Note: Descriptions are shown in the official language in which they were submitted.
13'~983~
HOECHST AKTIENGESELLSCHAFT HOE 86/F271J Dr.KL/mu
Description
Fusion proteins with a eukaryotic balla~t portion
Fusion proteins having a C- or N-terminal portion essen-
tially corresponding to the first 100 amino acids of inter-
leukin-2 have already been proposed (German Patent Appli-
cation P 3,541,856.7). The interleukin-2 portion in these
may be derived from mammalian interleukin-2, for example
from mouse or rat interleukin-2, which are discLosed in
European Patent Application with the publication number
(hereinafter "EP-A") 0,091,539, but preferably from human
interleukin-2. These fusion proteins are surprisingly
stable in the host cell and can, by reason of their low
solubility, easily be separated from the soluble proteins
intrinsic to the host.
In a further development of this inventive concept, it
has now been found, surprisingly, that even considerably
smaller portions of the interleukin-2 molecule are suit-
able as "ballast" portion for fusion proteins of this
type. The invention is defined in the patent claims.
Preferred embodiments are explained in detail hereinafter.
It is particularly advantageous to start from the syn-
thetic gene for human interleukin-2 (hereinafter "IL-2")
which is described in EP-A 0,163,249 and depicted in the
addendum. This synthetic gene contains a number of unique
restriction cleavage sites which permit the DNA coding for
IL-2 to be broken do~n into "manageable" segments. Using
these segments it is possible by the modular principle to
tailor the ballast portion for fusion proteins, the solu-
bility of the fusion proteins obtained ranging from high
to low depending on the combination of the segments and
depending on the nature of the desired protein.
Thus the invention allows the solubility to be directed
to~ards that which is most advantageous for the possible
1339894
~ or desired working up of the product, that is to say high
solubility when the product is to be purified by chroma-
tography, for example using an antibody column, or low
solubility if, for pre-purification, the soluble proteins
intrinsic to the host are to be removed, for example by
centrifugation.
A particular advantage of the invention is that it is
possible to prepare fusion proteins having a very small
"ballast portion", since this results in the relative
- yield of desired protein being considerably increased.
Another advantage of the invention is that the '!baLlast
portion" can be constructed in such a way that it impairs
the spatial structure of the desired protein as little as
possible and thus, for example, does not prevent folding
up .
Cleavage of the fusion proteins results in not only the
desired protein but also the "ballast portion", that is
to say the IL-2 derivative. This may have IL-2 activity
(T-cell proliferation test) or bind to IL-2 receptors.
The "modular principle" according to the invention can
thus also be used to produce, as "by-products" IL-2 deriva-
tives which have the biological activities of IL-2 to a
greater or lesser extent.
Particularly advantageous embodiments of the invention
are explained hereinafter with reference to the synthetic
gene described in EP-A 0,163,249. This gene is cut at the
5' end with the restriction endonuclease EcoRI and at the
3' end with SalI. Apart from the three unique restriction
cleavage sites for the enzymes PstI, XbaI and SacI, which
were used to construct this gene, the locations of the
unique cleavage sites for MluI and PvuI are also favorable.
When the sequences located between these cleavage sites
are designated A to F, the synthetic gene can be repre-
sented diagrammatically as follows
- 3 - I 3 39 894
(EcoRI)-A-PstI-B-MluI-C-XbaI-D-SacI-E-PvuI-F-(SalI)
The segments A to F are thus particularly suitable "units"
for the modular system according to the invention. Thus,
in this representation the "ballast portion" for the fu-
sion proteins described in German Patent Application
P 3,541,856.7 corresponds to the segments A to E, and
that for the bifunctional protein having the entire IL-2
gene, ~hich is mentioned in the same application, corre-
sponds to all the segments A to F. In contrast, the geneconstructs according to the invention relate to other
combinations of the segments A to F, preferably having
fe~er than 4 of these segments, the segment A coding for
the N-terminal end of the fusion protein. ~he arrangement
of the other segments is arbitrary, use optionally being
made of appropriate adaptors or linkers. Appropriate
adaptor or linker sequences can also be introduced at the
C-terminal end of the "ballast portion", and in this case
they can code for amino acids or short amino acid sequences
~hich permit or facilitate the cleavage off, enzymatically
or chemically, of the "ballast portion" from the desired
protein. The adaptor or linker sequences can, of course,
also be used to tailor the "ballast portion" for a parti-
cular fusion protein, for example to achieve a desired
solubility. In this context, it has emerged, surprisingly,
that the solubility of the fusion proteins does not depend
on the molecular size but that, on the contrary, even rel-
atively small molecules may have lo~ solubility. Thus,
with knowledge of these relationships, ~hich are explained
in detail in the examples, those skilled in the art are
able ~ithout great experimental effort to obtain fusion
proteins according to the invention ~ith a small "ballast
portion" and having particular desired properties.
Thus, if the desired protein is a eukaryotic protein the
fusion proteins obtained according to the invention are
composed exclusively or virtually exclusively of eukaryo-
tic protein sequences. However, surprisingly, these
fusion proteins are not recognized as foreign proteins by
133989~
-~ 4
- the prokaryotic host cells, and are not rapidly degraded
by proteases intrinsic to the host. This degradation takes
place particularly often in the case of proteins which are
foreign to the host and coded for by cDNA sequences which
are to be expressed in bacteria. It has now emerged that
cDNA sequences can be expressed very effectively if they
are "embedded" in the segments according to the invention.
It is possible to construct specific vectors for this pur-
pose, which contain between the sequences according to the
invention a polylinker sequence which has several cloning
sites for the cDNA sequences. ~here the cDNA which has
been cloned in contains no stop codon the polypeptide
sequence coded for by the cDNA sequence is additionally
protected by the polypeptide for which the C-terminal
segment codes.
The fusion proteins can be cleaved chemically or enzymati-
cally in a manner known per se. The choice of the suit-
able method depends, in particular, on the amino acid
sequence of the desired protein. If the latter contains,
for example, no methionine it is possible for the connect-
ing element to denote Met, in-which case chemical cleavage
with cyanogen chloride or bromide is carried out. If there
is a cysteine at the carboxyl terminal end of the connect-
ing element, or if the connecting element represents Cys,then it is possible to carry out a cysteine-specific enzy-
matic cleavage or chemical cleavage, for example after
specific S-cyanylation. If there is a tryptophan at the
carboxyl terminal end of the bridging element, or if the
connecting element represents Trp, then chemical cleavage
with N-bromosuccinimide can be carried out.
Desired proteins which do not contain Asp - Pro in their
amino acid sequence and are sufficiently stable to acid
can, as fusion proteins with this bridging element, be
cleaved proteolytically in a manner known per se. This
results in proteins which contain N-terminal proline or
C-terminal aspartic acid. It is therefore also possible
in this way to synthesize modified proteins.
1339~9~
-- 5
The Asp-Pro bond can be made even more labile to acid if
- this bridging element is (Asp)n-Pro or Glu-(Asp)n-Pro,
n denoting 1-to 3.
Examples of enzymatic cleavages are likewise known, it
also being possible to use modified enzymes of improved
specificity (cf. C.S. Craik et al., Science 228 (1985)
291-297). If the desired eukaryotic peptide is proinsulin,
then the chosen sequence is advantageously a peptide
sequence in which an amino acid which can be split off
with trypsin (Arg, Lys) is bonded to the N-terminal amino
acid (Phe) of proinsulin, for example Ala-Ser-Met-Thr-Arg,
since in this case the arginine-specific cleavage can be
carried out with the protease trypsin.
If the desired protein does not contain the amino acid
sequence
Ile-Glu-Gly-Arg,
then the fusion protein with the appropriate bridging
element can be cleaved with factor Xa (EP-A 0,025,190 and
0,161,973).
The fusion protein is obtained by expression in a suit-
able expression system in a manner known per se. All
known host-vector systems are suitable for this purpose,
that is to say, for example, mammalian cells and micro-
organisms, for example yeasts and, preferably, bacteria,
in particular E. coli.
The DNA sequence which codes for the desired protein is
incorporated in a known manner into a vector ~hich ensures
satisfactory expression in the selected expression system.
In bacterial hosts, it is advantageous to select the pro-
moter and operator from the group comprising lac, tac,
trp, PL or PR of phage ~, hsp, omp or a synthetic pro-
moter as proposed in, for example, German Offenlegungs-
schrift 3,430,683 (EP-A 0,173,149). The tac promoter-
operator-sequence is advantageous and is now commercially
- 6 - 1339894
- avai.able (for example expression vector pKK223-3,
Pharmacia, "Molecular Biologicals, Chemicals and Equip-
ment for Molecular Biology", 1984, page 63).
S In the expression of the fusion protein according to the
invention it may prove advantageous to modify individual
tripLets for the first few amino acids downstream of the
ATG start codon in order to prevent any base-pairing at
the mRNA level. Modifications of this type, as well as
modifications, deletions or additions of individual amino
acids in the IL-2 protein portion, are familiar to those
skilled in the art, and the invention likewise relates to
them. Elimination of cysteine or replaçement of cysteine
by other amino acids, in order to prevent formation of
undesired disulfide bridges, as is disclosed in, for ex-
ample, EP-A 109,748, may be mentioned by way of example.
Figures 1 to 13 illustrate in the manner of a flow diagram
the processes of the syntheses described in the examples
having the same numbers. To facilitate comprehension, the
preparation of the starting materials and intermediates
has been depicted in Figures A to C. For the sake of
clarity the reference numbers in Figures 1 to 13 each
start a new decade, thus (11) in Figure 1. Reference
numbers of starting materials to which the present appli-
cation does not relate end with zero, thus, for example,
(20) in Figure 2. The figures are not drawn to scale, in
particular the scale is expanded appropriately in the
region of the polylinker sequences. IL-2 sequences are
defined by thick lines, and structural genes for desired
proteins are emphasized in other ways.
Figure A gives an overview of the segments A to F accord-
ing to the invention and of the combination of segments
A and B. The starting material is the plasmid p159/6,
whose preparation is described in detail in EP-A 0,163,249
and which is defined by Figure 5 in this publication.
Figure 3 shows the expression plasmid pE~1000, whose
- 7 _ 13398~4
preparation is described in German Patent Application
P 3,541,856.7 and is shown in Figure 1 therein. This
plasmid is opened in the polylinker sequence by appropri-
ate double digestion, this resulting in the linearized
S plasmids (Ex1) to (Ex4).
Figure C shows the preparation of the pUC12 derivative
p~226 and of the expression plasmid p~Z26-1, both of which
contain segments A and F separated by a polylinker se-
quence.
Figure 1 shows the preparation of the pUC12 derivativepKH40 and of the expression plasmid pK40, which code for
fusion proteins in which the protein sequence correspond-
ing to segment A, that is to say the first 22 amino acidsof IL-2, is followed by the bridging element Thr-Arg,
with subsequently the amino acid sequence of proinsulin.
Figure 2 shows the construction of the plasmid pSL11 and
of the expression plasmid pSL12, which code for polypep-
tides in which the segment A is followed by a bridging
element corresponding to polylinker sequences t2) and (20a),
with subsequently the amino acid sequence of proinsulin.
Figure 3 shows the construction of the expression plasmid
pK50 which codes for a polypeptide in which segments A
and B, that is to say the first 38 amino acids of IL-Z,
are dlrectly followed by the amino acid sequence of pro-
insul ln.
30Figure 4 shows the construction of the expression plasmid
pKS1 which codes for a polypeptide in which segments A
and B are followed by a bridging element corresponding to
sequences (42) and (41), to which is connected the amino
acid sequence of proinsulin.
Figure 5 shows the construction of the expression plasmid
pK52 which differs from pKS1 by the inserted MluI linker
(51) which codes for the amino acid sequence which permits
- - 8 - 1~3~9~
cleavage with factor Xa. pKS2 can also be obtained from
pKS0 (Figure 3) by cleavage with MluI and introduction of
the said MluI linker.
S Figure 6 shows the construction of the expression plasmid
pK53 from pKS1 (Figure 4), likewise by introduction of
the MluI linker.
Figure 7 shows the construction of the expression plasmid
pSL14 from pSL12 (Figure 2) by introduction of the frag-
ment C into the polylinker. This results in direct attach-
ment of the segment C to the segment A. In the following
polylinker the first two amino acids (each Glu) corre-
spond to amino acids 60 and 61 of IL-2. Thus the IL-2
portion is composed of amino acids 1 to 22 and 37 to 61.
The subsequent amino acid sequence corresponds to that
which is coded for by the plasmid pSL12 (Figure 2).
Figure 8 shows the construction of the expression plasmid
pPH31 which codes for a fusion protein in which segments
A to C are followed by a bridging element which is re-
presented by sequence (81), with subsequently the amino
acid sequence of proinsulin.
Figure 9 shows the construction of the plasmid pK192 which
codes for a fusion protein in which segements A and B are
followed by methionine and, thereafter, the amino acid
sequence of hirudin.
Figure 10 shows the construction of the plasmid pW214
which codes for a fusion protein in which segments A and
B are followed by the amino acid sequence which permits
cleavage with factor Xa, with subsequently the amino acid
sequence of granulocyte/macrophage colony stimulating
factor (CSF).
Figure 11 shows the construction of the expression plasmid
p~233 which codes for a fusion protein in which segments
A and C (corresponding to amino acids 1 to 22 and 37 to
- 9 - 1339894
61 of IL-2) are followed by the bridging element Leu-Thr-
Ile-Asp-Asp-Pro, with subsequently the amino acid sequence
of CSF.
Figure 12 shows the construction of the expression plasmid
p~234 which codes for a fusion protein having the follow-
ing amino acid sequence: Segment A (amino acids 1 to 22)
is followed by a bridging element Thr-Arg, then by segment
D (amino acids 59 to 96 of IL-2), by Thr-Asp-Asp-Pro as
a further connecting element, and finally by CSF.
Figure 13 shows the construction of the plasmids pH200
and pH201 and of the expression plasmid pH202. These
plasmids have a polylinker located between segments A and
F or A, P and F, into whose numerous cleavage sites
foreign DNA can be cloned. These plasmids are particularly
suitable for cloning cDNA sequences.
The invention is explained in detail in the examples which
follow, in which the numbering coincides with that in the
figures. Unless otherwise stated, percentage data relate
to weight.
Example A
The starting plasmid p159/6 is described in EP-A 0,163,249
(Figure S). The sequence defined there as "IL-2" or in
the text as "DNA sequence I" is in Figure A divided into
segments A to F which are bounded by cleavage sites for
the enzymes EcoRI, PstI, MluI, XbaI, SacI, PvuI and SalI.
Double digestion with the appropriate enzymes results in
the segments (A) to (F) or adjoined segments, for example
the segment (A,B) with EcoRI and MluI.
Example E
The preparation of the expression plasmid pEW1000 has been
proposed in the (not prior-published) German Patent Appli-
cation P3,541,856.7 (Figure 1). This plasmid is a
1339891
- 10 -
derivative of the plasmid ptac11 (Amann et al., Gene 25
(1983) 167 - 178) into whose recognition site for EcoRI
has been incorporated a synthetic sequence which contains
a SalI cleavage site. In this way the expression plasmid
S pKK177.3 is obtained. Insertion of the lac repressor
(Farabaugh, Nature 274 (1978) 765 - 769) results in the
plasmid pJF118. This is opened at the unique restriction
cleavage site for AvaI, and is, in a known manner, short-
ened by about 1000 bp by exonuclease treatment and is
ligated. This results in the plasmid pEW1000. Opening
this plasmid in the polylinker using the enzymes EcoRI
and HindIII, SalI, PstI or SmaI results in the linearized
expression plasmids (Ex1), (Ex2), (Ex3) and (Ex4).
Example C
The commercially available plasmid pUC12 is opened with
EcoRI and SalI, and the linearized plasmid (1) is iso-
lated. Ligation of (1) with the segment (A), the synthe-
tic linker sequence (2) and the segment (F) results inthe plasmid p~226 (3).
The strain E. coli 79/02 is transformed in a known manner
with the plasmid DNA from the ligation mixture. The cells
are plated out on agar plates which contain isopropyl-B-D-
thiogalactopyranoside (IPTG), S-bromo-4-chloro-3-indolyl-
B-D-galactopyranoside (X-gal) and 20 ~g/ml ampicillin (Ap).
The plasmid DNA is obtained from white clones, and the
formation of the plasmid (3) is confirmed by restriction
analysis and DNA sequence analysis.
The small EcoRI-HindIII fragment (4) is cut out of the
plasmid (3) and is isolated. This fragment is ligated
with the linearized expression plasmid (Ex1) in a T4 DNA
ligase reaction. The resulting plasmid pW226-1 (S) is
characterized by restriction analysis.
Competent cells of the strain E. coli Mc 1061 are trans-
formed with DNA from the plasmid pW 226-1. Clones which
133989~
- 11 -
are resistant to ampicillin are isolated on Ap-containing
agar plates. The plasmid DNA is reisolated from Mc 1061
cells and then characterized anew by restriction analysis.
Competent cells of the E. coli strain W 3110 are now
transformed with plasmid DNA isolated from E. coli Mc 1061
cells. E. coli ~ 3110 cells are always used for expression
hereinafter. All the expression experiments in the stated
examples are carried out in accordance with the following
conditions.
An overnight culture of E. coli cells which contain the
plasmid (5) is diluted in the ratio of about 1:100 with
LB medium (J.H. Miller, Experiments in Molecular Genetics,
Cold Spring Harbor Laboratory, 1972) which contains 50
~g/ml ampicillin, and growth is followed by measurement
of the OD. ~hen the OD is 0.5 the culture is adjusted to
1 mM in IPTG and, after 150 to 180 minutes, the bacteria
are spun down. The bacteria are boiled in a buffer mix-
ture (7M urea, 0.1% SDS, 0.1 M sodium phosphate, pH 7.0)
for 5 minutes, and samples are applied to a SDS gel
electrophoresis plate. After electrophoresis, bacteria
which contain the plasmid (5) produce a protein band which
corresponds to the size of the expected protein (6 kD).
The stated induction conditions apply to shake cultures;
for larger fermentations appropriate modifications of the
OD values and, where appropriate, slight variations in
the IPTG concentrations are advantageous.
The resulting protein shows no biological activity in a
cell proliferation test with an IL-2-dependent cell line
(CTLL 2).
Example 1
The plasmid (3) is opened with MluI and SalI, and the two
resulting fragments are separated by gel electrophoresis.
The larger fragment (11) is isolated.
- 12 - 1 ~ 3 9 ~ 9 4
The synthetic oligonucleotide (12) is ligated with the
blunt-ended DNA (13) coding for proinsulin (Wetekam et al.,
~ene 19 (1982) 179 - 183), this resulting in DNA sequence
(14). The latter is cut with MluI and SalI, this result-
ing in DNA sequence (15). The latter is now ligated witht~7e fragment (11), this resulting in formation of the
pLasmid pKH40 (16). The latter is characterized by re-
striction analysis.
The plasmid (16) is digested with EcoRI and HindIII, and
the small fragment (17) is isolated by gel electrophoresis.
Ligation with the linearized expression plasmid (Ex1) re-
sults in the expression plasmid pK40 (18). Expression as
indicated in Example C results in a protein which, after
cell disruption, is found in the soluble fraction of
cellular protein. The Western blot technique is used to
demonstrate that the proinsulin sequence is intact.
Example 2
The starting material is the plasmid pPH30 which is de-
picted in the (not prior-published) German Patent Appli-
cation P 3,541,856.7, in Figure 3c. Within the meaning
of the present invention, in Figure 2 the IL-2 part-
sequence is shown as "A-E" (20). The end of this se-
quence and the bridging element up to the proinsulin se-
quence is shown as (20a) in Figure 2.
The plasmid (20) is digested with PvuI and HindIII, and
the smalL fragment (22) is isolated. In addition, the
plasmid (3) is opened with EcoRI and PvuI, and the small
fragment (23) is isolated. Moreover, the vector pUC12 is
digested with EcoRI and HindIII, and the large fragment
(21) is isolated. Ligation of the fragments (21), (23)
and (22) results in the plasmid pSL11 (24).
The plasmid (24) is digested with HindIII and partially
with EcoRI, and the fragment (25) which contains the
segment A and the proinsulin gene is isolated. Ligation
1339894
- 13 -
of (25) into the linearized expression plasmid (Ex1) re-
sults in the expression plasmid pSL12 (26).
Expression as indicated in Example C and subsequent work-
ing up results in a soluble fusion protein. ~estern blot
analysis with insulin antibodies confirms that this pro-
tein contains the intact insulin sequence.
Example 3
The plasmid ptrpED5-1 (30) (Hallewell et al., Gene 9 (1980)
27-47) is used for amplification of the proinsulin gene.
The plasmid is opened with HindIII and SalI, and the
large fragment (31) is isolated. The fragment (31) is
ligated with DNA sequence (14), this resulting in the
plasmid pH106/4 (32).
The plasmid (32) is digested with SalI and MluI, and the
small fragment (15) is isolated. The linearized expres-
sion plasmid (Ex2), the segment (A,B) and the fragment(15) are now ligated, this resulting in the expression
plasmid pK50 (33).
Expression of the coded fusion protein is carried out as
indicated in Example C. The cells are then spun down
from the culture broth and ruptured in a French press.
The protein suspension is now centrifuged to separate it
into its soluble and insoluble protein constituents. The
two fractions are analyzed by gel electrophoresis in a
known manner on 17.5% SDS polyacrylamide gels and subse-
quently by staining the proteins with the dyestuff Coo-
massie blue. It is found, surprisingly, that the fusion
protein is located in the insoluble sediment. Western
blot analysis with insulin antibodies confirms that in-
tact proinsulin is present in the fusion protein.
The sediment from the French press disruption can now im-
mediately be used further for isolation of proinsulin.
- - 14 - I 3 3 9 8 9 ~
Example 4
The starting material is the plasmid pPH20 (40) which is
depicted in German Patent Application P 3,541,856.7, in
S Figure 3c. Cutting this plasmid with EcoRI, filling in
the protruding ends and cutting with HindIII results in
the fragment (41), from which the DNA sequence of the part
of (40) which is of interest here can be seen.
Ligation of the linearized expression plasmid (Ex4) with
the segment (A,B) the synthetic oligonucleotide (42) and
the fragment (41) results in the plasmid pK51 (43).
Example 5
Ligation of the linearized expression plasmid (Ex2) with
the segment (A,B), the synthetic oligonucleotide (51) and
the DNA sequence (15) results in the plasmid pK52 (52).
The correct orientation of the oligonucleotide (51) is
established by sequence analysis. The plasmid codes for
a fusion protein which contains the amino acid sequence
which corresponds to the oligonucleotide (51) and thus
can be cleaved by activated Factor Xa.
The plasmid (52) can also be obtained in the following
manner:
Partial cutting of the plasmid (33) with MluI and ligation
of the resulting opened plasmid (53) with the DNA sequence
(51) likewise results in the plasmid pK52.
Example 6
Partial cutting of the plasmid (43) with MluI and ligation
of the resulting linearized plasmid (61) with the synthe-
tic DNA sequence (51) results in the plasmid pK53 (62).The latter likewise codes for a fusion protein which can
be cleaved with activated factor Xa. The correct orient-
ation of the sequence (51) is established, as in Example 5,
by DNA sequence analysis.
1339894
- 15 -
Example 7
The plasmid (26) is cleaved with XbaI and partially with
MluI, and the large fragment (71) is isolated. Ligation
with the segment (C) results in the plasmid pSL14 (72).
After expression and cell disruption, the fusion protein
is found in the soluble fraction of cellular protein.
Example 8
1 0
The plasmid (20) is cleaved with XbaI and partially with
EcoRI, and the protruding ends are fiLled in, this result-
ing in DNA sequence (81). Ligation under blunt end con-
ditions results in the plasmid pPH31 (82). The fusion
protein is found in the insoluble fraction of cellular
protein.
Example 9
The starting material used is the plasmid (90) which is
described in EP-A 0,171,024 (Figure 3). This plasmid is
- reacted with SalI and then with AccI, and the small frag-
ment (91) is isolated. The latter is ligated with the
synthetic oligonucleotide (92), this resulting in DNA
sequence (93). The latter is cut with MluI, this result-
ing in DNA fragment (94).
The plasmid (33) is digested with MluI, partially, and
~ith SalI, and the large fragment (95) is isolated. The
latter is ligated with the DNA sequence (94), this result-
ing in the expression plasmid pK192 (96). The latter codes
for a fusion protein in which the first 38 amino acids of
IL-2 are followed by methionine and then by the amino acid
sequence of hirudin. The fusion protein is found in the
soluble fraction of cellular protein.
Example 10
The starting material used is the plasmid pHG23 (100)
- 16 - I~ 3 9 8 9 ~
which is described in EP-A 0,183,350 and which is gener-
ally accessible from the American Type Culture Collection
under No. ATCC 39000. This plasmid is cut with SfaNI,
the protruding ends are filled in, then reaction with
PstI is carried out, and the small fragment (101) is iso-
lated. Ligation of the linearized expression plasmid
(Ex3) with the segment (A,~), the synthetic oligonucleo-
tide (102) and the fragment (101) results in the express-
ion plasmid p~214 (103). This plasmid codes for a fusion
protein in which the first 38 amino acids of IL-2 are
followed by the sequence which is derived from the oligo-
nucleotide (102) and which allows the molecule to be
cleaved with factor Xa, with subsequently the am;no acid
sequence of CSF. After cell disruption, the fusion pro-
tein is found in the insoluble fraction of cellular pro-
tein.
Example 11
The starting plasmid p~216 (110) is proposed in German
Patent Application P 3,545,568.3 (Figure 2b). In this
plasmid, the IL-2 sequence corresponding to segments A
to E (PvuI cleavage site) is followed by a linker which
codes for the amino acids Asp-Asp-Pro, immediately followed
by the amino acid sequence for CSF. The connecting se-
quence between IL-2 and CSF allows the fusion protein to
be cleaved proteolytically.
The sequence (111) is isolated from the plasmid (110) by
cutting with PvuI and HindIII.
The plasmid t3) is cut with MluI and XbaI, and the large
fragment (112) is isolated. The latter is ligated with
the segment (C), this resulting in the plasmid pW227 (113).
This plasmid is reacted with EcoRI and HindIII, and the
short fragment (114) is isolated. If this fragment is
ligated with the linearized expression plasmid (Ex1) the
result is the plasmid p~227-1 (115). The plasmid codes
for a protein which is derived from IL-2 but which has no
133989~
- 17 -
IL-2 activity.
The plasmid (113) is additionally cut ~ith EcoRI and Pvu~,
and the short fragment (116) is isolated. Ligation of
the linearized expression plasmid (Ex1) with the fragments
(116) and (111) results in the expression plasmid pW233
(117). The latter codes for an insoluble fusion protein
~hich, by reason of the abovementioned linker, can be
cleaved proteolytically.
1 0
Example 12
The plasmid (3) is cut with XbaI and SacI, and the large
fragment (121) is isolated. Ligation with the segment
(D) results in the plasmid pW228 (122). The latter is
cut ~ith EcoRI and HindIII, and the small fragment (123)
is isolated. Ligation of the linearized expression plas-
mid (Ex1) ~ith the fragment (123) results in the express-
ion plasmid pW228-1 (124). This plasmid codes for a bio-
logically inactive IL-2 derivative. The plasmid is diges-
ted ~ith EcoRI and PvuI, and the short fragment (125) is
isolated. Ligation of the linearized expression plasmid
(Ex1) with the fragments (125) and (111) results in the
expression plasmid p~234 (126). The latter codes for a
sparingly soluble fusion protein which can likewise be
cleaved proteolytically.
Example 13
For the construction of plasmids which are suitable, in
particular, for the expression of cDNA sequences, initially
the polylinker sequence (131) is synthesized.
Ligation of the linearized plasmid (1) ~ith the segment
(A), the polylinker sequence (131) and segment (F) results
in the plasmid pH200 (132).
The plasmid (132) is reacted ~ith EcoRI and MluI, and the
large fragment (133) is isolated. Ligation of the latter
1339894
- 18 -
with the segment (A,B) results in the plasmid pH201 (134).
The plasmid (134) is reacted with EcoRI and HindIII, a~nd
tne short fragment (135) is isolated. Ligation of this
fragment with the linearized expression plasmid (Ex1) re-
sults in the expression plasmid pH 202 (136).
The plasmid (136) is opened with BamHI, and the cDNA which
is to be expressed is introduced into the linearized plas-
mid via a commercially available BamHI adaptor. Depend-
ing on the orientation of the cDNA, every third sequence
is attached to (A,B) in the reading frame. If the cDNA
sequence contains no stop codon the polypeptide sequence
for which it codes is additionally protected by the amino
acid sequence corresponding to the segment (F).
If the cDNA is not connected in the correct reading frame,
a shift of the reading frame is brought about by,
for example, cleaving the cDNA-containing (original or
multiplied) plasmids with MluI or XbaI (as long as the
cDNA does not contain cleavage sites for these enzymes)
and filling in the protruding ends by a Klenow polymerase
reaction.
- 19 - 1339894
Addendum
Triplet No. 0 1 ~ 2
Amino acid Met Ala Pro
Nucleotide No. 1 10
Cod. strand 5 ' AA TTC ATG GCG CCG
Non-cod. strand 3' G TAC CGC GGC
(EcoRI)
3 4 5 6 7 8 9 10 11 12
Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu
ACC TCT TCT TCT ACC AAA AAG ACT CAA CTG
TGG AGA AGA AGA TGG TTT TTC TGA GTT GAC
13 14 15 16 17 18 19 20 21 22
Gln Leu Glu His Leu Leu Leu Asp Leu Gln
CAA CTG GAA CAC CTG CTG CTG GAC CTG CAG
GTT GAC CTT GTG GAC GAC GAC CTG GAC GTC
PstI
Z3 24 25 26 27 28 29 30 31 32
Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
100
ATG ATC CTG AAC GGT ATC AAC AAC TAC AAA
TAC TAG GAC TTG CCA TAG TTG TTG ATG TTT
33 34 35 36 37 38 39 40 41 42
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe
110 120 130
AAC CCG AAA CTG ACG CGT ATG CTG ACC TTC
TTG GGC TTT GAC TGC GCA TAC GAC TGG AAG
MluI
- 20 - 1339894
43 44 45 46 47 48 49 50 51 52
Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu
140 150 160
AAA TTC TAC ATG CCG AAA AAA GCT ACC GAA
TTT AAG ATG TAC GGC TTT TTT CGA TGG CTT
53 54 55 56 57 58 59 60 61 62
Leu Lys His Leu Gln Cys Leu Glu Glu Glu
170 180 190
CTG AAA CAC CTC CAG TGT CTA GAA GAA GAG
GAC TTT GTG GAG GTC ACA GAT CTT CTT CTC
XbaI
63 64 65 66 67 68 69 70 71 72
Leu Lys Pro Leu Glu Glu Val Leu Asn Leu
200 210 220
CTG AAA CCG CTG GAG GAA GTT CTG AAC CTG
GAC TTT GGC GAC CTC CTT CAA GAC TTG GAC
73 74 75 76 77 78 79 80 81 82
Ala Gln Ser Lys Asn Phe His Leu Arg Pro
230 240 250
GCT CAG TCT AAA AAT TTC CAC CTG CGT CCG
CGA GTC AGA TTT TTA AAG GTG GAC GCA GGC
83 84 85 86 87 88 89 90 91 92
Arg Asp Leu Ile Ser Asn Ile Asn Val Ile
260 270 280
CGT GAC CTG ATC TCT AAC ATC AAC GTT ATC
GCA CTG GAC TAG AGA TTG TAG TTG CAA TAG
93 94 95 96 97 98 99 100 101 102
Val Leu Glu Leu Lys Gly Ser Glu Thr Thr
290 300 310
GTT CTG GAG CTC AAA GGT TCT GAA ACC ACG
CAA GAC CTC GAG TTT CCA AGA CTT TGG TGC
SacI
.
133989~
- 21 -
103 104 105 106 107 108 109 110 111 112
Phe Met Cys Glu Tyr Ala Asp Glu ~ Thr Ala
320 330 340
TTC ATG TGC GAA TAC GCG GAC GAA ACT GCG
AAG TAC ACG CTT ATG CGC CTG CTT TGA CGC
113 114 115 116 117 118 119 120 121 122
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile
350 360 370
ACG ATC GTT GAA TTT CTG AAC CGT TGG ATC
TGC TAG CAA CTT AAA GAC TTG GCA ACC TAG
PvuI
123 124 125 126 127 128 129 130 131 132
Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu
380 390 400
ACC TTC TGC CAG TCG ATC ATC TCT ACC CTG
TGG AAG ACG GTC AGC TAG TAG AGA TGG GAC
133 134 135
Thr
410
ACC TGA TAG 3 '
TG6 ACT ATC AGC T S'
( S a l I )
