A macrophage colony stimulating factor, M-CSF.gamma., which is a primary translation product of an alternative mRNA splicing event and is a precursor to biologically active M-CSF. DNA sequences encoding M-CSF.gamma. and recombinant expression vectors comprising the DNA sequences.

BACKGROUND OF THE INVENTION 
The present invention relates to macrophage colony stimulating factors 
(M-CSF) and, more specifically, to macrophage colony stimulating 
factor-.gamma. (M-CSF.gamma.). 
Colony stimulating factors are proteins that influence the growth and 
differentiation of cells responsible for the formation of blood in the 
body and have traditionally been defined by their ability to stimulate 
growth of colonies of bone marrow cells in semi-solid media. Macrophage 
colony stimulating factor ("M-CSF") is a subclass of colony stimulating 
factors and plays a role in the regulation of immune responses by 
potentiating the proliferation and differentiation of macrophages from 
immature hematopoietic progenitor cells, and inducing effector functions 
of mature macrophages including secretion of interferon-.gamma., tumor 
necrosis factor and non-M-CSF colony stimulating activities. Native 
mammalian M-CSF is a glycosylated, disulfide-linked homodimer with 
molecular weights ranging from 45 to 90 kilodaltons (kDa). Disulfide bond 
formation is required for biological activity. 
Recently, two human M-CSF complementary DNAs (cDNA) encoding distinct M-CSF 
proteins have been isolated. Kawasaki et al. (Science 230:291, 1985) first 
reported the isolation of a cDNA clone from a human pancreatic carcinoma 
cell line, MIA-PaCa. This cDNA, when expressed in COS-7 cells, produced 
M-CSF activity as judged by its ability to cause proliferation and 
monocytic colony formation from murine bone marrow cells. The cDNA encoded 
a protein of 256 amino acids that included a 32 amino acid signal sequence 
and a putative transmembrane region of 23 hydrophobic amino acids near its 
carboxyl end. It was proposed that this protein was synthesized as a 
membrane-bound precursor that was then proteolytically cleaved, releasing 
mature M-CSF. 
A second M-CSF cDNA was isolated by Wong et al. (Science 235:1504, 1987), 
who reported that this cDNA encoded a protein of 554 amino acids. The 
larger coding region was due to an in-frame insertion of 894 bp after 
amino acid 181. The coding regions from both cDNAs share the same amino- 
and carboxyl-terminal amino acids including the signal sequence and 
transmembrane regions. The larger cDNA, upon expression in COS-7 cells, 
was found to yield a biologically active protein as judged by its ability 
to form monocytic colonies from human and murine bone marrow cells. It was 
suggested that the two M-CSF cDNAs were formed by alternate splicing of 
mRNA. In order to distinguish the two distinct proteins identified and 
isolated by Kawasaki et al. and Wong et al., they are referred to herein 
as M-CSF.alpha. and M-CSF.beta., respectively. 
The existence of several distinct but related species of M-CSF now raises 
the possibility that these and possibly other different forms may be 
responsible for mediating the different biological activities described 
above. In order to fully elucidate the biological role of M-CSF activity, 
it is thus necessary to identify and characterize the factors responsible 
for M-CSF activity. 
SUMMARY OF THE INVENTION 
The present invention is directed to yet a third species of M-CSF, 
designated herein as M-CSF.gamma.. In one aspect of the invention, a DNA 
sequence is provided comprising a single open reading frame nucleotide 
sequence encoding human M-CSF.gamma.. Preferably, such DNA sequences are 
selected from the group consisting of (a) cDNA clones having the 
nucleotide sequence of FIG. 2; (b) DNA sequences capable of hybridization 
to the clones of (a) under moderately stringent conditions and which 
encode M-CSF.gamma. molecules; and (c) DNA sequences which are degenerate 
as a result of the genetic code to the DNA sequences defined in (a) and 
(b) and which encode M-CSF.gamma.. The present invention also provides 
recombinant expression vectors comprising the DNA sequences defined above, 
and recombinant M-CSF.gamma. molecules produced using the recombinant 
expression vectors. 
In another aspect of the present invention, homogeneous human M-CSF.gamma. 
protein compositions are provided having a molecular size of about 44 
kilodaltons (kDa). 
These and other aspects of the present invention will become evident upon 
reference to the following detailed description and attached drawings.

DETAILED DESCRIPTION OF THE INVENTION 
Definitions 
"Macrophage colony stimulating factor" and "M-CSF" refer to a protein 
capable of inducing biological activity as defined by the ability of 
transfected COS cell supernatants to stimulate proliferation and colony 
formation of murine bone marrow cells, as well as formation of monocytic 
colonies from human bone marrow cells. The particular assays used for this 
determination are set forth below in Example 6. 
"M-CSF.gamma." refers to a protein which is a 438 amino acid precursor to 
M-CSF and is a primary translation product of an alternative mRNA splicing 
event. After translation, M-CSF.gamma. is inserted into the plasma 
membrane and cleaved, releasing a soluble biologically active M-CSF 
monomer subunit protein of about 44 kDa. M-CSF.gamma. has an amino acid 
sequence as set forth in FIG. 2. Prior to being inserted into the plasma 
membrane, M-CSF.gamma. has a theoretical molecular weight of 47,890 
including the leader sequence; without the leader sequence, the 
theoretical molecular weight is 44,638. 
"Substantially identical" and "substantially similar," when used to define 
amino acid sequences, mean that a particular subject sequence, for 
example, a mutant sequence, varies from a reference sequence by one or 
more substitutions, deletions, or additions, the net effect of which does 
not result in an adverse functional dissimilarity between reference and 
subject sequences. For purposes of the present invention, amino acid 
sequences having greater than 30 percent similarity are considered to be 
substantially similar, and amino acid sequences having greater than 80 
percent similarity are considered to be substantially identical. In 
defining nucleic acid sequences, all subject nucleic acid sequences 
capable of encoding substantially similar amino acid sequences are 
considered substantially similar to a reference nucleic acid sequence, and 
all nucleic acid sequences capable of encoding substantially identical 
amino acid sequences are considered substantially identical to a reference 
sequence. For purposes of determining similarity, truncation or internal 
deletions of the reference sequence should be disregarded. Sequences 
having lesser degrees of similarity, comparable biological activity, and 
equivalent expression characteristics are considered to be equivalents. 
"Recombinant," as used herein, means that a protein is derived from 
recombinant (e.g., microbial or mammalian) expression systems. "Microbial" 
refers to recombinant proteins made in bacterial or fungal (e.g., yeast) 
expression systems. As a product, "recombinant microbial" defines a 
protein essentially free of native endogenous substances and unaccompanied 
by associated native glycosylation. Protein expressed in most bacterial 
cultures, e.g., E. coli, will be free of glycan; protein expressed in 
yeast will have a glycosylation pattern different from that expressed in 
mammalian cells. 
"Biologically active," as used throughout the specification as a 
characteristic of M-CSF, means either that a particular molecule shares 
sufficient amino acid sequence similarity with the embodiments of the 
present invention disclosed herein to be capable of causing proliferation 
of monocytic colony formation in murine bone marrow cells and forming 
monocytic colonies from human bone marrow cells. 
"DNA sequence" refers to a DNA polymer, in the form of a separate fragment 
or as a component of a larger DNA construct, which has been derived from 
DNA isolated at least once in substantially pure form, i.e., free of 
contaminating endogenous materials and in a quantity or concentration 
enabling identification, manipulation, and recovery of the sequence and 
its component nucleotide sequences by standard biochemical methods, for 
example, using a cloning vector. Such sequences are preferably provided in 
the form of an open reading frame uninterrupted by internal nontranslated 
sequences, or introns, which are typically present in eukaryotic genes 
(i.e., a single open reading frame nucleotide sequence). However, it will 
be evident that genomic DNA containing the relevant sequences could also 
be used. Sequences of non-translated DNA may be present 5' or 3' from the 
open reading frame, where the same do not interfere with manipulation or 
expression of the coding regions. "Nucleotide sequence" refers to a 
heteropolymer of deoxyribonucleotides. DNA sequences encoding the proteins 
provided by this invention are assembled from cDNA fragments and short 
oligonucleotide linkers, or from a series of oligonucleotides, to provide 
a synthetic gene which is capable of being expressed in a recombinant 
transcriptional unit. 
"Recombinant expression vector" refers to a plasmid comprising a 
transcriptional unit comprising an assembly of (1) a genetic element or 
elements having a regulatory role in gene expression, for example, 
promoters or enhancers, (2) a structural or coding sequence which is 
transcribed into mRNA and translated into protein, and (3) appropriate 
transcription and translation initiation and termination sequences. 
Structural elements intended for use in yeast expression systems 
preferably include a leader sequence enabling extracellular secretion of 
translated protein by a host cell. Alternatively, where recombinant 
protein is expressed without a leader or transport sequence, it may 
include an N-terminal methionine residue. This residue may optionally be 
subsequently cleaved from the expressed recombinant protein to provide a 
final product. 
"Recombinant microbial expression system" means a substantially homogeneous 
monoculture of suitable host microorganisms, for example, bacteria such as 
E. coli or yeast such as S. cerevisiae, which have stably integrated a 
recombinant transcriptional unit into chromosomal DNA or carry the 
recombinant transcriptional unit as a component of a resident plasmid. 
Generally, cells constituting the system are the progeny of a single 
ancestral transformant. Recombinant expression systems as defined herein 
will express heterologous protein upon induction of the regulatory 
elements linked to the DNA sequence or synthetic gene to be expressed. 
The synthesis of human M-CSF is more complex than the other hematopoietic 
growth factors. The gene for M-CSF transcribes multiple RNA species that 
range in size from 1.5 to 4.4 kb. After translation, M-CSF polypeptides 
are glycosylated, react with each other to form dimers and are inserted 
into the plasma membrane. The membrane-bound precursor is then cleaved, 
releasing secreted M-CSF. The two known cDNAs that encode biologically 
active M-CSF, namely M-CSF.alpha. and M-CSF.beta., encode proteins of 256 
and 554 amino acids, respectively. M-CSF.gamma. of the present invention 
encodes a protein of 438 amino acids. While not being bound to any 
particular theory, it is believed that the cDNAs are likely a result of 
alternative RNA splicing within the coding region of the gene as judged by 
the presence of consensus splice sequences at the divergence points 
between the cDNAs. It is possible that the M-CSF.beta. mRNA could be 
spliced to give either the M-CSF.alpha. or M-CSF.gamma. mRNAs by using 
alternative donor sites spliced to the same acceptor site. Alternatively, 
all three mRNAs could be produced by alternative splicing of a common 
precursor. The proteins encoded by M-CSF.alpha., M-CSF.beta. and 
M-CSF.gamma. have a common amino terminus of 149 amino acids including a 
32 amino acid signal sequence and a common 75 amino acid carboxyl-terminus 
including a membrane-spanning region. Each of the M-CSF molecules is 
represented by a form anchored to the plasma membrane. M-CSF.beta. and 
M-CSF.gamma. have an additional 298 and 182 amino acids, respectively, as 
compared to M-CSF.alpha.. This insertion is upstream and adjacent to the 
membrane spanning region. It is within this region that the membrane-bound 
precursors are likely to be proteolytically processed. 
Isolation of cDNAs Encoding M-CSF.gamma. 
In order to identify the coding sequence of human M-CSF.gamma., a DNA 
sequence encoding human M-CSF.gamma. was isolated from a cDNA library 
prepared by reverse transcription of polyadenylated mRNA isolated from 
mitogen-stimulated human pancreatic tumor cells, MIA-PaCa-2, which are 
known to produce significant levels of human M-CSF. The library was probed 
with an M-CSF probe ([s]M-CSF.alpha.) consisting of nucleotides 97 to 544 
and 1439 to 1467 (FIG. 2). This fragment was assembled from 16 synthetic 
oligonucleotides and contains the first 158 amino acids of M-CSF.alpha.. 
Restriction mapping and DNA sequence analysis of eight hybridizing clones 
revealed three classes of M-CSF cDNAs (FIGS. 1A and 2). Two of the classes 
represent the M-CSF.alpha. (three isolates) and M-CSF.beta. (four 
isolates) cDNAs isolated previously by Kawaski et al. and Wong et al. 
However, a new class of M-CSF cDNA, referred to here as M-CSF.gamma. (one 
isolate), was found that encodes a primary translation product 
intermediate in size to M-CSF.alpha. and M-CSF.beta.. 
The M-CSF.gamma. cDNA encoded a protein of 438 amino acids, 182 amino acids 
larger than encoded by M-CSF.alpha. and 116 amino acids smaller than 
encoded by M-CSF.beta.. M-CSF.gamma. appears to be a result of an in-frame 
insertion of 546 bp (FIG. 2, #2 arrows) into M-CSF.alpha. at the same 
location as the 894 bp insertion forming M-CSF.beta. (FIG. 2, #1 arrows). 
Analysis of the DNA sequence (FIG. 2) shows that these 546 bp are 
identical to those found in M-CSF.beta., indicating that the three cDNAs 
are probably a result of alternative splicing of M-CSF mRNA. The 
nucleotide sequences surrounding the insertions are similar to the 
consensus sequences found for mRNA donor, .sup.C.sub.A G/GT.sup.A.sub.G GT 
and acceptor, (.sup.T.sub.C).sub.n N.sup.T.sub.C AG/G splice sites, as 
described by Mount (Nucl. Acids Res. 10:457, 1982). Splicing of the mRNA 
for M-CSF.beta. between the #1 and #2 arrows would form M-CSF.alpha. and 
M-CSF.gamma., respectively. 
In its nucleic acid embodiments, the present invention provides DNA 
sequences comprising a single open reading frame nucleotide sequence 
encoding human M-CSF.gamma.. M-CSF.gamma. DNAs are preferably provided in 
a form which is capable of being expressed in a recombinant 
transcriptional unit under the control of mammalian, microbial, or viral 
transcriptional or translational control elements. For example, a sequence 
to be expressed in a microorganism will contain no introns. In preferred 
aspects, the DNA sequences comprise at least one, but optionally more than 
one sequence component derived from a cDNA sequence or copy thereof. Such 
sequences may be linked or flanked by DNA sequences prepared by assembly 
of synthetic oligonucleotides. However, synthetic genes assembled 
exclusively from oligonucleotides could be constructed using the sequence 
information provided herein. Exemplary sequences include those 
substantially identical to the nucleotide sequences depicted in FIG. 2. 
Alternatively, the coding sequences may include codons encoding one or 
more additional amino acids located at the N-terminus, for example, an 
N-terminal ATG codon specifying methionine linked in reading frame with 
the nucleotide sequence. Due to code degeneracy, there can be considerable 
variation in nucleotide sequences encoding the same amino acid sequence; 
exemplary DNA embodiments are those corresponding to the sequence of 
nucleotides in FIG. 2. Other embodiments include sequences capable of 
hybridizing to the sequence of FIG. 2 under moderately stringent 
conditions (50.degree. C., 2.times.ssc) and other sequences degenerate to 
those described above which encode M-CSF.gamma.. 
Recombinant Expression of M-CSF cDNAs 
In order to compare the molecular weight and biological activity of the 
protein encoded by M-CSF.gamma. cDNA to those encoded by M-CSF.alpha. and 
M-CSF cDNAs, the coding regions for M-CSF.alpha., M-CSF.beta. and 
M-CSF.gamma., were inserted into the mammalian expression vector, pDC201. 
The resulting plasmids, designated pDCCSF.alpha., pDCCSF.beta., 
pDCCSF.gamma., were transfected into COS-7 monkey kidney cells. After 72 
hr, the cultures were labeled for 24 hr with .sup.35 S-Met and .sup.35 
S-Cys and M-CSF specific proteins immunoprecipitated from the supernatants 
with a rabbit anti-M-CSF polyclonal antiserum. Immunoprecipitates were 
then subjected to SDS-PAGE under reducing conditions and protein bands 
visualized by autoradiography. The proteins synthesized by M-CSF.beta. and 
M-CSF.gamma. cDNAs had a molecular size of 44 kDa while M-CSF.alpha. 
synthesized a protein of molecular size 28 kDa (FIG. 3, lanes f, d, and 
j). Thus, M-CSF.beta. and M-CSF.gamma. cDNAs produced proteins of similar 
size even though M-CSF.gamma. cDNA encoded a precursor protein that was 
116 amino acids smaller. This indicates that processing of the membrane 
bound precursor probably occurs upstream of Thr-363 (FIG. 2) since this 
residue is the last amino acid common to both cDNAs upstream of the 
transmembrane region. In order to prove the membrane localization of the 
M-CSF precursors, COS-7 cells were transfected with pDCCSF.alpha., 
pDCCSF.beta. and pDCCSF.gamma. and stained with the rabbit-anti-M-CSF 
antiserum as described in FIG. 4. In all three cases, strong surface 
staining of approximately 20% of the transfected cells was seen. No 
staining was seen with a preimmune rabbit-serum or on COS-7 cells 
transfected with the pDC201 vector alone. 
Supernatants from the M-CSF cDNA transfections were also tested for M-CSF 
activity in murine and human bone marrow proliferation and colony 
formation assays. As can be seen from FIG. 5, all three M-CSF cDNAs 
produce proteins that were active on murine bone marrow cells in 
proliferation and monocytic colony assays. In addition they were active in 
the human bone marrow monocytic colony assay although at a much lower 
level than in the mouse assay. Surprisingly, no activity was detected in 
the human bone marrow proliferation assay. 
Several observations can be made from analysis of the proteins released by 
COS-7 cells transfected with the genomic clone of M-CSF, M-CSF.alpha., 
M-CSF.beta., M-CSF.gamma. cDNAs, and a mutated form of M-CSF.alpha., 
[s]M-CSF.alpha., that lacks the membrane-spanning region. First, the two 
predominant proteins produced by M-CSF.alpha., M-CSF.beta. and 
M-CSF.gamma. (44 kDa and 28 kDa) are also produced by the genomic M-CSF 
clone indicating that the three cDNA species are probably not artifactual. 
The same two M-CSF proteins are found in supernatants of MIA-PaCa-2 cells, 
a source of non-recombinant human M-CSF. Secondly, the cleavage site for 
the proteins synthesized by M-CSF.beta. and M-CSF.gamma. must be upstream 
of Thr-363 as they both synthesize a protein of similar size (44 kDa). The 
amino acids that are present in M-CSF.beta. but not in M-CSF.gamma. are 
therefore not required for processing of the protein. This region includes 
two potential N-linked glycosylation sites (FIG. 2). Third, the 
transmembrane region which may be involved in the normal processing of 
M-CSF, is not required to synthesize a biologically active secreted 
protein. This was illustrated by construction of a mutant form of M-CSF, 
[s]M-CSF.alpha., which lacks the transmembrane region. This cDNA encodes a 
secreted protein indistinguishable in its activity from those produced by 
M-CSF.alpha., M-CSF.beta. and M-CSF.gamma.. Fourth, a large portion of the 
coding region in M-CSF.alpha., M-CSF.beta. and M-CSF.gamma., is not 
required for biological activity (as defined by in vitro bone marrow 
assays) in that a protein with only 159 amino acids (encoded by [ 
s]CSFM-CSF.alpha., M-CSF.beta. and M-CSF.alpha.) is fully active. In fact 
cDNA encoding for as few as 147 amino acids (amino acids 33 to 179, FIG. 
2) has been constructed. When transformed into yeast cells this cDNA 
yields an M-CSF protein with full biological activity. 
The three membrane bound precursors may present different proteolytic 
cleavage sites that are cleaved by different proteases, located at 
different sites in the body, releasing secreted M-CSF. Alternatively, the 
membrane-bound M-CSF molecules could function as cell-surface ligands. 
While no conclusive evidence is presently available, the membrane bound 
M-CSF may be biologically active, requiring direct cell-to-cell contact 
for binding to M-CSF receptors on adjacent cells to initiate a biological 
response. The three different M-CSF cDNAs would extend the 
receptor-binding site of M-CSF (within the amino-terminal 147 amino acids) 
various distances from the cell membrane. 
All of the reported cDNA sequences for M-CSF result in the production of 
biologically active M-CSF proteins by COS-7 cells. The data in FIG. 5 also 
confirm that the recombinant human M-CSF.gamma. has much less biological 
activity on human bone marrow cells than they have on comparable 
populations of murine bone marrow cells. In colony assays, human M-CSF 
appears to be 50-200 fold more active on murine bone marrow then on human 
bone marrow. In addition, while human M-CSF(s) will cause murine bone 
marrow cells to proliferate, they have no mitogenic effect on bone marrow 
cells of human origin, contrary to their previously reported role in bone 
marrow development. In contrast to M-CSF, human GM-CSF (FIG. 5) stimulates 
colony formation and exhibits potent mitogenic activity on human bone 
marrow. The inability of M-CSF to stimulate significant proliferation of 
human bone marrow cells can also be seen by direct analysis of the 
colonies formed in soft agar. In contrast to the large size of the 
colonies stimulated in murine bone marrow by human M-CSF(s) or the large 
size of colonies produced in response to GM-CSF stimulation of human 
marrow (&gt;50-2000 cells), the colonies generated by M-CSF stimulation of 
human marrow contain far fewer cells (.ltoreq.50 cells). These data raise 
the question of the role of M-CSF in human hematopoiesis. In the murine 
system it is clear that M-CSF is a potent colony stimulating factor which 
is required for survival, growth, and differentiation of the mononuclear 
macrophage lineage. It is possible that in humans, M-CSF has lost many of 
these functions and its main role is as an effector of mature macrophages. 
Alternatively, perhaps its main role is not that of a soluble cytokine but 
that of a cell membrane family of molecules, the expression of which may 
direct cell to cell interactions important to the regulation of 
hematopoiesis. 
The present invention also provides expression vectors for producing useful 
quantities of purified M-CSF.gamma.. The vectors can comprise synthetic or 
cDNA-derived DNA fragments encoding human M-CSF.gamma. or bioequivalent 
homologues operably linked to regulatory elements derived from mammalian, 
bacterial, yeast, bacteriophage, or viral genes. Useful regulatory 
elements are described in greater detail below. Following transformation, 
transfection or infection of appropriate cell lines, such vectors can be 
induced to express recombinant protein. 
Human M-CSF.gamma. can be expressed in mammalian cells, yeast, bacteria, or 
other cells under the control of appropriate promoters. Cell-free 
translation systems could also be employed to produce human M-CSF.gamma. 
using RNAs derived from the DNA constructs of the present invention. 
Appropriate cloning and expression vectors for use with bacterial, fungal, 
yeast, and mammalian cellular hosts are described by Pouwels et al. 
(Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985), the relevant 
disclosure of which is hereby incorporated by reference. 
Various mammalian cell culture systems can be employed to express 
recombinant protein. Examples of suitable mammalian host cell lines 
include the COS-7 lines of monkey kidney cells, described by Gluzman (Cell 
23:175, 1981), and other cell lines capable of expressing an appropriate 
vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. 
Mammalian expression vectors may comprise nontranscribed elements such as 
an origin of replication, a suitable promoter and enhancer, and other 5' 
or 3' flanking nontranscribed sequences, and 5' or 3' nontranslated 
sequences, such as necessary ribosome binding sites, a polyadenylation 
site, splice donor and acceptor sites, and termination sequences. DNA 
sequences derived from the SV40 viral genome, for example, SV40 origin, 
early promoter, enhancer, splice, and polyadenylation sites may be used to 
provide the other genetic elements required for expression of a 
heterologous DNA sequence. Additional details regarding the use of 
mammalian high expression vector to produce recombinant human M-CSF.gamma. 
are provided in Examples 2 and 3 below. Exemplary vectors can be 
constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 
1983). 
A useful system for stable high level expression of M-CSF.gamma. cDNAs in 
C127 murine mammary epithelial cells can be constructed substantially as 
described by Cosman et al. (Mol. Immunol. 23:935, 1986). 
Yeast systems, preferably employing Saccharomyces species such as S. 
cerevisiae, can also be employed for expression of the recombinant 
proteins of this invention. Yeast of other genera, for example, Pichia or 
Kluyveromyces, have also been employed as production strains for 
recombinant proteins. 
Generally, useful yeast vectors will include origins of replication and 
selectable markers permitting transformation of both yeast and E. coli, 
e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 
gene, and a promoter derived from a highly expressed yeast gene to induce 
transcription of a downstream structural sequence. Such promoters can be 
derived from yeast transcriptional units encoding highly expressed genes 
such as 3-phosphoglycerate kinase (PGK), .alpha.-factor, acid phosphatase, 
or heat shock proteins, among others. The heterologous structural sequence 
is assembled in appropriate reading frame with translation initiation and 
termination sequences, and, preferably, a leader sequence capable of 
directing secretion of translated protein into the extracellular medium. 
Optionally, the heterologous sequence can encode a fusion protein 
including an N-terminal identification peptide (e.g., 
Asp-Tyr-Lys-(Asp).sub.4 -Lys) or other sequence imparting desired 
characteristics, e.g., stabilization or simplified purification of 
expressed recombinant product. 
Useful yeast vectors can be assembled using DNA sequences from pBR322 for 
selection and replication in E. coli (Amp.sup.r gene and origin of 
replication) and yeast DNA sequences including a glucose-repressible 
alcohol dehydrogenase 2 (ADH2) promoter. The ADH2 promoter has been 
described by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et 
al. (Nature 300:724, 1982). Such vectors may also include a yeast TRP1 
gene as a selectable marker and the yeast 2.mu. origin of replication. A 
yeast leader sequence, for example, the .alpha.-factor leader which 
directs secretion of heterologous proteins from a yeast host, can be 
inserted between the promoter and the structural gene to be expressed (see 
Kurjan et al., U.S. Pat. No. 4,546,082; Kurjan et al., Cell 30:933 (1982); 
and Bitter et al., Proc. Natl, Acad. Sci. USA 81:5330, 1984). The leader 
sequence may be modified to contain, near its 3' end, one or more useful 
restriction sites to facilitate fusion of the leader sequence to foreign 
genes. 
Suitable yeast transformation protocols are known to those skilled in the 
art; an exemplary technique is described by Hinnen et al. (Proc. Natl. 
Acad. Sci. USA 75:1929, 1978), selecting for Trp+ transformants in a 
selective medium consisting of 0.67% yeast nitrogen base, 0.5% casamino 
acids, 2% glucose, 10 .mu.g/ml adenine and 20 .mu.g/ml uracil. 
Host strains transformed by vectors comprising the ADH2 promoter may be 
grown for expression in a rich medium consisting of 1% yeast extract, 2% 
peptone, and 1% glucose supplemented with 80 .mu.g/ml adenine and 80 
.mu.g/ml uracil. Derepression of the ADH2 promoter occurs upon exhaustion 
of medium glucose. Crude yeast supernatants are harvested by filtration 
and held at 4.degree. C. prior to further purification. 
Useful expression vectors for bacterial use are constructed by inserting a 
DNA sequence encoding human M-CSF.gamma. together with suitable 
translation initiation and termination signals in operable reading phase 
with a functional promoter. The vector will comprise one or more 
phenotypic selectable markers and an origin of replication to ensure 
amplification within the host. Suitable prokaryotic hosts for 
transformation include E. coli, Bacillus subtilis, Salmonella typhimuium, 
and various species within the genera Pseudomonas, Streptomyces, and 
Staphylococcus, although others may also be employed as a matter of 
choice. 
Expression vectors are conveniently constructed by cleavage of cDNA clones 
at sites close to the codon encoding the N-terminal residue of the mature 
protein. Synthetic oligonucleotides can then be used to "add back" any 
deleted sections of the coding region and to provide a linking sequence 
for ligation of the coding fragment in appropriate reading frame in the 
expression vector, and optionally a codon specifying an initiator 
methionine. 
As a representative but nonlimiting example, useful expression vectors for 
bacterial use can comprise a selectable marker and bacterial origin of 
replication derived from commercially available plasmids comprising 
genetic elements of the well-known cloning vector pBR322 (ATCC 37017). 
Such commercial vectors include, for example pKK223-3 (Pharmacia Fine 
Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis., 
USA). These pBR322 "backbone" sections are combined with an appropriate 
promoter and the structural sequence to be expressed. 
A particularly useful bacterial expression system employs the phage 
.lambda.P.sub.L promoter and cI857 thermolabile repressor. Plasmid vectors 
available from the American Type Culture Collection which incorporate 
derivative of the .lambda.P.sub.L promoter include plasmid pHUB2, resident 
in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident in E. coli RR1 
(ATCC 53082). Other useful promoters for expression in E. coli include the 
T7 RNA polymerase promoter described by Studier et al. (J. Mol. Biol. 
189:113, 1986), the lacZ promoter described by Lauer (J. Mol. Appl. Genet. 
1:139-147, 1981) and available as ATCC 37121, and the tac promoter 
described by Maniatis (Molecular Cloning: A Laboratory Manual, Cold Spring 
Harbor Laboratory, 1982, p 412) and available as ATCC 37138. 
Following transformation of a suitable host strain and growth of the host 
strain to an appropriate cell density, the selected promoter is 
derepressed by appropriate means (e.g., temperature shift or chemical 
induction) and cells cultured for an additional period. Cells are 
typically harvested by centrifugation, disrupted by physical or chemical 
means, and the resulting crude extract retained for further purification. 
Cells are grown, for example, in a 10 liter fermenter employing conditions 
of maximum aeration and vigorous agitation. An antifoaming agent (Antifoam 
A) is preferably employed. Cultures are grown at 30.degree. C. in the 
superinduction medium disclosed by Mott et al. (Proc. Natl. Acad. Sci. USA 
82:88, 1985), alternatively including antibiotics, derepressed at a cell 
density corresponding to A.sub.600 =0.4-0.5 by elevating the temperature 
to 42.degree. C., and harvested from 2-20 hours, preferably 3-6 hours, 
after the upward temperature shift. The cell mass is initially 
concentrated by filtration or other means, then centrifuged at 
10,000.times.g for 10 minutes at 4.degree. C. followed by rapidly freezing 
the cell pellet. 
Preferably, purified human M-CSF.gamma. or bioequivalent homologues are 
prepared by culturing suitable host/vector systems to express the 
recombinant translation products of the synthetic genes of the present 
invention, which are then purified from culture media. 
Recombinant protein produced in bacterial culture is usually isolated by 
initial extraction from cell pellets, followed by one or more 
concentration, salting-out, aqueous ion exchange or size exclusion 
chromatography steps. Finally, high performance liquid chromatography 
(HPLC) can be employed for final purification steps. Microbial cells 
employed in expression of recombinant mammalian M-CSF.gamma. can be 
disrupted by any convenient method, including freeze-thaw cycling, 
sonication, mechanical disruption, or use of cell lysing agents. 
Fermentation of yeast which express mammalian M-CSF.gamma. as a secreted 
protein greatly simplifies purification. Secreted recombinant protein 
resulting from a large-scale fermentation can be purified by methods 
analogous to those disclosed by Urdal et al. (J. Chromatog. 296:171, 
1984). This reference describes two sequential, reversed-phase HPLC steps 
for purification of recombinant human GM-CSF on a preparative HPLC column. 
In its various embodiments, the present invention provides substantially 
homogeneous recombinant human M-CSF.gamma. polypeptides free of 
contaminating endogenous materials, with or without associated 
native-pattern glycosylation. The native human M-CSF molecule is recovered 
from cell culture extracts as a glycoprotein having an apparent molecular 
weight of about 45 kDa. M-CSF.gamma. expressed in mammalian expression 
systems, e.g., COS-7 cells, has a theoretical molecular weight of 47,890 
including the leader sequence. 
Recombinant M-CSF.gamma. proteins within the scope of the present invention 
also include N-terminal methionyl human M-CSF.gamma.. Additional 
embodiments include soluble truncated versions wherein certain regions, 
for example, the transmembrane region with associated terminal portions, 
and intracellular domains, are deleted. Also contemplated are human 
M-CSF.gamma. expressed as fusion proteins with a polypeptide leader 
comprising the sequence Asp-Tyr-Lys-(Asp.sub.4)-Lys, or with other 
suitable peptide or protein sequences employed as aids to expression in 
microorganisms or purification of microbially-expressed proteins. 
Bioequivalent homologues of the proteins of this invention include various 
analogs, for example, truncated versions of M-CSF.gamma. wherein internal 
residues or sequences not needed for biological activity are deleted. 
Other analogs contemplated herein are those in which one or more cysteine 
residues have been deleted or replaced with other amino acids, for 
example, neutral amino acids. Other approaches to mutagenesis involve 
modification of adjacent dibasic amino acid residues to enhance expression 
in yeast systems in which KEX2 protease activity is present, or 
modification of the protein sequence to eliminate one or more N-linked 
glycosylation sites. 
As used herein, "mutant amino acid sequence" refers to a polypeptide 
encoded by a nucleotide sequence intentionally made variant from a native 
sequence. "Mutant protein" or "analog" means a protein comprising a mutant 
amino acid sequence. "Native sequence" refers to an amino acid or nucleic 
acid sequence which is identical to a wild-type or native form of a gene 
or protein. The terms "KEX2 protease recognition site" and 
"N-glycosylation site" are defined below. The term "inactivate," as used 
in defining particular aspects of the present invention, means to alter a 
selected KEX2 protease recognition site to retard or prevent cleavage by 
the KEX2 protease of S. cerevisiae, or to alter an N-glycosylation site to 
preclude covalent bonding of oligosaccharide moieties to particular amino 
acid residues by the cell. 
Site-specific mutagenesis procedures can be employed to inactivate KEX2 
protease processing sites by deleting, adding, or substituting residues to 
alter Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of 
these adjacent basic residues. Lys-Lys pairings are considerably less 
susceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg to 
Lys-Lys represents a conservative and preferred approach to inactivating 
KEX2 sites. The resulting analogs are less susceptible to cleavage by the 
KEX2 protease at locations other than the yeast .alpha.-factor leader 
sequence, where cleavage upon secretion is intended. 
Many secreted proteins acquire covalently attached carbohydrate units 
following translation, frequently in the form of oligosaccharide units 
linked to asparagine side chains by N-glycosidic bonds. Both the structure 
and number of oligosaccharide units attached to a particular secreted 
protein can be highly variable, resulting in a wide range of apparent 
molecular masses attributable to a single glycoprotein. Attempts to 
express glycoproteins in recombinant systems can be complicated by the 
heterogeneity attributable to this variable carbohydrate component. For 
example, purified mixtures of recombinant glycoproteins such as human or 
murine granulocyte-macrophage colony stimulating factor (GM-CSF) can 
consist of from 0 to 50% carbohydrate by weight. Miyajima et al. (EMBO J. 
5:1193, 1986) reported expression of a recombinant murine GM-CSF in which 
N-glycosylation sites had been mutated to preclude glycosylation and 
reduce heterogeneity of the yeast-expressed product. 
The presence of variable quantities of associated carbohydrate in 
recombinant glycoproteins complicates purification procedures, thereby 
reducing yield. In addition, should the glycoprotein be employed as a 
therapeutic agent, a possibility exists that recipients will develop 
immune reactions to the yeast carbohydrate moieties, requiring therapy to 
be discontinued. For these reasons, biologically active, homogeneous 
analogs of immuno-regulatory glycoproteins having reduced carbohydrate may 
be desirable for therapeutic use. 
Functional mutant analogs of human M-CSF.gamma. having inactivated 
N-glycosylation sites can be produced by oligonucleotide synthesis and 
ligation or by site-specific mutagenesis techniques as described below. 
These analog proteins can be produced in a homogeneous, 
reduced-carbohydrate form in good yield using yeast expression systems. 
N-glycosylation sites in eukaryotic proteins are characterized by the 
amino acid triplet Asn-A.sup.1 -Z, where A.sup.1 is any amino acid except 
Pro, and Z is Ser or Thr. In this sequence, asparagine provides a side 
chain amino group for covalent attachment of carbohydrate. Such a site can 
be eliminated by substituting another amino acid for Asn or for residue Z, 
deleting Asn or Z, or inserting a non-Z amino acid between A.sup.1 and Z, 
or an amino acid other than Asn between Asn and A.sup.1. Preferably, 
substitutions are made conservatively; i.e., the most preferred substitute 
amino acids are those having physicochemical characteristics resembling 
those of the residue to be replaced. Similarly, when a deletion or 
insertion strategy is adopted, the potential effect of the deletion or 
insertion upon biological activity should be considered. 
In addition to the particular analogs described above, numerous DNA 
constructions including all or part of the nucleotide sequences depicted 
in FIG. 2, in conjunction with oligonucleotide cassettes comprising 
additional useful restriction sites, can be prepared as a matter of 
convenience. Mutations can be introduced at particular loci by 
synthesizing oligonucleotides containing a mutant sequence, flanked by 
restriction sites enabling ligation to fragments of the native sequence. 
Following ligation, the resulting reconstructed sequence encodes an analog 
having the desired amino acid insertion, substitution, or deletion. 
Alternatively, oligonucleotide-directed site-specific mutagenesis 
procedures can be employed to provide an altered gene having particular 
codons altered according to the substitution, deletion, or insertion 
required. By way of example, Walder et al. (Gene 42:133, 1986); Bauer et 
al. (Gene 37:73, 1985); Craik (Biotechniques, January 1985, 12-19); Smith 
et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); 
and U.S. Pat. No. 4,518,584 disclose suitable techniques, and are 
incorporated by reference herein. 
Mutational Analysis of M-CSF 
As mentioned above, soluble M-CSF is processed from a membrane-bound 
precursor. To test whether the transmembrane region is required for 
synthesis of an active M-CSF protein, a mutant M-CSF.alpha. gene, 
[s]M-CSF.alpha., that lacks the carboxyl-terminal 66 amino acids of 
M-CSF.alpha. including the transmembrane region was constructed. The 
mutant M-CSF gene was inserted into pDC201 resulting in pDC[s]CSF.alpha.. 
The plasmid was transfected into COS-7 cells and supernatants were 
collected and analyzed as before. Fluorescent staining of COS-7 cells 
containing this construct failed to detect membrane bound forms of M-CSF. 
However, SDS-PAGE shows that [s]M-CSF.alpha. yields proteins of 18 kDa, 22 
kDa and 28 kDa (FIG. 3, lane l) that are present in the supernatant. Two 
of the three protein bands are likely a result of glycosylation at one or 
two of the potential N-linked glycosylation sites (Asn-154 and Asn-182, 
FIG. 2). The protein band at 18 kDa agrees well with the predicted size of 
nonglycosylated [s]M-CSF.alpha. (18.5 kDa). The fully glycosylated form of 
[ s]M-CSF.alpha. (28 kDa) is similar in size to the protein synthesized by 
M-CSF.alpha. (FIG. 3, lane j). These results support the theory that the 
transmembrane region present in the precursor protein encoded by 
M-CSF.alpha. may indeed be absent from the secreted product. The proteins 
synthesized by [s]M-CSF.alpha. were also active in the murine bone marrow 
assay and human bone marrow colony assay but not the human bone marrow 
proliferation assay (FIG. 5). These results indicate that the 
transmembrane region of M-CSF.alpha., which may be involved in the normal 
processing of M-CSF, is not required for secretion of a biologically 
active protein. 
Isolation and Expression of a Genomic M-CSF Clone 
Transcription of the gene for human M-CSF results in several mRNA species 
encoding at least three different M-CSF precursor proteins. When expressed 
in COS-7 cells, glycosylated subunit proteins of 44 kDa or 28 kDa are 
released. In order to determine if additional M-CSF related proteins 
exist, a genomic clone of M-CSF was isolated and expressed in COS-7 cells. 
A human genomic .lambda.-phage library (Lawn et al., Cell 15:1157, 1978) 
was screened with the [s]M-CSF.alpha. probe resulting in the isolation of 
two distinct classes of clones, .lambda.CSF-1-1 and .lambda.CSF-1-12, with 
inserts of 11.9 kbp and 13.0 kbp respectively. Restriction mapping and 
Southern hybridization analysis using synthetic oligonucleotide probes 
specific for the 5'- and 3'-non-coding regions of M-CSF indicated that the 
two .lambda. clones overlapped and contained all of the coding exons of 
the M-CSF gene (FIG. 1B). Together, the .lambda.-phage clones defined 17.3 
kbp of the M-CSF gene. They appeared however to lack the majority of the 
3'-non-coding exons. Using the common XhoI site, DNA fragments from 
.lambda.CSF-1-1 and .lambda.CSF-1-12 that contained the coding exons of 
the M-CSF gene were inserted into the mammalian cell expression vector, 
pMLSV (Cosman et al., Nature 312:768, 1984), resulting in the plasmid, 
pMLSV/genomic-CSF-1. After transfection into COS-7 cells, supernatants 
were analyzed as described above. As can be seen from the autoradiogram in 
FIG. 3 lane h, the genomic clone of M-CSF produced two forms of M-CSF with 
molecular sizes of 44 kDa and 28 kDa that were identical in size to the 
proteins encoded by M-CSF.alpha., M-CSF.beta. and M-CSF.gamma. cDNAs, 
although at lower levels (longer exposure time). Supernatants from the 
genomic M-CSF transfection were also found to be active in the murine and 
human bone marrow colony assays and the murine bone marrow proliferation 
assay, although at reduced levels (FIG. 5). The lower protein production 
probably reflects decreased plasmid replication in the COS-7 cells due to 
the large size of the pMLSV/genomic-CSF-1 plasmid (23.5 kbp). As before, 
no activity was detected in the human bone marrow proliferation assay. 
These results indicate that the predominant forms of M-CSF are proteins of 
44 kDa and 28 kDa. 
These results were further substantiated by analysis of mitogen stimulated 
MIA-PaCa-2 cells. MIA-PaCa-2 is a human pancreatic tumor cell line known 
to synthesize M-CSF. Mia-PaCa-2 cells were labeled for 24 hr with .sup.35 
S-Met and .sup.35 S-Cys, supernatants collected and M-CSF specific 
proteins immunoprecipitated. SDS-PAGE analysis revealed two predominant 
protein bands of 44 kDa and 28 kDa (FIG. 3, lane n). These proteins were 
the same size as those synthesized by M-CSF.alpha., M-CSF.beta., 
M-CSF.gamma. and the genomic clone of M-CSF. 
The following examples are offered by way of illustration, and not by way 
of limitation. 
Example 1: Isolation of M-CSF cDNA and Genomic Clones 
For mRNA isolation, human pancreatic tumor cells, Mia-PaCa-2 (ATCC 
#CRL1420) (Wu et al., J. Biol. Chem. 254:6226, 1979), were grown in RPMI 
1640 with 0.292 mg/ml glutamine, 0.2 g/l penicillin, and 0.2 g/l 
stretomycin and 10% fetal calf serum to one day preconfluence and then 
stimulated for two days with phorbol myristate acetate (PMA, 50 ng/ml). 
Procedures for mRNA purification and cDNA synthesis have been described by 
Cosman et al., Nature 312:768, 1984, and March et al., Nature 315:641, 
1985, and are incorporated herein by reference. The cDNA was modified with 
EcoRI linkers, cloned into .lambda.gt10, packaged in vitro, and used to 
infect E. coli strain C600 hfl.sup.- according to the procedure described 
by Huynh et al. (DNA Cloning: A Practical Approach, IRL Press, Oxford, 
1985) (.lambda.gt10, packaging kits and bacterial hosts purchased from 
Stratagene). Independent plaques (5.times.10.sup.5) were blotted onto 
nitrocellulose filters (Schleicher and Schuell, Keene, N.H.), and 
hybridized with a .sup.32 P-labeled human M-CSF probe. The probe was 
.sup.32 P-labeled by nick-translating a 420 bp PstI/XbaI DNA fragment of 
[s]M-CSF.alpha. which consists of nucleotides 141 to 544 and 1439 to 1467 
(FIG. 2). The [s]M-CSF.alpha. gene was assembled from 16 synthetic 
oligonucleotides and contains the first 158 amino acids of M-CSF.alpha.. 
Filters were incubated 24 hr at 37.degree. C. with 5.times.10.sup.5 cpm/ml 
probe in Stark's hybridization buffer (see Wahl et al., Proc. Natl. Acad. 
Sci. USA 76:3683, 1979) consisting of 50% formamide, 5.times.SSC (0.75M 
NaCl/0.075M tri-sodium citrate pH 7), 50 mM KH2PO4 pH 6.5, ficoll, 
polyvinylpyrrolidone, and bovine serum albumin at 0.04% each, 0.1% sodium 
dodecyl sulfate (SDS), 20 mM EDTA, and 150 .mu.g/ml salmon sperm DNA. 
Filters were then washed extensively in 6.times.SSC at room temperature 
followed by successive washes at 55.degree. C. in 6.times.SSC, 
2.times.SSC/0.1% SDS, and finally 0.5.times.SSC/0.1% SDS, prior to 
autoradiography. After rescreening positive plaques, cDNA inserts were 
isolated and sub-cloned into pGEMBL18 for DNA sequencing according to the 
techniques of Hattori and Sakaki (Anal. Biochem. 152:272, 1986) and Sanger 
et al. (Proc. Natl. Acad. Sci. USA 74:3463, 1977). Plasmid pGEMBL18 is a 
derivative of pGEMBL18 (see Dente et al., Nucl. Acids Res. 11:1645, 1983), 
in which the promoters for Sp6 and T7 polymerases flank the multiple 
cloning site. Sequence analysis was done by programs designed by the 
University of Wisconsin Genetics Computer Group described by Devereux et 
al. (Nucl. Acids Res. 12:387, 1984). 
A human .lambda.-phage genomic library (1.times.10.sup.6 plaques) described 
by Lawn et al. (Cell 15:1157, 1978), was screened with a .sup.32 P-labeled 
M-CSF probe as described above except that hybridizations were conducted 
at 55.degree. C. in 6.times.SSC and washes were performed at 68.degree. C. 
in 1.times.SSC. DNA from positive plaques was isolated and characterized 
by restriction mapping and Southern blot analysis as described by Maniatis 
et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor 
Laboratory Press, New York, 1982) using .sup.32 P-labeled oligonucleotide 
probes complementary to the 5' non-coding and 3' non-coding regions 
(nucleotides 106-124 and 968-987, respectively) of M-CSF.alpha.. The 
oligonucleotides were .sup.32 P-labeled with T4 polynucleotide kinase. 
Example 2: Construction of M-CSF Expression Plasmids 
Plasmids, designed to synthesize and secrete M-CSF, were constructed by 
inserting DNA fragments encoding M-CSF.alpha., M-CSF.beta., M-CSF.gamma. 
and the genomic sequences of M-CSF into the mammalian expression plasmids, 
pDC201 or pMLSV. Plasmid pDC201 is a derivative of pMLSV. pDCCSF.beta. was 
constructed by blunt-ending the 1720 bp EcoRI fragment (from the .lambda. 
phage cDNA isolate, .lambda.-13) containing the entire coding region of 
M-CSF.beta. and inserting it into the SmaI site of pDC201. As the cDNA 
isolate of M-CSF.gamma. (.lambda.-8) lacked the first 140 amino acids, we 
constructed a hybrid between M-CSF.beta. and M-CSF.gamma. by substituting 
DNA sequence downstream of the NcoI site in M-CSF.beta. with sequence 
downstream of the NcoI site from M-CSF.gamma. (FIG. 1A). This was 
accomplished by ligating together: a 1500 bp SfiI/NcoI fragment from 
pDCCSF.beta. (SfiI site is from pDC201 and is upstream of the M-CSF.beta. 
coding region) containing amino acids 1-214, a 650 bp NcoI/EcoRI (blunted) 
fragment from .lambda.-8 containing the carboxyl 224 amino acids of 
M-CSF.gamma. and a 4500 bp SfiI/SmaI fragment from pDC201, resulting in 
plasmid pDCCSF.gamma.. For construction of an expression vector containing 
M-CSF.alpha., we used a synthetic version of M-CSF.alpha. which was 
constructed from twenty oligonucleotides. This was accomplished by 
ligating the 670 bp BstXI/XbaI fragment containing the distal 214 amino 
acids of mature M-CSF.alpha. to the signal sequence of the interleukin-2 
receptor contained in a SstI/XbaI fragment of pN1/N4-S with the aid of 
oligonucleotides encoding the first 10 amino acids of mature M-CSF.alpha.. 
The coding region of M-CSF.alpha. together with the signal sequence of the 
interleukin-2 receptor was then excised by XbaI digestion, blunt-ended, 
and ligated to the SmaI site of pDC201 resulting in plasmid pDCCSF.alpha.. 
In a similar manner the truncated version of M-CSF.alpha., 
[s]M-CSF.alpha., was inserted into pDC201 resulting in plasmid 
pDC[s]CSF.alpha.. 
The genomic expression vector pMLSV/genomic-CSF-1 was constructed as 
follows. A fragment encoding the 3' portion of the CSF-1 gene from the 
XhoI site to a ClaI site in the .lambda. arm (from .lambda.CSF-1-12) was 
subcloned into a plasmid having a polylinker containing XhoI, AccI and 
NotI sites. Insertion between the XhoI and AccI sites allowed the CSF-1-12 
3' end fragment to be re-isolated as a XhoI-NotI fragment. This was 
ligated together with a SpeI/XhoI fragment from .lambda.CSF-1-1 into the 
mammalian expression plasmid, pMLSV, that had been cut with XbaI and NotI. 
The XbaI and NotI sites are present in a polylinker in between the SV40 
early promoter and the SV40 splicing/polyadenylation signals. 
Example 3: Transfection of COS-7 cells and analysis of M-CSF 
COS-7 cells (10 cm plates) were transfected by a standard DEAE-dextran 
method, such as that described previously by Cosman et al. (Nature 
312:768, 1984) with the pDCCSF.gamma. vector DNA three days prior to 
collection of media for M-CSF assays or labeling. For labeling, each plate 
of cells was washed twice with phosphate buffered saline and incubated in 
3 ml of MEM without methionine and cysteine. After the addition of .sup.35 
S-methionine and .sup.35 S-cysteine (100 .mu.Ci "Translabel", Amersham) 
the cells were incubated for 24 hours at 37.degree. C. The media, 
containing secreted M-CSF species, was removed and centrifuged for 5 min 
in a microfuge. Proteinase inhibitors were added to the supernatants at 
final concentrations of 5 mM EDTA, 5 mM EGTA, 10 .mu.g/ml soybean trypsin 
inhibitor, 10 .mu.M leupeptin, 10 .mu.M pepstatin A, 10 .mu.M 
o-phenanthroline, and 25 mM benzamidine-HCl. A preclearing reaction was 
performed by addition of 5 .mu.l of preimmune rabbit serum and 50 .mu.l of 
a 20% suspension of protein A Sepharose. After incubation at 4.degree. C. 
on a rocker for 30 min., the suspension was centrifuged in a microfuge for 
5 min. The supernatants were incubated with 5 .mu.l of preimmune serum or 
5 .mu.l of anti-M-CSF rabbit serum and 70 .mu.l of a 20% suspension of 
protein A Sepharose for 4 hours at 4.degree. C. The immunoprecipitates 
were then washed four times in RIPA buffer (50 mM HEPES, pH 8.0, 150 mM 
NaCl, 0.5% sodium deoxycholate, 0.5% nonidet P-40, and 0.1% SDS), and 
boiled in SDS sample buffer containing 1 mM dithiothreitol. Following 
centrifugation, the supernatants were removed and stored at -20.degree. C. 
until electrophoresis on a 15% SDS-polyacrylamide gel. 
MIA-PaCa-2 cells were labeled as above and the resulting media, containing 
radiolabeled M-CSF, was harvested and processed identically to 
supernatants from transfected COS-7 cells. 
Example 4: Preparation of Anti-M-CSF Rabbit Serum 
Adult female New Zealand rabbits were obtained from R and R Rabbitry 
(Sultan, Wash.). All immunizations were given intradermally. Rabbits 
initially received 100 .mu.g of purified yeast generated recombinant 
[s]M-CSF.alpha. emulsified 1:1 in complete Freund's adjuvant. In all 
subsequent immunizations the antigen was emulsified 1:1 in incomplete 
Freund's adjuvant. The rabbits received 100 .mu.g on day 22, 250 .mu.g on 
days 41 and 58, and 200 .mu.g on days 79, 156, and 192. Bleeds were taken 
10 days after every injection and the serum titers to [s]M-CSF.alpha. 
determined by dot blot analysis as described by Conlon et al. (J. Immunol. 
135:328, 1985). A final bleed was obtained on day 205. At a 1:130,000 
dilution, this serum could detect 25 ng of [s]M-CSF.alpha. by 
immunodot-blot. 
Example 5: Fluorescent Staining of Transfected COS-7 Cells 
COS-7 cells were transfected as described above and cultured on glass 
slides (1.times.3 in, Lab-Tek, Naperville, Ill.) for 72 hr. Rabbit 
antisera, directed to M-CSF, was diluted 1:1000 in RPMI 1640 containing 2% 
bovine serum albumin, 20 mM Hepes, and 0.2% sodium azide, added to the 
cell monolayers for 60 min at 4.degree. C., and washed three times with 
cold RPMI 1640. Goat anti-rabbit IgG, to which fluorescin isothiocyanate 
had been coupled (TAGO, Burlingame, Calif.) was then diluted 1:50 and 
added to the cell monolayers. After 30 min., monolayers were washed with 
RPMI 1640 and examined on a Leitz Dialux 20EB fluorescence microscope. 
Photographs were taken using an Olympus photomicrographic system model 
PM-10AD with Kodak Ektrachrome P800/1000 film. Controls included the use 
of preimmune rabbit serum as well as monolayers of COS-7 cells transfected 
with non-M-CSF containing DNA. 
Example 6: Bone Marrow Assays 
Procedures for preparation of human bone marrow and human bone marrow 
colony assays have been described by Cantrell et al. (Proc. Natl. Acad. 
Sci. USA 82:6250, 1985). For the human bone marrow proliferation assay, 
serial threefold dilutions of sample were made in 96-well tissue 
culture-treated microtiter plates, so that the final volume in each well 
was 50 .mu.l. Thereafter, 50 .mu.l of the bone marrow cell suspension 
(2.5.times.10.sup.5 cells/mL) was added to each well. Plates were 
incubated for 96 hr. at which time 25 .mu.l of medium containing 80 
.mu.Ci/ml 3H-thymidine (.sup.3 H-Tdr, 80 Ci/mM, New England Nuclear 
NET-027Z) was added to each well and incubation continued for an 
additional 5 hr. The contents of each well were harvested into glass fiber 
strips using a multiple automated sample harvester and radionuclide 
incorporation was assessed by liquid scintillation counting. Units of 
activity were determined as the inverse of the dilution which yielded 50% 
of maximal level of .sup.3 H-Tdr incorporated in response to stimulation 
with a known standard (purified recombinant human GM-CSF). 
Murine bone marrow colony assays were performed with COS-7 cell 
supernatants in a traditional semi-solid agar assay using 10.sup.5 bone 
marrow cells per ml (see Stanley, Meth. Enzymol., Academic Press, New 
York, 1985). The CFU-C per ml was defined as the reciprocal of the 
dilution multiplied by the number of colonies observed at one half the 
maximal response. Colonies were all of macrophage lineage. For the murine 
bone marrow proliferation assay, bone marrow derived macrophages (BMM) 
were obtained as described by Tushinski et al. (Cell 28:71, 1982). 
Briefly, C.sub.3 H/Hej bone marrow cells (1.times.10.sup.6 /ml) were 
seeded into tissue culture flasks (Falcon) at a density of 
2.9.times.10.sup.5 cells/cm.sup.2 in the presence of 1000 units/ml of 
partially purified M-CSF from L929 fibroblast conditioned medium. The 
culture media consisted of alpha MEM (Gibco) supplemented with 15% fetal 
calf serum, 0.292 mg/ml glutamine, 0.02 mg/ml asparagine, 
5.times.10.sup.-5 M 2-mercaptoethanol, 0.2 g/l penicillin and 0.2 g/l 
streptomycin. After three days the nonadherent cells were harvested and 
used in a proliferation assay. COS-7 cell supernatants were serially 
diluted into 96 well microtiter plates. Then BMM, at 2.times.10.sup.5 /ml, 
were added to the samples in a final volume of 100 .mu.l. 
Following a five hour incubation at 37.degree. C. in 10% CO.sub.2, the 
cultures were pulsed with 2 .mu.Ci .sup.3 H-Tdr. After a total incubation 
time of 24 hours, the cultures were harvested onto glass fiber filters and 
.sup.3 H-Tdr measured by liquid scintillation counting. One unit of 
activity was defined as the amount of sample necessary to induce 50% of 
maximal .sup.3 H-Tdr incorporated as compared to a known standard 
(conditioned media from murine L929 cell). 
Example 7: Expression of M-CSF.gamma. in Yeast 
For expression of human or murine M-CSF.gamma. in yeast, a yeast expression 
vector derived from pIXY120 is constructed as follows. pIXY120 is 
identical to pY.alpha.HuGM (ATCC 53157), except that it contains no cDNA 
insert and includes a polylinker/multiple cloning site with an NcoI site. 
This vector includes DNA sequences from the following sources: (1) a large 
SphI (nucleotide 562) to EcoRI (nucleotide 4361) fragment excised from 
plasmid pBR322 (ATCC 37017), including the origin of replication and the 
ampicillin resistance marker for selection in E. coli; (2) S. cerevisiae 
DNA including the TRP-1 marker, 2.mu. origin of replication, ADH2 
promoter; and (3) DNA encoding an 85 amino acid signal peptide derived 
from the gene encoding the secreted peptide .alpha.-factor (See Kurjan et 
al., U.S. Pat. No. 4,546,082). An Asp718 restriction site was introduced 
at position 237 in the .alpha.-factor signal peptide to facilitate fusion 
to heterologous genes. This was achieved by changing the thymidine residue 
at nucleotide 241 to a cytosine residue by oligonucleotide-directed in 
vitro mutagenesis as described by Craik, Biotechniques: 12 (1985). A 
synthetic oligonucleotide containing multiple cloning sites and having the 
following sequence was inserted from the Asp718 site at amino acid 79 near 
the 3' end of the .alpha.-factor signal peptide to a SpeI site in the 
2.mu. sequence: 
##STR1## 
pBC120 also varies from pY.alpha.HuGM by the presence of a 514 bp DNA 
fragment derived from the single-standed phage f1 containing the origin of 
replication and intergenic region, which has been inserted at the Nru1 
site in the pBR322 sequence. The presence of an f1 origin of replication 
permits generation of single-stranded DNA copies of the vector when 
transformed into appropriate strains of E. coli and superinfected with 
bacteriophage f1, which facilitates DNA sequencing of the vector and 
provides a basis for in vitro mutagenesis. To insert a cDNA, pIXY120 is 
digested with Asp718 which cleaves near the 3' end of the .alpha.-factor 
leader peptide (nucleotide 237) and, for example, NcoI which cleaves in 
the polylinker. The large vector fragment is then purified and ligated to 
a DNA fragment encoding the protein to be expressed. 
To create a secretion vector for expressing human M-CSF.gamma., a cDNA 
fragment including the complete open reading frame encoding hM-CSF.gamma. 
is cleaved with an appropriate restriction endonuclease proximal to the 
N-terminus of the mature protein. An oligonucleotide or oligonucleotides 
are then synthesized which are capable of ligation to the 5' and 3' ends 
of the hM-CSF.gamma. fragment, regenerating any codons deleted in 
isolating the fragment, and also providing cohesive termini for ligation 
to pIXY120 to provide a coding sequence located in frame with respect to 
an intact .alpha.-factor leader sequence. 
The resulting expression vectors are then purified and employed to 
transform a diploid yeast strain of S. cerevisiae (XV2181) by standard 
techniques, such as those disclosed in EPA 0165654, selecting for 
tryptophan prototrophs. The resulting transformants are cultured for 
expression of an hM-CSF.gamma. protein as a secreted or extracted product. 
Cultures to be assayed for hM-CSF.gamma. expression are grown in 20-50 ml 
of YPD medium (1% yeast extract, 2% peptone, 1% glucose) at 37.degree. C. 
to a cell density of 1-5.times.10.sup.8 cells/ml. To separate cells from 
medium, cells are removed by centrifugation and the medium filtered 
through a 0.45.mu. cellulose acetate filter prior to assay. Supernatants 
produced by the transformed yeast strain, or extracts prepared from 
disrupted yeast cells, are assayed for the presence of hG-CSF.gamma. using 
binding assays as described above.