Note: Descriptions are shown in the official language in which they were submitted.
134119
HOECHST AKTIENGESELLSCHAFT HOE 85/F 293 Dr.KL/ml
GM-CSF protein, its derivatives, the preparation of
proteins of this type, and their use
Human granulocyte macrophage colony-stimulating factor
(GM-CSF) is a glycoprotein with a molecular weight of
about 23,000 dalton. The cDNA sequence and the expres-
sion of the glycoprotein in mammalian cells have
already been disclosed (G.G. Wong et al., Science 228
(1985>, 810-815, D. Metcalf, Science 229 (1985),
16-22).
It has now been found, surprisingly, that the expres-
sion of human GM-CSF protein, called "CSF" hereinafter,
in bacteria results in a biologically active product.
Thus the invention relates to CSF for use in medical
treatment and to the use for the preparation of medi-
cements.
The invention furthermore relates to the preparation
of CSF by expression in bacteria, in particular in E.
coli. In particular, it is possible to use for this
purpose the published cDNA sequences which can be ob-
tained in a manner known per se, preferably by synthe-
sis.
The invention additionally relates to expression
vectors for use in bacteria, in particular in E. coli,
which contain, in a suitable arrangement ("operatively
linked to">, a DNA coding for CSF or a CSF fusion pro-
tein.
The invention additionally relates to biologically
active derivatives of CSF which can be obtained by
modifications, which are known per se, of the ONA
sequences. Thus, for example, it is possible to in-
corporate cleavage sites in the construction of
~3'~~19~
- - 2 -
vectors for fusion proteins which, after elimination of
the CSF protein, have C-terminal and N-terminal modifi-
cations in the amino acid sequence. Furthermore, the
invention relates to the use of proteins of this type
in medical treatment and to their use for the prepara-
tion of medicaments, and to medicaments which contain
CSF protein and its biologically active derivatives, in
particular medicaments for the stimulation of prolifer-
ation of hemopoietic cells and for promotion of the
formation of granulocytes and macrophages.
Further aspects of the invention and its preferred em-
bodiments are illustrated in detail below and are
defined in the patent claims.
The invention is furthermore illustrated by Figures 1
to 15, each of which explains, mostly in the form of a
flow diagram, the processes of the examples of the same
numbers. These figures are not to scale, in particular
the scale has been "expanded" in the region of the
polylinkers.
Thus, Figure 1 and its continuations 1a and 1b show the
preparation of the vector pW 225 which is used for the
direct expression of (Met-)CSF. The figures which fol-
low relate to vectors which result in the expression of
fusion proteins in which a "ballast" protein, which is
derived from a part-sequence of human interleukin-2,
hereinafter "IL-2" or "eIL-2", is located at the N-
terminal end in front of the CSF amino acid sequence:
Figure 2 and its continuations 2a and 2b show the prep-
aration of the vector pW 216 which codes for a fusion
protein from which is obtained, by acid cleavage, a CSF
derivative which is extended at the N-terminal end by
the amino acid proline.
Figure 3 shows the synthesis of the vector pW 240 which
codes for a fusion protein which results, after acid
1341187
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cleavage, in a CSF derivative which has proline in place
of the first amino acid (alanine).
Figure 4 relates to the preparation of the vector pW 241
which codes for a fusion protein which results, after
S acid cleavage, in a CSF derivative in which the first
amino acid (alanine) is missing.
Figure 5 demonstrates the preparation of the vector pW
242 which codes for a fusion protein which results,
after acid cleavage, in a CSF derivative in which the
first five amino acids have been eliminated.
Figure 6 relates to the preparation of the vector pW 243
which codes for a fusion protein which results, after
acid cleavage, in a CSF derivative in which the first
seven amino acids are missing.
Figure 7 shows the synthesis of the vector pW 244 which
codes for a fusion protein with which is obtained, after
acid cleavage, a CSF derivative in which the first 11
amino acids have been eliminated.
Figure 8 and its continuation 8a show the synthesis of
the vector pW 246. This codes for a fusion protein in
' which two modified sequences, denoted "CSF'", follow
the IL-2 part-sequence. Acid cleavage results in a CSF
derivative in which proline is located at the N-termi-
nal end in front of the first amino acid proline and in
which the last amino acid has been replaced by aspartic
acid.
Figure 9 shows the synthesis of the vector pW 247 which
codes for a fusion protein in which three CSF' sequen-
ces follow the IL-2 part-sequence. Acid cleavage
results in the CSF derivative characterized in Figure 8
being obtained.
1341197
- 4 -
Figure 10 and its continuation Figure 10a show the pre-
paration of the hybrid plasmids pS 200 to 204 which
contain synthetic CSF DNA part-sequences, the plasmid
pS 200 containing "synthesis block I", shown in Appen-
dix I, plasmid pS 201 containing "synthesis block II"
shown in Appendix II, plasmid pS 202 containing "syn-
thesis block III" shown in Appendix III, plasmid pS 203
containing the entire synthetic gene, and pS 204 repre-
senting an expression plasmid which likewise contains
the entire synthetic CSF DNA sequence. Expression and
acid cleavage result in the same CSF derivative as des-
cribed in Figure 2 being obtained.
Figure 11 and its continuation Figure 11a show the
synthesis of the expression plasmid pS 207 which codes
for a fusion protein which provides, after cleavage
with N-bromosuccinimide, a CSF derivative in which Trp
in each of positions 13 and 122 has been replaced by
His.
Figure 12 shows a synthetic DNA part-sequence which
permits the preparation of a CSF derivative in which
Ile in position 100 has been replaced by Thr.
Figure 13 and its continuation Figure 13a show the
synthesis of the expression plasmid pS 210 which codes
for a fusion protein which provides, after cleavage
with cyanogen bromide, a CSF derivative in which all
methionine residues have been replaced by neutral
amino acids, namely by Ile in position 36 and by Leu
in positions 46, 79 and 80.
Figure 14 shows a synthetic DNA sequence which permits,
in accordance with the synthesis scheme in Figure 13,
the preparation of a CSF derivative in which Met in
position 36 has been replaced by Ile, and Met in posi-
tion 46 has been replaced by Leu, and a single Leu
residue is present in place of amino acids 79 and 80.
134t19~
- 5 -
Figure 15 shows a synthetic DNA whose use in
the synthesis scheme shown in Figure 13 permits the
preparation of a CSF derivative in which Met in posi-
tion 36 has been replaced by Ile and in position 46 has
been replaced by Leu, and in which the two amino acids
in positions 79 and 80 have been deleted.
Figure 16 shows synthesis block 1 containing 6 DNA part
sequenc es (Ia/Ib, Ic/Id, Ie/If, Ig/Ih, Ii/Ik, I1/Im). The
compatible ends of the part-sequences are indicated by
angular lines within the DNA-sequence. The numbering above
the upper strand of the DNA refers to the nucleotides, the
numbering in brackets below the protein sequence refers to
the amino acids.
Figure 17 shows synthesis block II containing 3 DNA part-
sequences (IIa/IIb, IIc/IId, IIe/IIf). Symbols and numbering
see legend of Figure 16. The numbering of synthesis block
II is a continuation of the numbering of synthesis block I.
Figure 18 shows synthesis block III containing 6 DNA part-
sequences (IIIa/IIIb, IIIc/IIId, IIIe/IIIf, IIIg/IIIh,
IIIi/III~, IIIk/III1). Symbols and numbering see legend of
Figure 16. The numbering of synthesis block III is a
continuation of the numbering of synthesis block II.
The possible variations explained in these figures and
examples are, of course, merely examples of the large
numbers of modifications which are possible according
to the invention. Thus, it is also possible in a man-
ner known per se to use other protein sequences,
especially bacterial, as the "ballast" portion of the
fusion proteins, and it is possible to use all custom-
ary methods for the linkage and cleavage of the fusion
proteins, it being possible for other CSF derivatives
with a modified amino acid sequence in the molecule or
'C
1341197
-- - Sa -
at both ends of the molecule to result. The choice of
the IL-2 sequence and the synthetic DNA sequences and
the cleavage of the fusion proteins should thus be
viewed merely as preferred embodiments of the invention
which can be varied in a manner knovn per se.
It has emerged that the "open reading frame" comprising
a ONA which codes for interleukin-2 is particularly
advantageous as an expression aid for the expression of
peptides and proteins, and that an N-terminal portion of
IL-2 which essentially corresponds to the first 100
amino acids is particularly well suited for the prepar-
ation of fusion proteins. The primary product obtained
in this way is a fusion protein which is composed
entirely or very predominantly of eukaryotic protein
sequences. Surprisingly, this protein is apparently
not recognized as being a foreign protein by the
proteases which are intrinsic to the host, nor is it
immediately degraded again. Another advantage is that
the fusion proteins according to the invention are
sparingly soluble or insoluble and thus can easily be
~C
1341197
- 6 -
removed, appropriately by centrifugation, from the
soluble proteins.
Since, according to the invention, the functioning of
the "ballast portion" of the fusion protein does not
depend on the IL-2 portion being a biologically active
molecule, it likewise does not depend on the exact
structure of the IL-2 portion. It suffices for this
purpose that essentially the first 100 N-terminal amino
acids are present.- Thus, it is possible, for example,
to carry out at the N-terminal end modifications which
permit cleavage of the fusion protein in the case where
the desired protein is located N-terminal thereto.
Conversely, modifications at the C-terminal end can be
carried out in order to permit or facilitate the elimi-
nation of the desired protein.
The natural DNA sequence coding for human IL-2 is dis-
closed in the European Patent Application with the pub-
lication number~"0,091,539. The literature quoted there
also relates to mouse and rat IL-2. These mammalian
DNAs can be used for the synthesis of the proteins ac-
cording to the invention. However, it is more appro-
priate to start from a synthetic ONA, particularly ad-
vantageously from the DNA for human IL-2 which has been
described in German Offenlegungsschrift 3,419,995 and in
the EP-A 0,163,249. This synthetic DNA not only has the
advantage that in its choice of codons it is suited to
the circumstances in the host which is used most fre-
quently, E. coli, but it also contains a number of
cleavage sites for restriction endonucleases at the
start and in the region of the 100th triplet, it being
possible to make use of these according to the inven-
tion. However, this does not rule out modifications to
the DNA being carried out in the region lying between
them, it being possible to make use of the other cleav-
age sites.
*in the following text "EP-A"
1341197
If use is made of the nucleases Ban II, Sac I or Sst I,
then the IL-2 part-sequence which is obtained codes for
about 95 amino acids. This length is, in general,
sufficient to obtain an insoluble fusion protein. If
S the lack of solubility is still inadequate, for example
in the case of a desired hydrophilic CSF derivative,
but it is not wanted to make use of cleavage sites
located nearer to the C-terminal end - in order to pro-
duce as little "ballast" as possible - , then the DNA
sequence can be extended at the N-terminal and/or C-
terminal end by appropriate adapters or linkers and
thus the "ballast" portion can be "tailored" to re-
,. quirements. Of course, it is also possible to use the
DNA sequence - more or less - up to the end and thus
generate biologically active IL-2 - modified where
appropriate - as "by-product".
Thus the invention relates to fusion proteins of the
general formula
Met - X - Y - Z or Met - Z - Y - X
(Ia) (Ib)
in which X essentially denotes the amino acid sequence
of approximately the first 100 amino acids of, prefer-
ably, human IL-2, Y denotes a direct bond in the case
where the amino acid or amino acid sequence adjacent to
the desired protein allows splitting off of the desired
protein, or else denotes a bridge member which is com-
posed of one or more genetically codable amino acids and
allows the splitting, and Z is a sequence of genetically
codable amino acids representing the desired CSF pro-
tein.
As is evident from formulae Ia and Ib - and as already
mentioned above too - it is possible to effect expres-
sion of the desired protein in front of or behind the
IL-2 portion. In order to simplify, hereinafter
1341197 '
_8_
essentially the second option, which corresponds to
the conventional method for the preparation of fusion
proteins, will be explained. Thus, although this
"classic" variant is described heretofore and herein-
after, this is not intended to rule out the other
alternative.
The cleavage of the fusion protein can be carried out
chemically or enzymatically in a manner known per se.
The choice of the suitable method depends, in particu-
lar, on the amino acid sequence of the desired protein.
If there is tryptophan or methionine at the carboxyl
terminal end of the bridge member Y, or if Y represents
Trp or Met, then chemical cleavage with N-bromosuccin-
imide or cyanogen halide can be carried out in the cases
where the particular C5F derivatives which are synthe-
sized do not contain these amino acids.
CSF and those of its derivatives which contain in their
amino acid sequence
Asp - Pro
and are sufficiently stable to acid can, as already
shown above, be cleaved proteolytically in a manner
known per se. This results in proteins which contain
proline at the N-terminal end or aspartic acid at the
C-terminal end being obtained. Thus, it is possible in
this way also to synthesize modified proteins.
The Asp-Pro bond can be made even more labile to acid
if this bridge member is (Asp)n-Pro or Glu-(Asp)n-Pro, n
denoting 1 to 3.
Examples for enzymatic cleavages are likewise known, it
also being possible to use modified enzymes having im-
proved specificity (cf. C.S. Craik et al., Science 228
(1985) 291-297).
134119
- 9 -
The fusion protein is obtained by expression in a
bacterial expression system in a manner known per se.
Suitable for this purpose are all known host-vector
systems, such as bacteria of the varieties Strepto-
myces, B. subtilis, Salmonella typhimurium or Serratia
marcescens, in particular E. coli.
The DNA sequence which codes for the desired protein
is incorporated in a known manner in a vector which
ensures good expression in the selected expression
system.
It is appropriate for this to select the promoter and
operator from the group comprising trp, lac, tac, P~ or
PR of phage ~, hsp, omp or a synthetic promoter as
proposed in, for example, German Offenlegungsschrift
3,430,683 or EP-A 0,173,149. The tac promoter-operator
sequence is advantageous, and this is now commercially
available (for example expression vector pKK223-3,
Pharmacia, "Molecular Biologi~cals, Chemicals and
Equipment for Molecular Biology", 1984, page 63).
It may prove to be appropriate in the expression of the
fusion protein according to the invention to modify
individual triplets for the first few amino acids after
the ATG start codon in order to prevent any base-
pairing at the level of the mRNA. Modifications of
this type, such as deletions or additions of individual
amino acids, are familiar to the expert, and the inven-
tion likewise relates to them.
Particularly advantageous CSF derivatives are those
containing N-terminal proline, since proteins of this
type are more stable to attack by proteases. The CSF
derivative which has the entire CSF amino acid se-
quence following the proline added to the N-terminal
end is particularly preferred. However, it has
emerged, surprisingly, that the variants of the CSF
- 10 - 13 4 1 1 9 ~
molecule obtained by elimination of the first 11 amino
acids also have biological activity.
Variants of the invention which are also advantageous
are those which initially result in fusion proteins
which contain the CSF sequence more than once, advan-
tageously twice or three times. By their nature, the
ballast portion in these fusion proteins is reduced,
and thus the yield of the desired protein is increased.
The plasmid pHG 23 which was obtained by incorporation
of the CSF cDNA sequence into the Pst I cleavage site
of pBR 322 has been deposited, in E. coli, at the
American Type Culture Collection under number ATCC
39900. The DNA sequence of this corresponds to the
variant described in Figure 3 (B) of Wong et al. The
incorporation made use of the Pst I cleavage site near
the 5' end, on the one hand, and of a Pst I site intro-
duced at the 3' end by GC tailing (EP-A 0,183,350>.
Example 1
Direct Expression of CSF
The commercially available vector pUC 12 is opened with
the restriction enzymes Sma I and Pst I, and the large
fragment (1) is isolated.
By cutting the cDNA sequence for CSF with the enzymes
Sfa NI and Pst I is obtained the fragment (2) which is
ligated with the synthetic linker (3) and then with the
pUC 12 fragment (1). The hybrid plasmid pW 201 (4)
which is thus obtained contains the CSF DNA sequence
following the start codon ATG.
The hybrid plasmid (4> is opened with Nco I, and the
protruding ends are filled in to give the blunt-ended
fragment (5). The vector pUC 12 is opened with the
1341197 .
- 11 -
enzyme Eco RI, whereupon the protruding ends are filled
in. This is followed by treatment with bovine alkaline
phosphatase, the pUC 12 derivative (6) being obtained.
Ligation of the fragments (5) and (6) results in vec-
tons which contain the CSF DNA sequence in both orienta-
tions being obtained. They are called pW 203 (7).
Using Eco RI and Rsa I on the vector (7> results in
isolation of the fragment (8) which contains the codons
for amino acids 63 to 127 of CSF. On the other hand,
cutting the vector (4) with Nco I and Rsa I results in
isolation of the fragment (9) which contains the codons
for amino acids 1 to 61 of CSF.
The plasmid pH 131/5 (German Offenlegungsschrift
3,514,113 or EP-A 0,198,415, Example 1, Figure 1) (10) is
cut with Pvu II, the small fragment is removed, and the
larger one is ligated to give the plasmid pPH 160 (11)
which is present in E. coli cells in a higher copy number
than pH 131/5. The plasmid (11) is opened with Nco I and
Eco RI, and the large fragment (12> is isolated.
The fragments (8), (9) and (12) are now ligated to
give the hybrid plasmid pW 206 (13). This restores the
codon for amino acid 62.
The commercially available plasmid pKK 65-10 (PL Bio-
chemical Inc.) is cleaved with Eco RI, and the fragment
(14) which contains the two terminators T1 and T2 is
isolated. This fragment (14> is inserted into the
plasmid (13) which has been opened with Eco RI, the
plasmid pW 225 (15) being obtained.
E. coli 24 bacteria which contain the plasmid (15) are
cultured in LB medium (J.H. Miller, Experiments in
Molecular Genetics, Cold Spring Harbor Laboratory,
1972) containing 30 to 50 ug/ml ampicillin at 37°C
1341197
- 12 -
overnight. The culture is diluted in the ratio 1:100
with M9 medium (J. M. Miller, op. cit.) which contains
200 Nm/l casamino acids and 1 Ng/l thiamine, and the
mixture is incubated at 37°C with continuous agitation.
At an OD600 = 0.5 or 1 indolyl-3-acrylic acid is
added to a final concentration of 15 Ng/l, and the mix-
ture is incubated for 2 to 3 hours or 16 hours respect-
ively. The bacteria are then removed by centrifug-
ation. The bacteria are boiled for five minutes in a
buffer mixture (7M urea, 0.1% SDS, 0.1 M sodium phos-
phate, pH 7.0), and samples are applied to an SDS gel
electrophoresis plate. It emerges that the protein
pattern of cells whose trp operon has been induced con-
tains a new protein, in the range of about 14,000-18,000
dalton, which is not found with non-induced cells.
The induction conditions which have been indicated
apply to shake cultures; for larger fermentations
appropriately modified OD values and, where appropri-
ate, slight variations in the inducer concentrations
are advantageous.
Example 2
ProO-CSF
The vector pUC 12 is opened with Eco RI and Pst I, and
the large fragment C16) is isolated. This fragment
(16) is ligated with the synthetic DNA fragment (17>
and the fragment (2) (Example 1; Figure 1). Competent
cells of E. coli JM 103 are transformed with the lig-
ation mixture, and the desired clones which contain the
plasmid pW 212 (18) are selected.
The fragment (19) which contains the CSF sequence is
cut out of the plasmid DNA using Pvu I and Pst I.
Insertion of the lac repressor (P. J. Farabaugh, Nature
274 (1978) 765-769) into the plasmid pKK 177-3 contain
- 13 _ 134 1 1g 7 '
the pUC 8 polylinker (Amann et al., Gene 25 (1983) 167;
EP-A 0,133,282) results in the plasmid pJF 118 (20> being
obtained (Fig. 2a; cf. German Patent Application P 35 26
995.2, Example 6, Fig. 6). The latter is opened at the
unique restriction site for Ava I, and is reduced in size
by about 1,000 by by exonuclease treatment in a manner
known per se. Ligation results in the plasmid pEW 1000
(21) being obtained, in which the lac repressor gene is
completely retained but which, because of the reduction
in size, is present in a markedly higher copy number than
the initial plasmid.
In place of the plasmid pKK 177-3, it is also possible
to start from the abovementioned commercially available
plasmid pKK 223-3, to incorporate the lac repressor, and
to shorten the resulting product analogously.
The plasmid pEW 1000 (21) is opened with the restric-
tion enzymes EcoR I and Sal I, and the fragment (22) is
isolated.
The plasmid p159/6 (23>, prepared as described in German
Offenlegungsschrift 3,419,995 (EP-A 0,163,249), Example 4
(Figure 5), is opened with the restriction enzymes Eco RI
and Sal I, and the small fragment (24), which contains
the IL-2 sequence, is isolated.
The hybrid plasmid pEW 1001 (25) is obtained by ligation
of the fragments (22) and (24).
On the one hand, the plasmid (25) is opened with Eco RI
and Pvu I, the fragment (26) which contains the largest
part of the IL-2 sequence being obtained. This part-
sequence is denoted "~IL2" in the figures.
On the other hand, the plasmid (25) is opened with Eco RI
and Pst I, and the large fragment (27) is isolated.
1341197
- 14 -
Ligation of the fragments (19>, (26) and (27), trans-
formation of competent E. coli 294 cells, and selection
results in clones which contain the plasmid pW 216 (28)
being obtained. The plasmid DNA is characterized by
restriction analysis and DNA sequence analysis.
An overnight culture of E. coli cells which contain the
plasmid (28) is diluted with LB medium (J. H. Milter,
op. cit.), which contains SO Ng/ml ampicillin, in the
ratio of about 1:100, and the growth is followed via
measurement of the 00. At OD - 0.5, the culture is ad-
justed to 1 mM in isopropyl s-galactopyranoside (IPTG)
and, after 150 to 180 minutes, the bacteria are removed
by centrifugation. The bacteria are boiled for five
minutes in a buffer mixture (7M urea, 0.1X SDS, 0.1 M
sodium phosphate, pH 7.0>, and samples are applied to
an SDS gel electrophoresis plate. Following electro-
phoresis, a protein band which corresponds to the size
of the expected fusion protein is obtained from bac-
teria which contain the plasmid (28). After disrup-
tion of the bacteria (French press; (R)Dyno mill) and
centrifugation, the fusion protein is located in the
sediment so that it is possible already to remove con-
siderable amounts of the other proteins with the super-
natant. Isolation of the fusion protein is followed by
acid cleavage to liberate the expected CSF derivative
which contains an additional N-terminal proline. This
shows activity in the biological test.
The induction conditions which have been indicated
apply to shake cultures; for larger fermentations
appropriately modified OD values and, where appropri-
ate, slight variations in the IPTG concentrations are
advantageous.
1341197 '
-15-
Example 3
Pro1-CSF(2-127)
Ligation of the fragments (2) (Figure 1) and (16) (Fig-
ure 2) with the synthetic DNA sequence (29) results
in the hybrid plasmid (30) which corresponds to the
plasmid (18) apart from the synthetic DNA sequence.
Pvu I and Pst I are used to cut out of the plasmid (30)
the fragment (31) which contains the CSF DNA sequence in
which, however, the codon for the first amino acid has
been replaced by a codon for proline. Ligation of the
fragment (31) with the fragments (26) and (27) results
in the hybrid plasmid pW 240 (32) being obtained. Ex-
pression in E. coli, which is carried out as in Example
2, provides a CSF derivative in which the first amino
acid has been replaced by proline. This derivative
also shows biological activity.
Example 4
CSF(2-127>
A plasmid which contains the CSF DNA sequence with a
Pst I restriction site at its 3' end, for example the
plasmid pHG 23 (ATCC 39900), is cleaved with Sfa NI,
and the linearized plasmid (34) is partially filled in
using Klenow polymerase and GTP. The protruding nuc-
leotide A is eliminated using S1 nuclease, and then the
fragment (35) is cut out with Pst I.
Ligation of the fragment (35) with the synthetic DNA
sequence (36) and the fragment (16) (Figure 2) results
in the plasmid (37), which is analogous to plasmid
(18), being obtained.
Pvu I and Pst I are used to cut the fragment (38) out
of the plasmid (37>. This fragment is ligated with the
1341197
- 1b -
fragments (26> and (27), by which means the plasmid
pW 241 (39) is obtained.
Expression as in Example 2 results in a fusion protein
which, after acid cleavage, provides a CSF derivative
missing the first amino acid. This derivative is bio-
logically active.
Example 5
CSF(6-127>
The plasmid (33) (or a corresponding plasmid which con-
tains the CSF DNA sequence) is first totally cleaved
with Pst I and then partially cleaved with Bst NI, and
the fragment (40) is isolated.
The synthetic DNA sequences (41> and (36) (Figure 4)
are first ligated to give the sequence (42), and the
latter is then ligated with the fragment (40) and the
fragment (16) (figure 2), the plasmid pW 212 (43> being
obtained.
Pvu I and Pst I are used to isolate from the plasmid
(43) the fragment (44) which contains the DNA sequence
for the CSF derivative. This fragment (44) is ligated
with the fragments (26) and (27), which results in the
hybrid plasmid pW 242 (45).
Expression as in Examples 2 results in a fusion protein
from which is obtained, after acid cleavage, a CSF
derivative missing the first five amino acids. This
product is also biologically active.
1341197
- 17 -
Example 6
CSF(8-127)
When first the synthetic DNA sequence (36) (Figure 4)
is ligated with the synthetic DNA sequence (46), and
thereafter the resulting DNA fragment (47) is ligated
with the fragments (40) and (16), then the hybrid plasmid
(48) is obtained. Pvu I and Pst I are used to cut out of
the latter the fragment (49) which contains the DNA
sequence for the CSF derivative. Ligation of the frag-
ments (49), (26) and (27) provides the hybrid plasmid pW
243 (SO) which corresponds to the plasmid (45) apart from
the shortened DNA sequence for the CSF derivative.
Expression as in Example 2 results in a fusion protein
which, after acid cleavage, provides a CSF derivative
missing the first seven amino acids. This derivative
is also biologically active.
Example 7
CSF(12-127)
When the synthetic DNA sequence (51) is ligated with
the fragments (33) and (16) then the hybrid plasmid
(52) is obtained. When Pvu I and Pst I are used to
cut out of the latter the sequence (53), which contains
the DNA sequence for the CSF derivative, and this frag-
ment (53) is ligated with the fragments (26) and (27>
then the hybrid plasmid pW 244 (54) which corresponds
to the plasmid (45> apart from the shortened CSF
sequence is obtained.
Expression as in Example 2 results in a fusion protein
which, after acid cleavage, provides a CSF derivative
from which amino acids 1 to 11 have been eliminated.
This shortened molecule is also biologically active.
1341197
- 18 -
Example 8
ProO-CSF(1-126)-Asp
The DNA sequence (19) (Figure 2) is partially cleaved
with Bst NI, and the fragment (55), which contains the
largest part of the CSF sequence, is isolated.
Cleavage of the plasmid (33) (Figure 4> (or of a cor-
responding plasmid which contains the CSF DNA sequence)
first with Pst I and then partially with Bst NI results
in the DNA sequence (56) which comprises the largest
part of the CSF sequence being obtained.
The DNA sequence (57) is synthesized which together with
the sequence (56) provides a DNA sequence which codes
for a CSF derivative in which the C-terminal glutamic
acid has been replaced by aspartic acid.
The vector pUC 13 is opened with Pst I and Sma I, and
the large fragment (58) is isolated. When this linea-
rized plasmid (58) is ligated with the fragments (56) and
(57), then the hybrid plasmid pW 245 (59) with the
modification of the C-terminal sequence is obtained.
Sfa NI and Pst I are used to cut out of the plasmid (59)
the fragment (60) which contains the modified CSF DNA
sequence. This fragment (60) is ligated with the syn-
thetic DNA sequence (61) and the fragment (55), the
DNA sequence (62) being obtained. The latter is lig-
ated with the DNA fragments (26) and (27) (Figure 2),
the hybrid plasmid pW 246 (b3> being obtained. This
plasmid is shown twice in Figure 8a, the lower repre-
sentation indicating the amino acid sequence of the
coded fusion protein.
Expression as in Example 2 results in a fusion protein
from which, after acid cleavage, is derived a CSF
1341 197
- 19 -
derivative which is extended by an N-terminal proline
and in which, additionally, the final amino acid has
been replaced by aspartic acid. This derivative is
biologically active.
Example 9
ProO-CSF(1-126)-Asp
The hybrid plasmid (63) (Figure 8) is cleaved with Eco
RI and Pst I, and the fragment which contains the two
modified CSF sequences following the IL-2 part-sequence
is isolated. This sequence (64) is partially cleaved
with Rsa I, and the two fragments (65) and (66> are iso-
lated. The fragment (66) is cleared with Bst NI, and
the fragment (67) is isolated. Ligation of the DNA se-
quences (27), (65), (67), (61) and (60) results in the
hybrid plasmid pW 247 (68) in which the ligated sequences
are arranged in the specified sequence.
Expression as in Example 2 provides a fusion protein
from which results, after acid cleavage, the same CSF
derivative as in Example 8.
Example 10
Synthetic gene (for ProO-CSF)
Processes known per se, for example the phosphite
method (German Offenlegungsschriften 3,327,007,
3,328,793, 3,409,9b6, 3,414,831 and 3,419,995) are used
to synthesize the three "synthesis blocks" I (CSF-I),
designated (69) in the figures, II (CSF-II), (70) in
the figures, and III (CSF-III), (71) in the figures.
The synthesized oligonucleotides Ia to Im, IIa to IIf
and IIIa to IIII are indicated in the nucleotide
25 sequence of these synthesis blocks (Appendix).
The choice of the nucleotides for the synthetic gene
1341197
_ Zo _
entailed provision not only of unique cleavage sites at
the points of union of the three synthesis blocks but
also of a number of unique restriction sites inside the
gene fragments. These are listed in the tables below.
These unique restriction sites can be used, in a manner
known per se, to exchange, add, or delete codons for
amino acids.
Synthesis Block I (CSF I)
Enzyme Recognition sequence Cut after nucleotide
no. (coding strand)
-- Nar I ('rG+CGCC 1
Hpa II C+CGG 4
Fiae II GGCGC+C 4
Nae I GCC+GGC 5
Pvu I CGAT+CG 13
Sal I G+TCGAC 24
Acc I GT+CGAC 25
Hinc II GTC+GAC 26
Hpa I/ GTT+AAC 48
Ainc II
Hha I GCG+C 66
Hint I G+AGTC 88
Nru I TCG+CGA 89
Xma III C+GGCCG 95
Sac II CCGC+CG
Eco R0 GAT+ATC 128
Synthesis Block II (CSF-II)
Enzyme Recognition sequence Cut after nucleotide
no. (coding strand)
AtlIII A+CATGT
MluI A+CGCGT
XhoI C+TCGAC ~?5
TaqI T+CGA ~~6
- - 21 - 1341 197
Synthesis Block I-I (CSF-II) (cont.)
Enzyme Recognition sequence Cut after nucleotide
no. (coding strand)
Hga I GACGC (5/10) ~~~
Ava I C+TCGAG 177
Alu I . AG+CT .180
Sac I/ GAGCT+C 182
Hgi AI
Stu I/ AGG+CCT 194
Hae I
Synthesis Block III (CSF-III)
Enzyme Cut after nucleotide
Recognition
sequence
no. (coding strand)
A~1 II C+TTAAG 217
Hae III GG+CC 224
Apa I GGGCC+C 22'7
Mnl I CCTC (7/7) 238
Nhe I G+CTAGC 241
Mae I C+TAG 242
Aha II GA+CGTC 280
Aat II GACGT+C 283
Sci NI G+CGC 287
Mst I TCG+GCA 288
Sau 3AI/ +GATC 296
Mbo I
Dpn I GA+TC 298
Asu II TT+CGAA 308
Aha III TTT+AAA 318
Ava II G+GTCC 382
Eco RII +CCAGG 384
Est NI/ CC+AGG 380
Scr FI
1341197
- 22 -
The three synthesis blocks were first individually
cloned, amplified in E. coli and re-isolated:
Synthesis block CSF-I (69) is incorporated in the pUC
12 derivative (16), the plasmid pS 200 (72) being
obtained.
pUC 12 is opened with the restriction enzymes Pst I and
Hind III and the linearized plasmid (73) is ligated
with synthesis block CSF-II (70), the plasmid pS 201
(74) being obtained.
pUC 13 is opened with Hind III and Sma I, and the lin-
earized plasmid (75> is ligated with CSF-III (71>, the
plasmid pS 202 (76> being obtained.
The re-isolated synthesis blocks (69), (70) and (71)
are now ligated in the vector pUC 12 (77) which has
been linearized with Eco RI and Sma I, the result being
the plasmid pS 203 (78). This hybrid plasmid is - as
the plasmids with the individual synthesis blocks -
amplified in E. coli 79/02, and the synthetic gene is
characterized by restriction analysis and sequence
analysis.
The plasmid (78) is cleaved with Pvu I partially and
with Bam HI, and and the small fragment (79) with the
complete CSF sequence is isolated.
The expression plasmid (21) is opened with Eco RI and
Bam HI, and the large fragment (80) is isolated. This
fragment (80) is now ligated with the fragment (26)
which contains the IL-2 part-sequence and the synthetic
gene (79). This results in the plasmid pS 204 (81)
which codes for a fusion protein in which the IL-2 part-
sequence is followed first by the bridge member which per
mits acid cleavage and then by the amino acid sequence
of CSF. Thus, acid cleavage results in a CSF derivative
which is extended by proline at the N-terminal end.
134119?
- 23 -
Example 11
CSF(1-12)His(14-121)His(123-127)
When the nucleotides in synthesis block I up to No. 48
(cleavage site for Hpa I) are replaced by the synthetic
sequences (82) and (83), then the result is a modified
synthesis block I which codes for a CSF I analog in
which there is Trp in front of the first amino acid
(Ala), and Trp in position 13 has been replaced by His.
The plasmid (72) (Figure 10) is opened with Eco RI and
Hpa I, and the large fragment (84) is isolated. The
Latter is now ligated with the synthetic fragments (82)
and (83>, the plasmid pS 205 (85> which codes for this
modified CSF I (CSF I') being obtained.
The plasmid (76) (Figure 10) is opened with Hind III
and Sal I, and the small (86) and large (87) fragments
are isolated. The small fragment (86) is then cut with
Taq I, and the fragment (88) is isolated.
The large fragment (87) is now ligated with (88> and with
the synthetic fragment (89) in which the codon for Trp
in position 122 has been replaced by His, the plasmid
pS 206 (90) which codes for the modified CSF III (CSF
III') being obtained. This plasmid is transformed into
E. coli, amplified, re-isolated, cut with Hind III and
Sal I, and the small fragment (91) which codes for CSF
III' is isolated.
The plasmid (85) is cut with Pvu I partially and with
Pst I, and the small fragment (92) which codes for CSF
I' is isolated.
When the fragments (22), (26), (92>, (70) and (91> are
now ligated then the plasmid pS 207 (93) is obtained.
This codes for a fusion protein in which the IL-2 part-
1341197
- 24 -
sequence is followed by a bridge member which contains
Trp immediately in front of the first amino acid of CSF
(Ala). Since Trp in positions 13 and 122 of the CSF
molecule have been replaced by His, it is now possible
to cleave the fusion protein with N-bromosuccinimide.
This results in the CSF derivative in which tryptophan
in both positions has been replaced by histidine.
Example 12
CSF(1-99)Thr(101-127)
When, in the synthesis of the synthesis block III,
oligonucleotides IIIe and IIIf are replaced by the
synthetic sequence (94) and the process is otherwise
carried out as in Example 10, then a CSF derivative in
which Ile in position 100 has been replaced by Thr
is obtained.
Example 13
CSF(1-35>Ile(37-45>Leu(47-78)Leu-Leu(81-127)
First the oligonucleotide (95) which contains in posi-
tion 36 the codon for Ile in place of Met, and the
oligonucleotide (96) in which the codon for Met in
position 46 has been replaced by a codon for Leu, are
synthesized.
The plasmid (72) (Figure 10) is then opened with Pvu I
and Xma III, and the fragment (97) is isolated.
In addition, the sequence (98) in which the codon for
Met is located in front of that for the first amino
acid is synthesized.
When the fragments (16), (98), (97), (95) and (96> are
now ligated then the plasmid pS 208 (99) is obtained.
This corresponds to the plasmid (72) but contains in
134119'
- 25 -
position 0 of the CSF I sequence the codon for Met, in
position 36 a codon for Ile, and in position 46 a Codon
for Leu.
In addition, the sequence (100) which in positions 79
and 80 codes for Leu in place of Met is synthesized.
When the plasmid (76) (Figure 10) is opened with Hind
III and Nhe I, and the large fragment (101) is isolated
and ligated with the synthetic sequence (100>, then the
plasmid pS 209 (102) which corresponds to the plasmid
(76) apart from replacement of the two codons in posi-
tions 79 and 80 in the CSF III sequence is obtained.
The plasmid (93) (Figure 11a) is now partially cut with
Pvu I and with Sal I, ahd the large fragment (103) is
isolated. The plasmid (99) is likewise partially
opened with Pvu I and with Pst I, and the small frag-
ment (104), which contains the modified CSF I sequence
is isolated. In addition, the plasmid (102) is opened
with Hind III and Sal I, and the small fragment (105),
which comprises the modified CSF III sequence is
isolated.
The fragments (103), (104), (70) and (105) are now
ligated, there being obtained the plasmid pS 210 (106)
which corresponds to the plasmid (93) (Figure 11a) but
codes for a CSF derivative which has Met in position 0
and in which, on the other hand, the four Met residues
have been replaced by the other amino acids.
When E. coli is transformed with the plasmid (106)
then, after inductian, a fusion protein is obtained
which can be cleaved with cyanogen halide resulting in
a CSF derivative which contains Ile in position 36 and
Leu in positions 46, 79 and 80.
134119
- 25 -
Example 14
CSF(1-35)Ile(37-45)Leu(47-78)Leu(81-127)
When the process is carried out as in Example 13, but
the synthetic sequence (107> is used in place of the
synthetic sequence (100), then a deletion product which
has Ile in position 36 and Leu in position 46, and in
which the amino acid Leu is present in place of amino
acids 79 and 80, is obtained.
Example 15
CSF(1-35)Ile(37-45)Leu(47-78)-(81-127)
When the process is carried out as in Example 13 but the
synthetic sequence (108) is used in place of the syn-
thetic sequence (100), then a deletion product which'
has Ile in position 36 and Leu in position 46, and in
which the amino acids in positions 79 and 80 have been
deleted, is obtained.
1~411g~
- 27 -
APPENDIX
Synthesis block I (CSF I) (69)
Ic
AAT TCG ATC GAC GAC CCG GCG CCG GCC CGA TCG CCG TCT CCG
GC TAG CTG CTG GGC CGC GGC CGG GCT AGC GGC AGA GGC
(Eco RI) Ile Asp Asp Pro Ala Pro Ala Arg Ser Pro Ser Pro
b (,) (5) ,~,
Z
.r- - le 50 Z
Ic ~
.
TCG ACC CAG CCC TGG GAA CAC GTT AAC GCG ATC CAG G GCG
AGC TGG GTC GGG ACC CTT GTG CAA TTG CGC TAG GTC CTT CGC
Ser Thr Gln Pro Trp Glu His Val Asn Ala Ile Gln Glu Ala
t5 (,o) c~5) (20) ~ f .
d
r
,oo
.
CGG CGT CTG CTG AAC CTG AGT CGC GAC ACG GCC GAA ATG
GCG
20 G(' GCA GAC GAC TTG GAC TCA GCG CTG TGC CGG CTT TAC
CGC
Arg Arg ?~eu Asn Zeu Ser Arg Asp Thr Ala Glu Met
Zeu Ala
(25) __~ (30) (35) .I k
X49
~t _ ~
25 . . .
~
AAC GAA ACC GTT GAA GTG ATA GAG ATG TTC GAC CTG CA
TCT
TTG CTT TGG CAA CTT CAC TAT CTC TAC AAG CTG G
AGA (Pst I)
Asn Glu Thr Val Glu Val Ile Glu Met Phe Asp(Zeu)
Ser
(40) (45) (50)
3 Im
13 4 1'~9~ .
_za_
Synthesis block II (CSF II) (70>
~ 5 0 a ,.
Jl G
(Pst I)G CCG ACA TGT CTC CAG ACG CGT CTC GAG CTC TAC
GAA
AC GTC CTT GGC TGT ACA GAG GTC TGC CTC GAG ATrr
GCA
GAG
(Gln)('rlu Pro Thr Cps Zeu Gln Thr Glu Leu Tar
Arg
Zeu
(50) (55) (60)
_ _
LL 6 ( I
~ ~
. ,~
c
200 214
yc j~'e ~.
AAA CAA GGC CTT CGT GGT CTG ACC A (Hind III)
TCT
TTT GTT CCG CCA GAC TGG TTC GA
GAA AGA
GCA
Las Gln Gly Gly Zeu Thr(Z~s)
heu Ser
Arg
(65) (?0) I( --,.
' 1341 19~
- 29 -
Synthesis block III (CSF III) (71)
215 250
a ~ -
.- -
. . . . .
AG CTT AAG GGG CCCCTC ACC ATG ATG GCT AGC CACTAC AAA
(Hind III)A TTC CCC GGGGAG TGG TAC TAC CGA TCG GTGATG ~_'TT
( Leu ) Gly ProLeu Thr Met Met Ala Ser HisTyr Lys ,
Lys
(72) (75) (80 ) (85)
,b
~
'
'' IZ
a
CAG CAC TGC CCG CCGACT CCG GAG ACG TCT TGC GCAACG CAG
GTC GTG ACG GGC GGCTGA GGC CTC TGC AGA ACG CGTTGC GTC
Gln His Cys Pro ProThr Pro Glu Thr Ser Cys AlaThr Gln
(90)~ (95)
~
300
. . ~~
.
ATC ATC ACC TTC GAATCT TTT AAA GAA AAC CTG AAGGAC TTT
TAG TAG TGG AAG CTTAGA AAA TTT CTT TTG GAC TTCCTG AAA
Ile Ile Thr Phe GluSer Phe Lys Glu Asn Leu LysAsp Phe
(100) ( 105) ( 110)
~~
350 ~ , ~ 391
~ z . ; ~ ., r--~' k
CTG CTT GTT ATA CCG TTC GAC TGT TGG GAG CCG GTC CAG GAA
GAC GAA CAA TAT GGC AAG CTG ACA ACC CTC GGC CAG GTC CTT
Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu
(115) (120) (125)
X11 ~C
Sal I Pst I
TGA TAG T~T GC GCC C
ACT ATC AGC TGA CGT CGG G
Stp Stp (Sma I)
