1 
The COS-7 system provides recombinant CSF-1 by permitting replication of 
and expression from the vector sequences. It is a transient expression 
system. 
The human CSF-1 sequence can also be stably expressed in procaryotic or 
eucaryotic systems. In general, procaryotic hosts offer ease of 
production, while eucaryotes permit the use of the native signal sequence 
and carry out desired post-translational processing. This may be 
especially important in the case of CSF-1 since the native protein is a 
dimer. Bacteria produce CSF-1 as a monomer, which would then be subjected 
to dimerizing conditions after extraction. 
Procaryotic Expression 
For procaryotic expression, the cDNA clone, or the genomic sequence with 
introns excised by, for example, site-specific mutagenesis, is altered to 
place an ATG start codon immediately upstream of the glutamic acid at the 
N-terminus, and a HindIII site immediately upstream of the ATG in order to 
provide a convenient site for insertion into the standard host expression 
vectors below. This can be done directly using insertion site-specific 
mutagenesis with a synthetic oligomer containing a new sequence 
complementary to the desired AAGCTTATG, flanked by nucleotide sequences 
complementary to the native leader and N-terminal coding sequences. 
For cDNA obtained using the method of Okayama and Berg, the DNA fragment 
containing the entire coding sequence is excised from pcCSF-17 or the 
corresponding mutein vector by digestion with XhoI (at sites retained from 
the host cloning vector), isolated by agarose gel electrophoresis, and 
recovered by electroelution. To carry out the mutagenesis, the host 
bacteriophage M13mp18 DNA is also treated with SalI and ligated with the 
purified fragment under standard conditions and transfected into frozen 
competent E. coli K12 strain DG98. The cells are plated on media 
containing 5.times.10.sup.-4 M isopropyl thiogalactoside (IPTG) obtained 
from Sigma Chem. (St. Louis, MO) and 40 .mu.g/ml X-gal. Non-complementing 
white plaques are picked into fresh media. Mini-cultures are screened for 
recombinant single strand phage DMA of the expected size, and the 
structure of the desired recombinant phage is confirmed using restriction 
analysis. 
A 34-mer complementary to the N-terminal and leader encoding portions of 
the CSF-1 sequence, but containing the complement to the desired AAGCTTATG 
sequence is synthesized and purified according to the procedures set forth 
in C.4. A portion of this 34-mer preparation is radiolabeled according to 
a modification of the technique of Maxam and Gilbert (Maxam, A., et al, 
Methods in Enzymology (1980) 68: 521, Academic Press) as set forth in C.4 
above. 
To perform the mutagenesis the above prepared recombinant bacteriophage is 
prepared in E. coli K12 strain DG98 and the single strand phage DNA 
purified. One pmole of single strand phage DNA and 10 pmoles of the above 
synthetic nucleotide primer (not kinased) are annealed by heating for 1 
min at 67.degree. C., and then 30 min at 37.degree. C. in 15 .mu.l 20 mM 
Tris-Cl, pH 8, 20 mM MgCl.sub.2, 100 mM NaCl, 20 mM 2-mercaptoethanol. The 
annealed DNA is incubated with DNA polymerase I (Klenow) and 500 .mu.M 
dNTPs for 30 min, 0.degree. C. and then brought to 37.degree. C. Aliquots 
(0.05 or 0.25 pmole) are removed after 5 min, 20 min, and 45 min, 
transformed into E. coli K12 strain DG98 and plated. 
After growth, the plates are chilled at 4.degree. C. and plaques lifted 
with PalI membranes obtained from Biodyne of S&S filters (1-2 min in the 
first filter, more than 10 min for the second filter). The filters are 
denatured in 2.5M NaCl, 0.5M NaOH (5 min). The denaturing medium is 
neutralized with 3M sodium acetate to pH 5.5, or with 1M Tris-Cl, pH 7.5 
containing 1M NaCl, the filters baked at 80.degree. C. in vacuo for 1 hr, 
and then prehybridized at high stringency. The filters are then probed 
with the kinased synthetic 34-mer prepared above at high stringency, 
washed, and autoradiographed overnight at -70.degree. C. 
The RF form of the desired mutated phage is treated with EcoRI, blunted 
with Klenow, and then digested with HindIII to excise the gene as a 
HindIII/blunt fragment. (In a strictly analogous manner, the CSF-1 
encoding sequence from pMCSF may be obtained and modified.) 
This fragment containing the human (or murine) CSF-1 encoding sequence is 
then ligated with HindIII/BamHI (blunt) digested pPLOP or pTRP3 (see 
below) to place the coding sequence containing the ATG start codon 
immediately downstream from the P.sub.L or trp promoter respectively. 
These resulting plasmids are transformed into E. coli MC1000 lambda 
lysogen or MM294, and the cells grown under non-inducing conditions and 
then induced by means appropriate to the promoter. The cells are harvested 
by centrifugation, sonicated and the liberated CSF-1 solubilized. The 
presence of human (or murine) CSF-1 is confirmed by subjecting the 
sonicate to the colony stimulating assay set forth above. 
In addition, the plasmid pFC54.t (ATCC 39789) which contains the P.sub.L 
promoter and the Bacillis thuringiensis positive retroregulatory sequence 
(as described in EPO Application Publication No. 717,331, published Mar. 
29, 1985) was used as a host vector. pFC54.5 was digested with 
HindIII/BamHI(blunt), and the desired coding sequences ligated into the 
vector using the HindIII/EcoRI(blunt) excised fragment from pcCSF-17 or 
the mutein encoding vectors described above. After transformation into E. 
coli MC1000 lambda lysogen, and induction, CSF-1 production was obtained 
and verified as described above. 
Finally, it was possible to improve the level of CSF-1 production from the 
foregoing constructs by altering the third nucleotide in each of the first 
six codons of the N-terminus. pFC54.5 containing the CSF-1 encoding 
fragment was digested with HindIII/BstXI, and the excised fragment (which 
contains the ATG and a short portion of the subsequent coding sequence) 
was replaced by a synthetic HindIII/BstXI segment wherein the first six 
codons have the sequence: GAAGAAGTTTCTGAATAT. The resulting analogous 
expression vector represents no change in the amino acid sequence encoded; 
however, the levels of expression are improved when this modified vector 
is used. 
Eucaryotic Expression 
The Okayama-Berg plasmid pcCSF-17, containing the cDNA encoding human CSF-1 
under control of the SV40 promoter, can also be used to effect stable 
expression in monkey CV-1 cells, the parent cell line from which the COS-7 
line was derived. The corresponding vectors encoding the muteins as 
described above can also be used in an exactly analogous way. The host 
monkey CV-1 cells were grown to confluence and then cotransformed using 10 
.mu.g pcCSF-17 and various amounts (1, 2, 5 and 10 .mu.g) of PRSV-NEO2 
(Gorman, C., et al, Science (1983) 221: 551-553) per 500,000 cells. The 
transformants were grown in DMEM with 10% FBS medium containing 100 
.mu.g/ml of G418 antibiotic, to which the pRSV-NEO2 plasmid confers 
resistance. The CV-1 cell line showed a G418 transformation frequency of 
10.sup.-5.12 colonies per 10.sup.6 cells per .mu.g DNA. 
The CV-1 cells were cotransformed as described above and selected in 
G418-containing medium. Resistant clones were tested for stability of the 
G418-resistant phenotype by growth in G418-free medium and then returned 
to G418-containing medium. The ability of these cultures to survive when 
returned to antibiotic-containing medium suggests that the pRSV-NEO2 DNA 
was integrated permanently into the cell genome. Since cells stably 
transformed with a marker plasmid have about 50% probability of having 
integrated the DNA of a cotransfecting plasmid, about half of these cells 
will also contain pcCSF-17 DNA in their chromosomal DNA. 
Several clones of the C418-resistant pools of CV-1 cells which were 
demonstrated to be stably transformed as above were picked and grown in 
duplicate flasks to near confluence. One flask of each duplicate was 
infected with SV-40 virus at a multiplicity of infection of 5, and the 
medium was harvested 6 days after infection for assay for CSF-1 using a 
radioimmunoassay. The immunoassay is based on competition of .sup.125 
I-labeled MIAPaCa CSF-1 for "Rabbit 52" polyclonal antiserum raised 
against purified human urinary CSF-1. 
One of the selected CV-1 clones showed 2335 U/ml production of CSF-1, 
according to this assay, whereas cells not infected with SV-40 showed less 
than 20 U/ml. Controls using COS-7 cells transformed with 10 .mu.g 
pcCSF-17 showed 2400 U/ml CSF-1 production without SV-40 infection. 
The CSF-1 producing CV-1 cell line contains the pcCSF-17 DNA stably 
integrated into its genome, and thus can be used for stable production of 
CSF-1 upon infection with SV-40. Infection is presumed to "rescue" the 
pcCSF-17 DNA from the genome, and provide the SV-40 T-antigen necessary 
for replication of the rescued DNA. Without SV-40 infection, the 
integrated pcCSF-17 DNA is not effectively expressed. 
Optimization of the expression of the CSF-1 encoding sequence by the CV-1 
(CSF-17) cell line showed 6500-8000 U/ml when measured by the 
radioimmunoassay six days after SV-40 infection using a multiplicity of 
infection of at least 1, and a 10% FBS medium. Studies on expression 
levels at a multiplicity of 10 showed comparable production, but 
production was reduced upon removal of the FBS from the medium on the 
second day after infection. 
In the alternative, appropriate control systems and host vectors permitting 
expression in eucaryotic hosts may be used to receive the CSF-1 encoding 
inserts. For example, CHO cells and suitable vectors may be used, as 
described in U.S. Ser. No. 438,991, filed Nov. 1, 1982, now adandoned 
assigned to the same assignee and incorporated herein by reference. 
E.6. Activity of CSF-1 
Additional definition of the activity of CSF-1 was provided using partially 
purified MIAPaCa CSF-1 or murine L cell CSF-1 as models for the 
CV-1-produced recombinant material. CSF-1 was shown to enhance the 
production of interferon and tumor necrosis factor (TNF) by induced human 
monocytes by up to 10-fold. CSF-1 also was demonstrated to stimulate 
macrophage antitumor toxicity. 
Stimulation of TNF Production by Human Monocytes 
MIAPaCa CSF-1 was purified from the supernatant by calcium phosphate gel 
filtration and lentil lectin chromatography. For assay of lymphokine 
production, peripheral blood-adherent cells were incubated in duplicate 
flasks containing 10.sup.7 cells each. One flask was treated with 1000 
U/ml CSF-1 purified as above. After 3 days, the cells were harvested, and 
washed, and resuspended at a cell concentration of 5.times.10.sup.5 /ml 
and plated in 24-well plates at 0.5 ml/well. The wells were treated with 
10 .mu.g/ml LPS and 20 ng/ml PMA for 48 hr and the supernatants were 
harvested for TNF assay. Cells treated with CSF showed TNF secretions 
approximately nine-fold higher than the untreated cells (1500 U/ml, 
compared to 162 U/ml). 
Stimulation of Interferon Production by Human Monocytes 
In an analogous experiment to determine the effect of CSF-1 on interferon 
production, peripheral blood-adherent cells were incubated for 3 days in 
the presence and absence of 1000 U/ml CSF-1, as described above, 
harvested, resuspended at 5.times.10.sup.5 /ml, and plated in a 25-well 
plate, as described above. The cells were induced for interferon 
production by addition of varying amounts of poly(I): poly(C). The 
supernatants were assayed for interferon production by their cytopathic 
effect on VSV-infected GM 2504 cells. The CSF-1-stimulated cells showed 
production of 100 U/ml when induced with 50 .mu.g/ml poly(I): poly(C), as 
described by McCormick, F., et al, Mol Cell Biol (1984) 4: 166, whereas 
comparably induced untreated cells produced less than 3 U/ml. 
Stimulation of Myeloid CSF Production by Human Monocytes 
Monocytes were incubated .+-.CSF-1 for 3 days and then induced for 
production of myeloid CSF as in Table 1. The three representative 
experiments shown used blood from different donors. 
TABLE 2 
______________________________________ 
Myeloid CSF (U/ml) 
Exp. 1 Exp. 2 Exp. 3 
Induction 
-CSF +CSF -CSF +CSF -CSF +CSF 
______________________________________ 
medium 0 0 0 0 0 0 
0.1 .mu.g/ml 
-- -- 0 0 0 80 .+-. 
LPS 17 
1 .mu.g/ml LPS 
0 700 .+-. 
40 .+-. 
200 .+-. 
103 .+-. 
377 .+-. 
72 20 20 12 57 
0.1 .mu.g/ml 
-- -- 617 .+-. 
993 .+-. 
1120 .+-. 
1280 .+-. 
LPS + 2 50 101 82 60 
ng/ml PMA 
1 .mu.g/ml LPS 
283 .+-. 
983 .+-. 
360 .+-. 
1400 .+-. 
537 .+-. 
1080 .+-. 
+ 2 ng/ml 
42 252 92 180 47 12 
PMA 
2 ng/ml -- 370 .+-. 
297 .+-. 
183 .+-. 
380 .+-. 
716 .+-. 
PMA 17 6 15 52 76 
______________________________________ 
Therefore, CSF-1 stimulates myeloid CSF production. 
Stimultion of Tumor Cell Killing by Murine Macrophage; Comparison to other 
Colony Stimulating Factors 
To assay macrophage stimulation, murine CSF-1 obtained from 
L-cell-conditioned medium, was used as a model for the recombinantly 
produced CSF-1 from pcCSF-17 in an assay which showed stimulation of the 
ability of murine macrophages to kill sarcoma targets. In this assay, 
normal 2 hr adherent C3H/HeN mouse peritoneal macrophages were incubated 
for 1 day in vitro with and without CSF-1 and then mixed at a 20:1 ratio 
with .sup.3 H-thymidine-labeled mouse sarcoma TU5 cells along with 10% v/v 
conA-induced (10 .mu.g/ml) spleen lymphokine (LK), which contains gamma 
interferon. The release of labeled thymidine over the following 48 hr was 
used as a measure of tumor cell killing. The effect of adding CSF-1 as 
murine L-cell-conditioned medium containing 1200 U/ml CSF-1 is shown in 
the following table. 
______________________________________ 
Treatment Increase Due 
DAY DAY Kill to CSF-1 
0.fwdarw.1 
1.fwdarw.3 % % 
______________________________________ 
-- -- 13 
-- LK 39 
-- CSF-1 + LK 49 26 
CSF-1 LK 51 31 
CSF-1 CSF-1 + LK 60 54 
-- -- 3 
-- LK 35 
-- CSF-1 + LK 47 34 
CSF-1 -- 7 
CSF-1 LK 49 40 
CSF-1 CSF-1 + LK 69 97 
______________________________________ 
Increase in the ability to kill the target cells was noted whether CSF-1 
was added during the preliminary 1 day of growth or during the period of 
induction; however, the most dramatic effects were observed with CSF-1 was 
present during both of these periods. 
The possibility of contaminating bacterial lipopolysaccharide (LPS) as the 
cause of stimulation of monocytes and macrophages was excluded: The LPS 
content of the applied CSF-1 was low (21 0.3 ng/3000 U CSF-1, by Limulus 
amoebocyte lysate assay); activity was removed by application to an 
anti-CSF-1 column; polymyxin B was used to neturalize LPS; the macrophages 
from C3H/HeJ mice respond to CSF-1 but not to LPS. 
CSF-GM was prepared from 6 mouse lungs obtained 5 hours after IV 
administration of 5 .mu.g LPS. The lungs were chopped and incubated for 3 
days in serum free medium, and the supernatant was depleted of CSF-1 using 
a YYG106 affinity column (CSF-1 content reduced from 270 U/ml to 78 U/ml). 
CSF-G was prepared from similarly treated LDI serum free medium. Both 
CSF-GM and CSF-G contents were assayed at 2000 U/ml by colony stimulating 
assay. 
The peritoneal macrophages were incubated with 40% of either of the 
foregoing media or with L-cell medium assayed at 2000 U/ml CSF-1 for 1 
day, and then incubated for 48 hours either with additional medium or with 
LK, and assayed for TU5 killing as described above. 
The results are shown in FIG. 6. While CSF-1 showed marked enhancement of 
toxicity to TU5, neither CSF-G nor CSF-GM had any effect. 
Stimulation of Murine Antiviral Activity 
Adherent murine thioglycolate-elicited mcarophages were incubated with 
CSF-1 for 3 days and infected with VSV overnight. Polymyxin B was added to 
test samples to block the LPS induction of interferon. The following table 
shows crystal violet staining of cells remaining adherent. 
TABLE 3 
______________________________________ 
Crystal Violet 
-Polymyxin B 
Treatment (mean) (S.D.) 
+Polymyxin B 
______________________________________ 
Medium/No VSV .158 .+-. .019 
Medium + VSV .0583 .+-. .02 
.049 .+-. .009 
CSF-1625 U/ml + VSV 
.139 .+-. .018 
.177 .+-. .04 
1250 + VSV .167 .+-. .06 
.205 .+-. .07 
2500 + VSV .160 .+-. .06 
.219 .+-. .04 
5000 + VSV .150 .+-. .03 
.202 .+-. .06 
______________________________________ 
CSF-1 treated cells, therefore, showed protection of the macrophage against 
VSV. 
E.7 Formulation of CSF-1 
The recombinantly produced human CSF-1 may be formulated for administration 
using standard pharmaceutical procedures. Ordinarily CSF-1 will be 
prepared in injectable form, and may be used either as the sole active 
ingredient, or in combination with other proteins or other compounds 
having complementary or similar activity. Such other compounds may include 
alternate antitumor agents such as adriamycin, or lymphokines, such as 
IL-1, -2, and -3, alpha-, beta-, and gamma-interferons and tumor necrosis 
factor. The effect of the CSF-1 active ingredient may be augmented or 
improved by the presence of such additional components. As described 
above, the CSF-1 may interact in beneficial ways with appropriate blood 
cells, and the compositions of the invention therefore include incubation 
mixtures of such cells with CSF-1, optionally in the presence of 
additional lymphokines. Either the supernatant fractions of such 
incubation mixtures, or the entire mixture containing the cells as well, 
may be used. 
F. Murine CSF-1 
An intronless DNA sequence encoding murine CSF-1 is prepared using a murine 
fibroblast cell line which produces large amounts of CSF-1. The L-929 
line, obtainable from ATCC, is used as a source for mRNA in order to 
produce a cDNA library. Using oligomeric probes constructed on the basis 
of the known murine N-terminal and CNBr-cleaved internal peptide sequence, 
this cDNA library is probed to retrieve the entire coding sequence for the 
murine form of the protein. Murine CSF-1 is believed to be approximately 
80% homologous to the human material because of the homology of the 
N-terminal sequences, the ability of both human and murine CSF-1 
preparations to stimulate macrophage colonies from bone marrow cells, and 
limited cross-reactivity with respect to radioreceptor and 
radioimmunoassays (Das, S. K., et al, Blood (1981) 58: 630). 
F.1. Protein Purification 
Murine CSF-1 was purified by standard methods similar to those that are 
disclosed by Stanley, E. R. et al, J Immunol Meth (1981) 42: 253-284 and 
by Wang, F. F., et al, J Cell Biochem (1983) 21: 263-275 of SDS gel 
electrophoresis as reviewed by Hunkapiller, M. W., et al, Science (1984) 
226: 304. 
Amino acids 1-39 of the murine sequence were obtained, taking advantage of 
cyanogen bromide cleavage at position 10 to extend the degradation 
procedure. An internal cleavage fragment from the mouse protein was also 
obtained and sequenced. 
Overall composition data for the mouse protein were also obtained as shown 
below. These data show correct relative mole % for those amino acids 
showing good recoveries; however the numbers are not absolute, as 
histidine and cysteine were not recovered in good yield. 
______________________________________ 
Amino Acid mole % residues/125 
______________________________________ 
Asp 20.1 25.1 
Glu 20.0 25.0 
His -- -- 
Ser 6.0 7.5 
Thr 5.9 7.4 
Gly 5.4 6.8 
Ala 6.8 8.5 
Arg 3.0 3.8 
Pro 6.7 8.4 
Val 5.3 6.6 
Met 1.1 1.4 
Ile 3.9 4.9 
Leu 8.5 10.6 
Phe 6.0 7.5 
Lys 3.5 4.4 
Tyr 4.1 5.1 
______________________________________ 
The conversion to residues/125 was based on an approximation of sequence 
length from molecular weight. 
F.2. Preparation of Murine CSF-1 cDNA 
The amino acid sequence 5-13 of the murine CSF-1 and the internal sequence 
were used as a basis for probe construction. 
Three sets of oligomers corresponding to the murine sequence were prepared. 
One sequence was prepared to encode "region A"--i.e., amino acids 9-13; 
another was prepared to "region B"--i.e., amino acids 5-9, as shown in 
FIG. 2; a third to encode positions 0-6 of an internal sequence, "region 
C". Because of codon redundancy, each of these classes of oligomers is 
highly degenerate. 
Thus, 15-mers constructed on the basis of region A number 48; 14-mers 
constructed on the basis of region B (deleting the last nucleotide of the 
codon for histidine) also number 48; 20-mers constructed on the basis of 
region C number 32. Alternatively stated, a 15-mer constructed so as to 
encode region A may have a mismatch in four of the fifteen positions; a 
particular 14-mer constructed with respect to region B may have a mismatch 
in six positions; a particular 20-mer constructed with respect to region C 
may have a mismatch in five positions. 
As described below, by suitable protocol design, an enriched messenger RNA 
fraction may be found for the production of the desired enriched murine 
cDNA library, and the precisely correct oligomers for use as probes also 
ascertained. 
Totla messenger RNA is extracted and purified from murine L-929 cells. 
Murine L-929 cells are cultured for 8 days on DME medium and then 
harvested by centrifugation. The total cytoplasmic ribonucleic acid (RNA) 
was isolated from the cells by the same protocol as set forth above for 
MIAPaCa mRNA. 
The mRNA is fractionated on gels for Northern blot as described in 
paragraph C.3. The 15-mer sequences corresponding to region A are divided 
into four groups of twelve each. Each of these groups was used to 
hybridize under low stringency both to control and to murine L-929 mRNA 
slabs and the resulting patterns viewed by radioautography. Under the low 
stringency conditions employed, hybridization occurs to fractions not 
containing the proper sequence, as well as those that do. Also, because 
the control cell line is different from that of the L-929 line in ways 
other than failure to produce CSF-1, hybridization occurs in a number of 
size locations not related to CSF-1 in the L-929 cell gels which are not 
present in the controls. 
Comparable sets of control and L-929 gels are probed with segregants of the 
48 14-mers representing region B and segregants of the 32 20-mers 
representing region C. Only the bands of messenger RNA which hybridize 
exclusively in the L-929 slabs for either regions A or B, and C probes are 
then further considered. 
The RNA band which continues to bind to one of the A region 15-mers mixture 
or one of the region B 14-mers mixture and one of the region C 20-mer 
mixture under conditions of increasingly higher stringency is selected. 
When the correct mRNA band is found, each of the groups of region A 15-mers 
is used to probe at various stringency conditions. The group binding at 
highest stringency presumably contains the correct 15-mer exactly to 
complement the mRNA produced. The correct 15-mer is ascertained by further 
splitting the preparation until a single oligomer is found which binds at 
the highest stringency. A similar approach is used to ascertain the 
correct 14-mer or 20-mer which binds to region B or C. These specific 
oligomers are then available as probes in a murine cDNA library which is 
prepared from the enriched mRNA fraction. 
The mRNA fraction identified as containing the coding sequence for CSF-1 is 
then obtained on a preparative scale. In this preparation, the poly 
A.sup.+ mRNA was fractionated on a sucrose gradient in 10 mM Tris-HCl, pH 
7.4, 1 mM EDTA, and 0.5% SDS. After centrifugation in a Beckman SW40 rotor 
at 30,000 rpm for 17 hr, mRNA fractions are recovered from the gradient by 
ethanol precipitation. RNA fractions recovered from the gradient were each 
injected into Xenopus oocytes in a standard translation assay and the 
products assayed for CSF-1 using radioimmunoassay with antibodies raised 
against murine CSF-1. Fractions for which positive results were obtained 
were pooled and used to construct the cDNA library. These same fractions 
hybridize to the oligomeric probes. 
Other methods of preparing cDNA libraries are, of course, well known in the 
art. One, now classical, method uses oligo dT primer, reverse 
transcriptase, tailing of the double stranded cDNA with poly dG, and 
annealing into a suitable vector, such as pBR322 or a derivative thereof, 
which has been cleaved at the desired restriction site and tailed with 
poly dC. A detailed description of this alternate method is found, for 
example, in U.S. Ser. No. 564,224, filed Dec. 20 1983, now U.S. Pat. No. 
4,518,584 and assigned to the same assignee, incorporated herein by 
reference. 
In the method used here, the enriched mRNA (5 .mu.g) is denatured by 
treatment with 10 mM methyl mercury at 22.degree. C. for 5 min and 
detoxified by the addition of 100 mM 2-mercaptoethanol (Payvar, F., et al, 
J. Biol Chem (1979) 254: 7636-7642). Plasmid pcDV1 is cleaved with KpnI, 
tailed with dTTP, and annealed to the denatured mRNA. This oligo dT primed 
mRNA is treated with reverse transcriptase, and the newly synthesized DNA 
strand tailed with dCTP. Finally, the unwanted portion of the pcDV1 vector 
is removed by cleavage with HindIII. Separately, pL1 is cleaved with PstI, 
tailed with dGTP, cleaved with HindIII, and then mixed with the poly T 
tailed mRNA/cDNA complex extended by the pcDV1 vector fragment, ligated 
with E. coli ligase and the mixture treated with DNA polymerase I (Klenow) 
E. coli ligase, and RNase H. The resulting vectors are transformed into E. 
coli K12 MM294 to Amp.sup.R. 
The resulting cDNA library is then screened using the oligomer probes 
identified as complementary to the mRNA coding sequence as described 
above. Colonies hybridizing to probes from regions A or B and C are picked 
and grown; plasmid DNA isolated, and plasmids containing inserts of 
sufficient size to encode the entire sequence of CSF-1 isolated. The 
sequence of the insert of each of these plasmids is determined, and a 
plasmid preparation containing the entire coding sequence including 
regions A and B at the upstream portion is designated pcMCSF. 
F.3 Expression of Murine CSF-1 DNA 
In a manner similar to that set forth above for the human cDNA, the murine 
cDNA is tested for transient expression in COS cells, and used for 
expression in stably transformed CV-1. In addition, the appropriate 
HindIII/ATG encoding sequences are inserted upstream of the mature protein 
by mutagenesis and the coding sequences inserted into pPLOP or pTRP3 for 
procaryotic expression. 
G. Host Vectors 
pPLOP is a host expression vector having the P.sub.L promoter and N gene 
ribosome binding site adjacent a HindIII restriction cleavage site, thus 
permitting convenient insertion of a coding sequence having an ATG start 
codon preceded by a HindIII site. The backbone of this vector is a 
temperature-sensitive high copy number plasmid derived from pCS3. pPLOP 
was deposited at ATCC on Dec. 18 1984, and has accession number 39947. 
pTRP3 is a host expression vector containing a trp promoter immediately 
upstream of a HindIII restriction site, thus permitting insertion of a 
coding sequence in a manner analogous to that above for pPLOP. The 
backbone vector for pTRP3 is pBR322. pTRP3 was deposited with ATCC on Dec. 
18 1984, and has accession number 39946. 
Construction of pPLOP 
Origin of Replication 
pCS3 provides an origin of replication which confers high copy number of 
the pPLOP host vector at high temperatures. Its construction is described 
extensively in U.S. Ser. No. 541,948, filed Oct. 14 1983, incorporated 
herein by reference. pCS3 was deposited June 3 1982 and assigned ATCC 
number 39142. 
pCS3 is derived from pEW27 and pOP9. pEW27 is described by E. M. Wong, Proc 
Natl Acad Sci (USA) (1982) 79: 3570. It contains mutations near its origin 
of replication which provide for temperature regulation of copy number. As 
a result of these mutations replication occurs in high copy number at high 
temperatures, but at low copy number at lower temperatures. 
pOP9 is a high copy number plasmid at all temperatures which was 
constructed by inserting into pBR322 the EcoRI/PvuII origin containing 
fragment from Col El type plasmid pOP6 (Gelfand, D., et al, Proc Natl Acad 
Sci (USA) (1978) 75: 5869). Before insertion, this fragment was modified 
as follows: 50 .mu.g of pOP6 was digested to completion with 20 units each 
BamHI and SstI. In order to eliminate the SstI 3' protruding ends and 
"fill in" the BamHI 5' ends, the digested pOP6 DNA was treated with E. 
coli DNA polymerase I (Klenow in a two-stage reaction first at 20.degree. 
C. for elimination of the 3' SstI protruding end and then at 9.degree. C. 
for repair at the 5' end. The blunt ended fragment was digested and 0.02 
pmole used to transform competent DG75 (O'Farrell, P., et al, J 
Bacteriology (1978) 134: 645-654). Transformants were selected on L plates 
containing 50 .mu./ml ampicillin and screened for a 3.3 kb deletion, loss 
of an SstI site, and presence of a newly formed BamHI site. 
One candidate, designated pOP7, was chosen and the BamHI site deleted by 
digesting 25 .mu.g of pOP7 with 20 units BamHI, repairing with E. coli DNA 
polymerase I fragment (Klenow), and religating with T4 DNA ligase. 
Competent DG75 was treated with 0.1 .mu.g of the DNA and transformants 
selected on L plates containing 50 .mu.g/ml ampicillin. Candidates were 
screened for the loss of the BamHI restriction site. pOP8 was selected. To 
obtain pOP9 the AvaI(repaired)/EcoRI Tet.sup.R fragment from pBR322 was 
prepared and isolated and ligated to the isolated PvuII(partial)/EcoRI 
3560 bp fragment from pOP8. 
Ligation of 1.42 kb EcoRI/AvaI(repair) Tet.sup.R (fragment A) and 3.56 kb 
EcoRI/PvuII Amp.sup.R (fragment B) used 0.5 .mu.g of fragment B and 4.5 
.mu.g of fragment A in a two-stage reaction in order to favor 
intermolecular ligation of the EcoRI ends. 
Competent DG75 was transformed with 5 .mu.l of the ligation mixture, and 
transformants were selected on ampicillin (50 .mu.g/ml) containing plates. 
pOP9, isolated from Amp.sup.R Tet.sup.r transformants, showed high copy 
number, colicin resistance, single restriction sites for EcoRI, BamHI, 
PvuII, HindIII, 2 restriction sites for HincII, and the appropriate size 
and HaeIII digestion pattern. 
To obtain pCS3, 50 .mu.g pEW27 DNA was digested to completion with PvuII 
and the EcoRI. Similarly, 50 .mu.g of pOP9 was digested to completion with 
PvuII and EcoRI and the 3.3 kb fragment was isolated. 
0.36 .mu.g (0.327 pmoles) pEW27 fragment and 0.35 .mu.g (0.16 pmoles) pOP9 
fragment were ligated and used to transform E. coli MM294. Amp.sup.R 
Tet.sup.R transformants were selected. Successful colonies were initially 
screened at 30.degree. C. and 41.degree. C. on beta-lactamase assay plate 
and then for plasmid DNA levels following growth at 30.degree. C. and 
41.degree. C. A successful candidate, designated pCS3, was confirmed by 
sequencing. 
Preparation of the P.sub.L N.sub.RBS Insert 
The DNA sequence containing P.sub.L phage promoter and the ribosome binding 
site for the N-gene (N.sub.RBS) was obtained from pFC5, and ultimately 
from a derivative of pKC30 described by Shimatake and Rosenberg, Nature 
(1981) 292: 128. pKC30 contains a 2.34 kb fragment from lambda phage 
cloned into the HindIII/BamHI vector fragment from pBR322. The P.sub.L 
promoter and N.sub.RBS occupy a segment in pKC30 between a BglII and HpaI 
site. The derivative of pKC30 has the BglII site converted to an EcoRI 
site. 
The BglII site immediately preceding the P.sub.L promoter was converted 
into an EcoRI site as follows: pKC30 was digested with BglII, repaired 
with Klenow and dNTPs and ligated with T4 ligase to an EcoRI linker 
(available from New England Biolabs) and transformed into E. coli K12 
strain MM294 lambda.sup.+. Plasmids were isolated from Amp.sup.R Tet.sup.R 
transformants and the desired sequence confirmed by restriction analysis 
and sequencing. The resulting plasmid, pFC3, was double-digested with PvuI 
and HpaI to obtain an approximately 540 bp fragment isolated and treated 
with Klenow and dATP, followed by Sl nuclease, to generate a blunt ended 
fragment with the 3' terminal sequence -AGGAGAA where the -AGGAGA portion 
is the N.sub.RBS. This fragment was restricted with EcoRI to give a 347 
base pair DNA fragment with 5'-EcoRI (sticky) and HinfI (partial repair, 
Sl blunt)-3' termini. 
To complete pFC5, p.beta.I-Z15 was used to create a HindIII site 3' of the 
N.sub.RBS. p.beta.I-Z15 was deposited Jan. 13 1984, ATCC No. 39578, and 
was prepared by fusing a sequence containing ATG plus 140 bp of .beta.-IFN 
fused to lac Z into pBR322. In p.beta.I-Z15, the EcoRI site of pBR322 is 
retained, and the insert contains a HindIII site immediately preceding the 
ATG start codon of .beta.-IFN. p.beta.I-Z15 was restricted with HindIII, 
repaired with Klenow and dNTPs, and then digested with EcoRI. The 
resulting EcoRI/HindIII (repaired) vector fragment was ligated with the 
EcoRI/HinfI (repaired) fragment above, and the ligation mixture used to 
transform MC1000-39531. Transformants containing the successful 
construction were identified by ability to grow on lactose minimal plates 
at 34.degree. but not at 30.degree.. (Transformations were plated on 
X-gal-Amp plates at 30.degree. and 34.degree. and minimal-lactose plates 
at 30.degree. and 34.degree.. Transformants with the proper construction 
are blue on X-gal-Amp plates at both temperatures, but on minimal lactose 
plates, grow only at 34.degree..) The successful construct was designated 
pFC5. 
Completion of pPLOP 
pCS3 was then modified to provide the P.sub.L and N.sub.RBS control 
sequences. pCS3 was digested with HindIII, and then digested with EcoRI. 
The vector fragment was ligated with an isolated EcoRI/HindIII from pFC5 
containing the P.sub.L N.sub.RBS and transformed into E. coli MM294. The 
correct construction of isolated plasmid DNA was confirmed by restriction 
analysis and sequencing and the plasmid designated pPLOP. 
Preparation of pTRP3 
To construct the host vector containing the trp control sequences behind a 
HindIII site, the trp promoter/operator/ribosome binding site sequence, 
lacking the attenuator region, was obtained from pVH153, obtained from C. 
Yanofsky, Stanford University. Trp sequences are available in a variety of 
such plasmids known in the art. pVH153 was treated with HhaI (which cuts 
leaving an exposed 3' sticky end just 5' of the trp promoter) blunt ended 
with Klenow, and partially digested with TaqI. The 99 bp fragment 
corresponding to restriction at the TaqI site, 6 nucleotides preceding the 
ATG start codon of trp leader, were isolated, and then ligated to EcoRI 
(repair)/ClaI digested, pBR322 to provide pTRP3. 
On Apr. 2 1985, Applicants have deposited with the American Type Culture 
Collection, Rockville, MD, USA (ATCC) the phage pHCSF-1 in E. coli DG98, 
accession no. 40177. On May 21 1985, pHCSF-1a, designated CMCC 2312 in the 
Cetus collection and pHCSF-1 .lambda. Charon 4A for deposit, was deposited 
with ATCC and has accession no. 40185. On June 14 1985, CSF-17 in E. coli 
MM294, designated CMCC 2347, was deposited with ATCC and has accession no. 
53149. In addition, the folllowing deposits were made with ATCC on the 
date of June 19 1986: 
______________________________________ 
Plasmid CMCC No. ATCC No. 
______________________________________ 
pCSF-asp59 2705 67139 
pCSF-gln52 2708 67140 
pCSF-pro52 2709 67141 
pCSF-Bam 2710 67142 
pCSF-BamBcl 2712 67144 
pCSF-Gly150 2762 67145 
______________________________________ 
These deposits were made under the provisions of the Budapest Treaty on the 
International Recognition of the Deposit of Microorganisms for the Purpose 
of Patent Procedure and the Regulations thereunder (Budapest Treaty). This 
assures maintenance of a viable culture for 30 years from date of deposit. 
The deposits will be made available by ATCC under the terms of the 
Budapest Treaty, and subject to an agreement between Applicants and ATCC 
which assures permanent and unrestricted availability upon issuance of the 
pertinent U.S. patent. The Assignee herein agrees that if the culture on 
deposit should die or be lost or destroyed when cultivated under suitable 
conditions, it will be promptly replaced upon notification with a viable 
specimen of the same culture. Availability of the deposits 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. 
These deposits were made for the convenience of the relevant public and do 
not constitute an admission that a written description would not be 
sufficient to permit practice of the invention or an intention to limit 
the invention to these specific constructs. Set forth hereinabove is a 
complete written description enabling a practitioner of ordinary skill to 
duplicate the constructs deposited and to construct alternative forms of 
DNA, or organisms containing it, which permit practice of the invention as 
claimed. 
The scope of the invention is not to be construed as limited by the 
illustrative embodiments set forth herein, but is to be determined in 
accordance with the appended claims.