Note: Descriptions are shown in the official language in which they were submitted.
1341203
HOECHST AKTIENGESELLSCHAFT HOE 85/F 263 Dr. KL/ml
EUKARYOTIC FUSION PROTEINS CONTAINING INTERLEUKIN-2 SEQUENCES
The invention relates to an "open reading frame" from a DNA which codes
for interleukin-2, and to the use of this DNA as an aid for the expression
of peptides and proteins.
In the preparation of eukaryotic proteins by genetic engineering, the
yield obtained in bacteria is frequently only low, especially in the case of
small proteins which have a molecular weight up to about 15,000 Daltons
and whose structures contain disulfide bridges. It is assumed that the
proteins which have been produced are rapidly degraded by proteases
intrinsic to the host. For this reason, it is expedient to construct gene
structures which code for fusion proteins, the undesired section of the
fusion protein being a protein which is intrinsic to the host and which,
after isolation of the primary product, is cleaved off by methods known
per se.
It had now been found, surprisingly, that an N-terminal section of
interleukin-2 which essentially corresponds to the first 100 amino acids is
especially well suited for the preparation of fusion proteins. Thus, the
primary product obtained is a fusion protein which is composed entirely
or very predominantly of eukaryotic protein sequences. Surprisingly, this
protein is apparently not recognized as foreign protein in the relevant
host organism, 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 straightforwardly be removed from the soluble
proteins, expediently by centrifugation.
Since it is unimportant according to the invention for the function of the
fusion protein as "ballast section" whether the interleukin-2 section
represents a biologically active molecule, nor is the exact structure of
the interleukin-2
13412p3
- 2 -
section of importance either. For this purpose it is suf-
ficient that essentially the first 100 N-terminal amino
acids are present. Thus, it is possible, for example,. to
undertake at the N-terminal end modifications which allow
cleavage of the fusion protein if the desired protein is
located N-terminal thereto. Conversely, it is possible to
undertake C-terminal modifications in order to make it
possible or easier to cleave off the desired protein if -
as customary - the latter is C-terminal bonded in the
fusion protein.
The natural DNA sequence coding for human interleukin-2,
"IL-2" in the text which follows, is known from the Euro-
pean Patent Application with the Publication No. EP-A1-
0,091,539. The literature cited there also relates to
IL-2 from mice and rats. This mammalian DNA can be used
for the synthesis of the proteins according to the inven-
tion. However, it is more expedient to start from a syn-
thetic DNA, and especially advantageously from the DNA for
human IL-2 which has been proposed in the (non-prior-
published) German Offenlegungsschrift 3,419,995 (cor-
responding to the European Patent Application published
under the No. 0,163,249). This synthetic DNA sequence is
depicted in the appendix (DNA sequence I). This synthetic
DNA not only has the advantage that its choice of codons
is suited to the circumstances in the host which is used
most often, E. coli, but it also contains a number of
cleavage sites for restriction endonucleases which can
be utilized according to the invention. Table 1 which
follows gives a selection of the suitable cleavage sites
at the start and in the region of the 100th triplet.
However, this does not rule out the possibility of
undertaking modifications in DNA in the intermediate
region, it being possible to make use of the other
cleavage sites listed in the abovementioned patent
application.
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TABLE 1
Restriction Recognition Position of the first
enzyme sequence nucleotide of the
recognition sequence
(coding strand)
5' 3'
Aha II, Ban I,
Hae II, Nar I, GGCGCC 8
Ban II, Sac I,
Sst I GAGCTC 291
Hha I GCGC 9
Hinf I GACTC 35
Pvu I CGATCG 346
Taq I TCGA 387
If use is made of the nucleases Ban II, Sac I or Sst I
then an IL-2 part-sequence which codes for about 95 amino
acids is obtained. This length is generally sufficient
to obtain an insoluble fusion protein. If the solubility
is still insufficiently low, for example in the case of
a desired hydrophilic eukaryotic protein, but it is not
intended to make use of the cleavage sites located nearer
to the C-terminal end - in order to produce as little
"ballast" as possible - then it is possible to extend the
DNA sequence at the N- and/or C-terminal end, by appropri-
ate adaptors or linkers, and thus "tailor" the "ballast"
section. Of course, it is also possible to use the DNA
sequence - more or less - right up to the end and thus
generate IL-2 which is biologically active, and optionally
modified, as a "byproduct" or generate a bifunctional pro-
tein which has the action of IL-2 in addition to the action
of the coded protein.
Thus the invention relates to fusion proteins of the
general formula
1341203
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Met - X - Y - Z or Met - Z - Y - X
(Ia) (Ib)
in which
X essentially denotes the amino acid sequence of approxi-
mately the first 100 amino acids of, preferably, human
IL-2,
Y denotes a direct bond if the amino acid or amino acid
sequence adjacent to the desired protein allows the
desired protein to be cleaved off, or otherwise denotes
a bridging element which is composed of one or more
genetically codable amino acids and permits the cleavage
off, and
Z is a sequence of genetically codable amino acids repre-
senting the desired protein.
As is evident from the formulae Ia and Ib - and as has
already been mentioned above - it is possible to bring
about the expression of the desired protein upstream or
downstream of the IL-2 section. For simplicity, in the
following text essentially the first option, which cor-
responds to the conventional method for the preparation
of fusion proteins, will be illustrated. Thus, although
this "classic" variant is described below, this is not
intended to rule out the other alternative.
The fusion protein 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 se-
quence of the desired protein. For example, if the latter
contains no methionine, Y can denote Met and then chemical
cleavage with cyanogen chloride or bromide is carried out.
If there is cysteine at the carboxyl terminal end of the
Linking element Y, or if Y represents Cys, then an enzy-
matic cysteine-specific cleavage or a chemical cleavage,
for example after specific S-cyanylation, can be carried
out. If there is tryptophan at the carboxyl terminal end
of the bridging element Y, or if Y represents Trp, then
chemical cleavage with N-bromosuccinimide can be carried out.
1341203
- 5 -
Proteins which do not contain
Asp - Pro
in their amino acid sequence and are sufficiently stable to
acid can be cleaved proteolytically in a manner known per se.
This results in proteins which contain N-terminal proline
and C-terminal aspartic acid respectively. It is thus also
possible in this way to synthesize modified proteins.
The Asp-Pro bond can be made 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 make use of modified enzymes with
improved specificity (cf. C.S. Craik et al., Science 228
(1985) 291-297). If the desired eukaryotic peptide is
proinsulin, then it is expedient to choose as sequence Y
a peptide sequence in which an amino acid which can be
cleaved off with trypsin (Arg, Lys) is bonded to the N-
terminal amino acid (Phe) of proinsulin, for example
Ala-Ser-Met-Thr-Arg, since it is then possible to carry
out the arginine-specific cleavage with the protease trypsin.
If the desired protein does not contain the amino acid
sequence
Ile-Glu-Gly-Arg,
then the fusion protein can be cleaved with factor Xa
(European Patent Applications with the Publication Nos.
0,025,190 and 0,161,973).
The fusion protein is obtained by expression in a suitable
expression system in a manner known per se. Suitable for
this purpose are all known host vector systems, that is
to say, for example, mammalian cells and microorganisms,
for example yeasts and, preferably, bacteria, in particular
1341243
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E. coli.
The DNA sequence which codes for the desired protein is
incorporated in a known manner in a vector which ensures
satisfactory expression in the selected expression system.
In bacterial hosts it is expedient to choose the promotor
and operator from the group lac, tac, trp, P~ or PR of
phage ~, hsp, omp or a synthetic promotor as proposed in,
for example, German Offenlegungsschrift 3,430,683 (Euro-
pean Patent Application with the Publication No. 0,173,149).
The tac promotor-operator sequence is advantageous and is
now commercially available (for example expression vector
pKK223-3, Pharmacia, "Molecular Biologicals, Chemicals
and Equipment for Molecular Biology", 1984, page 63).
It may prove expedient in the expression of the fusion
protein according to the invention to modify some of the
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 modi-
fications, deletions or additions of individual amino acids
in the IL-2 protein section, are familiar to the expert
and the invention likewise relates to them.
The invention is illustrated in detail in the examples which
follow and in the figures. In these,
Figure 1, and its continuation Figure 1a, relate to the
synthesis of the plasmid pK360 which codes for a fusion
protein which has the hirudin sequence;
Figure 2, and its continuation Figure 2a, relate to the
synthesis of the plasmid pK410 which likewise codes for a
fusion protein having the amino acid sequence of hirudin,
Figure 3, and its continuations Figures 3a to 3c, relate
to the construction of the plasmids pPH15, 16, 20 and 30
which code for fusion proteins which contain the amino acid
1341203
_ 7 _
sequence of monkey proinsulin,
Figure 4 relates to the synthesis of the plasmid pPH100
which codes for a fusion protein having the amino acid
sequence of hirudin,
Figure 5, and its continuation Figure 5a, relate to the
construction of the plasmid pK370 which codes for a fusion
protein having the amino acid sequence of hirudin, and
Figure 6, and its continuation Figure ba, relate to the
synthesis of the plasmid pKH101 which codes for a fusion
protein having the amino acid sequence of monkey proinsulin.
In general, the figures are not drawn to scale; in par-
ticular, the scale has been "stretched" in depicting the
polylinkers.
cYeMOi c
The plasmid pJF118 (1) is obtained by insertion of the
lac repressor (P. J. Farabaugh, Nature 274 (1978) 765-769)
into the plasmid pKK 177-3 (Amann et al., Gene 25 (1983)
167) (Fig. 1; cf. German Patent Application P 35 26
995.2, Example 6, Fig. 6). pJF118 is opened at the unique
restriction site for Ava I and is shortened by about
1,000 by in a manner known per se by exonuclease treatment.
Ligation results in the plasmid pEW 1000 (2) (Figure 1)
in which the lac repressor gene is fully retained but
which, by reason of the shortening, is present in a dis-
tinctly higher copy number than the starting plasmid.
In place of the plasmid pKK177-3, it is also possible to
start from the abovementioned commercially available plas-
mid pKK223-3, to incorporate the lac repressor, and to
shorten the resulting product analogously.
The plasmid pEW 1000 (2) is opened with the restriction
enzymes EcoR I and Sal I (3).
1341203
_8-
The plasmid (4) which codes for hirudin and has been pre-
pared as in German Offenlegungsschrift 3,429,430 (Euro-
pean Patent Application with the Publication No. 0,171,024>,
Example 4 (Figure 3>, is opened with the restriction en-
zymes Acc I and Sal I, and the small fragment (5) which
mostly contains the hirudin sequence is isolated.
The plasmid p159/6 (6), prepared as in German Offenlegungs-
schrift 3,419,995 (European Patent Application with the
Publication No. 0,1b3,249), Example 4 (Figure 5), is opened
with the restriction enzymes Eco RI and Pvu I, and the
small fragment (7) which contains most of the IL-2 sequence
is isolated. This part-sequence and other shortened IL-2
sequences in the text which follows are identified by
"~IL2" in the figures.
Thereafter the sequences (3), C5), (7> and the synthetic
DNA sequence (8; Figure 1a) are treated with T4 ligase.
The plasmid pK3b0 (9) is obtained.
Competent E. coli cells are transformed with the ligation
product and plated out on NA plates which contain 25 ug/ml
ampicillin. The plasmid DNA of the clones is character-
ized by restriction and sequence analysis.
An overnight culture of E. coli cells which contain the
plasmid (9) is diluted in the ratio of approximately
1:100 with LB medium (J. H. Miller, Experiments in Molecu-
lar Genetics, Cold Spring Harbor Laboratory, 1972) which
contains 50 ug/ml ampicillin, and the growth is monitored
by measurement of the OD. When the OD is 0.5, the shake
culture is adjusted to 1 mM isopropyl S-galactopyrano-
side (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 electro-
phoresis plate. Bacteria which contain the plasmid (9)
provide after electrophoresis a protein band which corres-
ponds to the size of the expected fusion protein.
1341203
- 9 -
Disruption of the bacteria (French Press; (R)Dyno mill)
and centrifugation results in the fusion protein being lo-
cated in the sediment so that considerable amounts of the
other proteins can now be removed with the supernatant.
After isolation of the fusion protein, cleavage with
cyanogen bromide results in Liberation of the expected
hirudin peptide. The latter is characterized after
isolation by protein sequence analysis.
The indicated induction conditions apply to shake cultures;
with larger fermentations appropriately modified OD values
and, where appropriate, slight changes in the IPTG con-
centrations are expedient.
EXAMPLE 2
The plasmid (4) (Figure 1) is opened with Acc I, and the
protruding ends are filled in with Klenow polymerase.
Then cleavage with Sac I is carried out, and the fragment
(10) which contains most of the hirudin sequence is iso-
lated.
The commercially available vector pUC 13 is opened with
the restriction enzymes Sac I and Sma I, and the large
fragment (11) is isolated.
Using T4 ligase, the fragments (10) and (11) are now li-
gated to give the plasmid pK 400 (12) (Fig. 2). The plas-
mid (12> is shown twice in Figure 2, the lower representa-
tion emphasizing the amino acid sequence of the hirudin
derivative which can thus be obtained.
The plasmid (4) (Figure 1) is opened with the restriction
enzymes Kpn I and Sal I, and the small fragment (13) which
contains the hirudin part-sequence is isolated.
The plasmid (12) is reacted with the restriction enzymes
Hinc II and Kpn I, and the small fragment (14) which con-
tains the hirudin part-sequence is isolated.
1341203
- 10 -
The plasmid (9) (Figure 1a) is partially cleaved with
EcoR I, the free ends are subjected to a fill-in reaction
with Klenow polymerase, and Sal I cleavage is carried out.
The derivative (15) of the plasmid pK360 is obtained.
S Ligation of the fragments (3), (13), (14) and (15) results
in the plasmid pK410 (16) which is shown twice in Figure
2a, the lower representation showing the amino acid se-
quence of the fusion protein and thus that of the hirudin
derivative obtained after acid cleavage.
Expression and working up as in Example 1 results in a new
hirudin derivative which has the amino acids proline and
histidine in positions 1 and 2. This hirudin derivative
has the same activity as the natural product, according
to German Offenlegungsschrift 3,429,430, which has the
amino acids threonine and tyrosine in these positions,
but is more stable to attack by aminopeptidases, which
may result in advantages for in vivo use.
CYSMPI F
The commercially available vector pBR 322 is opened with
Bam H I, this resulting in the linearized plasmid (17).
The free ends are partially filled in by use of dATP, dGTP
and dTTP, and the protruding nucleotide G is split off
with S1 nuclease, this resulting in the pBR 322 derivative
(18>.
The Hae III fragment (19) from monkey proinsulin (Wetekam
et al., Gene 19 (1982) 181) is ligated with the modified
plasmid (18), this resulting in the plasmid pPH 1 (20).
Since the insulin part-sequence has been inserted into the
tetracycline resistance gene, the clones which contain
this plasmid are not resistant to tetracycline and thus
can be identified.
The plasmid (20) is opened with Bam HI and Dde I, and the
small fragment (21) is isolated.
1341203
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In addition, the Dde I-Pvu II part-sequence (22) from the
monkey proinsulin sequence is isolated.
The vector pBR 322 is opened with Bam HI and Pvu II, and
the linearized plasmid (23) is isolated.
Ligation of the insulin part-sequences (21) and (22) with
the opened plasmid (23> results in the plasmid pPHS (24>.
The latter is opened with Bam HI and Pvu II, and the small
fragment (25) is isolated.
The DNA sequence (26) to make up the insulin structure
is synthesized.
The commercially available vector pUC 8 is opened with the
enzymes Bam HI and Sal I, and the remainder of the plasmid
(27) is isolated. The latter is ligated with the DNA
sequences (25) and (26) to give the plasmid pPH 15 (28).
The latter is opened with Sal I and the protruding ends
are filled in. Bam HI is used to cleave the DNA sequence
(30) off the resulting plasmid derivative (29).
The commercially available vector pUC 9 is opened with the
enzymes Bam HI and Sma I, and the large fragment (31) is
isolated. The latter is ligated with the DNA sequence
(30), this resulting in the plasmid pPH16 (32).
The plasmid (32> is opened with Sal I, and the linearized
plasmid (33> is partially filled in with dCTP, dGTP and
dTTP, and the remaining nucleotide T is cleaved off with
S1 nuclease. The resulting plasmid derivative (34) is
treated with Sam HI, and the protruding single strand is
removed from the product (35) with S1 nuclease, this re-
sulting in the plasmid derivative (36).
The blunt ends of the plasmid derivative (35) are cyclized
to give the plasmid pPH 20 (37).
Competent E. coli Hb 101 cells are transformed with the
~3~~zo3
- 12 -
ligation mixture and plated out on selective medium. Clones
which contain the desired plasmid express proinsulin, and
28 of 70 clones tested radioimmunologically contained de-
tectable proinsulin. The plasmids are also characterized
by DNA sequence analysis. They contain DNA which codes
for arginine upstream of the codon for the first amino
acid of the B chain (Phe).
The plasmid (37) is cleaved with Hind III, the protruding
ends are filled in, and then cleavage with Dde I is car-
vied out. The small fragment (38) is isolated.
The plasmid (28) (Figure 3a) is cleaved with Sal I and
Dde I, and the small fragment (39) is separated off.
The plasmid (9) (Figure 1a) is initially cleaved with Acc I,
the free ends are filled in, and then partial cleavage
with Eco RI is carried out. The fragment (40) which con-
tains the shortened IL-2 sequence is isolated.
The linearized plasmid (3) (Figure 1) and the DNA segments
(38), (39) and (40) are now ligated to give the plasmid
pPH 30 (41). This plasmid codes for a fusion protein which
has, downstream of amino acids 1 to 114 of IL-2, the fol-
lowing amino acid sequence:
Asp-Phe-Met-Ile-Thr-Thr-Tyr-Ser-Leu-Ala-Ala-Gly-Arg.
The arginine which is the last amino acid in this bridging
element Y makes it possible to cleave off the insulin
chains with trypsin.
It is also possible starting from plasmid (9) (Figure 1a)
to obtain plasmid (41) by the following route:
(9) is opened with Acc I, the protruding ends are filled
in, then cleavage with Sal I is carried out, and the re-
suiting plasmid derivative (42) is ligated with the seg-
ments (3), (38) and (39).
13 41203
- 13 -
cYeMai G 4
The plasmid (6) (Figure 1> is opened with the restriction
enzymes Taq I and Eco RI, and the small fragment (43) is
isolated. This fragment is ligated with the synthesized
DNA sequence (44) and the segments (3) and (5) to give
the plasmid pPH 100 (45). This plasmid codes for a fusion
protein in which the first 132 amino acids of IL-2 are
followed by the bridging element Asp-Pro and then by the
amino acid sequence of hirudin. Thus proteolytic cleavage
provides a modified, biologically active IL-2' which con-
tains Asp in place of Thr in position 133, and a hirudin
derivative which contains an N-terminal Pro upstream of the
amino acid sequence of the natural product. This product
is also biologically active and, compared with the natural
product, is more stable to attack by proteases.
The IL-2' hirudin fusion protein also has biological activ-
ity:
Biological activity was found in a cell proliferation test
using an IL-2-dependent cell line (CTLL2).
Furthermore, after denaturation in 6 M guanidinium hydro-
chloride solution followed by renaturation in buffer solu-
tion (10 mM tris-HCI, pH 8.5, 1 mM EDTA), high IL-2 acti-
vity was found. In addition, the coagulation time of acid-
treated blood to which thrombin had been added was in-
creased after addition of the fusion protein.
Thus a bifunctional fusion protein is obtained.
cYeMm c S
The commercially available vector pUC 12 is opened with
the restriction enzymes Eco RI and Sac I. Into this linear-
ized plasmid <46) is inserted an IL-2 part-sequence which
has been cleaved out of the plasmid (6) (Figure 1) with
the restriction enzymes Eco RI and Sac I. This sequence
(47) comprises the complete triplets for the first 94
1341203
- 14 -
amino acids of IL-2. Ligation of (46) and (47) results
in the plasmid pK 300 (48>.
The plasmid (9) (Figure 1a) is opened with Eco RI, the
protruding ends are filled in, and then cleavage with
Hind III is carried out. The small fragment (49) which
contains part of the polylinker from pUC 12 downstream of
the DNA sequence coding for hirudin is isolated.
The plasmid (48) is opened with the restriction enzymes
Sma I and Hind III, and the large fragment (50) is iso-
fated. Ligation of (50> with (49) results in the plasmid
pK 301 (51>.
The ligation mixture is used to transform competent E.
coli 294 cells. Clones which contain the plasmid (51) are
characterized by restriction analysis. They contain DNA
in which the codons for the first 96 amino acids of IL-2
are followed by codons for a bridging element of 6 amino
acids and, thereafter, the codons for hirudin.
The plasmid (51) is reacted with Eco RI and Hind III, and
the fragment (52) which contains the DNA sequence for the
said eukaryotic fusion protein is isolated.
The plasmid (2) (Figure 1) is opened with Eco RI and Hind
III. The resulting linearized plasmid (53) is ligated
with the DNA sequence (52), this resulting in the plasmid
pK 370 (54).
When expression of the plasmid (54) is effected in E. coli
as in Example 1, the fusion protein obtained has the first
96 amino acids of IL-2 followed by the bridging element
Ala-Gln-Phe-Met-Ile-Thr
and, thereafter, the amino acid sequence of hirudin.
X341203
- 15 -
EXAMPLE 6
Using the restriction enzymes Eco RI and Hind III, the DNA
segment which codes for monkey proinsulin is cleaved out
of the plasmid (41) (Example 3; Figure 3c), and the pro-
s truding ends are filled in. The DNA segment (55) is ob-
tained.
The plasmid (48) (Example 5, Figure 5) is opened with
Sma I and treated with bovine alkaline phosphatase. The
resulting linearized plasmid (56) is ligated with the DNA
segment (SS), this resulting in the plasmid pK 302 (57).
E. coli 294 cells are transformed with the ligation mix-
ture, and clones containing the desired plasmid are charac-
terized first by restriction analysis and then by sequence
analysis of the plasmid DNA.
Using Eco RI and Hind III, the segment (58) which codes
for IL-2 and monkey proinsulin is cleaved out of the
plasmid (57).
The plasmid (2) (Example 1, Figure 1) is likewise cleaved
with Eco RI and Hind III, and the segment (58) is ligated
into the linearized plasmid (3). The plasmid pKH 101 (59)
is obtained.
Expression as in Example 1 results in a fusion protein
in which the first 96 amino acids of IL-2 are followed by
a bridging element of 14 amino acids (corresponding to
Y in DNA segment (5$)), which is followed by the amino
acid sequence of monkey proinsulin.
1341203
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APPENDIX I: DNA sequence of interleukin-2
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
3 4 5 6 ? 8 9 10 11 12
Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu
20 30 40
ACC TCT TCT TCT ACC AAA AAG ACT CAA CTG
TGG AGA AGA AGA TGG TTT TTC TGA GTT GAC
13 14 15 16 1? 18 19 20 21 22
Gln Leu Glu His Leu Leu Leu Asp Leu Gln
5p 60 ?0
CAA CTG GAA CAC CTG CTG CTG GAC CTG CAG
GTT GAC CTT GTG GAC GAC GAC CTG GAC GTC
23 24 25 26 2? 28 29 30 31 32
Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys
80 90 100
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
1341243
-,7-
43 44 45 46 47 48 49 50 5, 52
Irys Phe Tyr Met Pro Zys Zys 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 5? 58 5g 60 61 62
Leu Zys His I,eu Gln Cys Zeu Glu Glu Glu
1?0 180 190
CTG AAA CAC CTC CAG TGT CTA GAA GAA GAG
GAC TTT GTG GAG GTC ACA GAT CTT CTT CTC
63 64 65 66 67 68 69 70 ?1 72
Zeu Zys Pro I~eu Glu Glu dal Zeu Asn Zeu
200 210 220
CTG AAA CCG CTG GAG GAA GTT CTG AAC CTG
GAC TTT GGC GAC CTC CTT CAA GAC TTG GAC
?3 ?4 ?5 ?6 7? ?e ?9 80 81 82
Ala Gln Ser I,ys Asn Phe His Zeu Arg Pro
,
23p 240 250
GCT CAG TCT AAA AAT TTC CAC CTG CGT CCG
CGA GTC AGA TTT TTA AAG GTG GAC GCA GGC
g3 84 85 86 87 88 6g 90 91 92
Arg Asp I,eu Ile Ser Asn Ile Asn Yal 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 Ireu I,ys 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
~3412A3
- 18 -
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 11? 118 119 120 121 122
Thr Ile Val Glu Phe Zeu 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
123 124 125 126 127 128 129 130 131 132
Thr Phe Cys Gln Ser Ile Ile Ser Thr Zeu
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'
TGG ACT ATC AGC T 5'
