U.S. patent application number 09/750913 was filed with the patent office on 2001-10-18 for recombinant preparation of calcitonin fragments and use thereof in the preparation of calcitonin and related analogs.
This patent application is currently assigned to Bio Nebraska, Inc.. Invention is credited to Frank, Julie A., Henriksen, Dennis B., Holmquist, Bart, Partridge, Bruce E., Stout, Jay S., Wagner, Fred W..
Application Number | 20010031856 09/750913 |
Document ID | / |
Family ID | 24385018 |
Filed Date | 2001-10-18 |
United States Patent
Application |
20010031856 |
Kind Code |
A1 |
Wagner, Fred W. ; et
al. |
October 18, 2001 |
Recombinant preparation of calcitonin fragments and use thereof in
the preparation of calcitonin and related analogs
Abstract
A process for the recombinant preparation of a calcitonin
fragment and the use of the fragment in the preparation of
calcitonin and related analogs is provided. The process includes
recombinantly forming a fusion protein which includes the
calcitonin fragment linked to a carbonic anhydrase. The
recombinantly formed fusion protein is subsequently cleaved to
produce a polypeptide which includes the calcitonin fragment. A
method for producing a calcitonin carba analog which includes
condensing a desaminononapeptide with the recombinantly formed
calcitonin fragment is also provided.
Inventors: |
Wagner, Fred W.; (Walton,
NE) ; Stout, Jay S.; (Lincoln, NE) ;
Henriksen, Dennis B.; (Lincoln, NE) ; Partridge,
Bruce E.; (Lincoln, NE) ; Holmquist, Bart;
(Lincoln, NE) ; Frank, Julie A.; (Lincoln,
NE) |
Correspondence
Address: |
Beth A. Burrous
FOLEY & LARDNER
Washington Harbor
3000 K Street, N.W, Suite 500
Washington
DC
20007-5109
US
|
Assignee: |
Bio Nebraska, Inc.
|
Family ID: |
24385018 |
Appl. No.: |
09/750913 |
Filed: |
January 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09750913 |
Jan 2, 2001 |
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09139819 |
Aug 25, 1998 |
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09139819 |
Aug 25, 1998 |
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08595868 |
Feb 6, 1996 |
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5962270 |
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Current U.S.
Class: |
530/307 ;
536/23.5 |
Current CPC
Class: |
C07K 2319/00 20130101;
C12N 9/88 20130101; C07K 14/585 20130101 |
Class at
Publication: |
530/307 ;
536/23.5 |
International
Class: |
C07H 021/04 |
Claims
What is claimed is:
1. A recombinant method of synthesizing a calcitonin fragment
comprising: (a) recombinantly forming a fusion protein which
includes a target sequence linked to a carbonic anhydrase through a
cleavage site; and (b) cleaving the fusion protein with a cleavage
reagent to produce a first polypeptide including the target
sequence, wherein the target sequence includes an amino acid
sequence of the formula: A.sub.10-A.sub.11-A.sub.1-
2-A.sub.13-A.sub.14-A.sub.15-A.sub.16-A.sub.17-A.sub.18-A.sub.19-A.sub.20--
A.sub.21-A.sub.22-A.sub.23-A.sub.24-A.sub.25-A.sub.26-A.sub.27-Gly-A.sub.2-
9-A.sub.30-A.sub.31-Pro-xx (SEQ ID NO:1) wherein A.sub.10 is Gly or
Ser, A.sub.11 is Lys, Thr or Ala, A.sub.12 is Leu or Tyr, A.sub.13
is Ser, Thr or Trp, A.sub.14 is Gln, Lys or Arg, A.sub.15 is Glu,
Asp or Asn, A.sub.16 is Leu or Phe, A.sub.17 is His or Asn,
A.sub.18 is Lys or Asn, A.sub.19 is Leu, Tyr or Phe, A.sub.20 is
Gln or His, A.sub.21 is Thr or Arc, A.sub.22 is Tyr or Phe,
A.sub.23 is Pro or Ser, A.sub.24 is Arg, Gly or Gln, A.sub.25 is
Thr or Met, A.sub.26 is Asp, Ala, Gly, or Asn, A.sub.27 is Val,
Leu, Ile, Phe, or Thr, A.sub.29 is Ala, Val, Pro or Ser, A.sub.30
is Gly, Val or Glu, A.sub.31 is Thr, Val or Ala, and -Xxx is --OH,
--NH.sub.2, an amino acid residue or a polypeptide group.
2. The method of claim 1 wherein -Xxx is amino acid residue having
an .alpha.--C(O)NH.sub.2 group.
3. The method of claim 1 wherein A.sub.10 is Gly; the cleavage site
includes Asn-A.sub.10; and the cleaving step includes contacting
the fusion protein with hydroxyl amine.
4. The method of claim 1 wherein -Xxx is --OH; and further
comprising reacting the first polypeptide with an amidating agent
to form a second peptide wherein -Xxx is --NH.sub.2.
5. The method of claim 1 wherein -Xxx includes an amino acid
residue; further comprising converting the Pro-XXX sequence to a
C-terminal Pro-NH.sub.2 residue.
6. The method of claim 5 wherein the converting step comprises
transamidating the -Xxx with an o-nitrobenzylamine to form a third
peptide; and photolyzing the third peptide to form a second peptide
including an amino acid sequence of the formula:
A.sub.10-A.sub.11-A.sub.-
12-A.sub.13-A.sub.14-A.sub.15-A.sub.16-A.sub.17-A.sub.18-A.sub.19-A.sub.20-
-A.sub.21-A.sub.22-A.sub.23-A.sub.24-A.sub.25-A.sub.26-A.sub.27-Gly-A.sub.-
29-A.sub.30-A.sub.31-Pro-NH.sub.2. (SEQ ID NO:2)
7. The method of claim 1 wherein the
A.sub.10-A.sub.11-A.sub.12-A.sub.13-A-
.sub.14-A.sub.15-A.sub.16-A.sub.17-A.sub.18-A.sub.19-A.sub.20-A.sub.21-A.s-
ub.22-A.sub.23-A.sub.24-A.sub.25-A.sub.26-A.sub.27-Gly-A.sub.29-A.sub.30-A-
.sub.31-Pro-Xxx (SEQ ID NO:1) sequence is:
7 Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr- (SEQ ID NO:6)
Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro-Xxx.
8. The method of claim 1 further comprising reacting the fusion
protein with a protecting reagent to form a protected fusion
protein which includes an amino acid residue having a protected
amino, hydroxyl or carboxy reactive side chain group.
9. The method of claim 8 comprising reacting the fusion protein
with a protecting reagent to form a protected fusion protein which
includes a Lys residue having a protected side chain amino
group.
10. The method of claim 1 wherein the cleaving step includes
contacting the fusion protein with the cleavage agent to form a
minifusion protein including an amino acid sequence having the
formula: Linker
Peptide-A.sub.10-A.sub.11-A.sub.12-A.sub.13-A.sub.14-A.sub.15-A.sub.16-A.-
sub.17-A.sub.18-A.sub.19-A.sub.20-A.sub.21-A.sub.22-A.sub.23-A.sub.24-A.su-
b.25-A.sub.26-A.sub.27-Gly-A.sub.29-A.sub.30-A.sub.31-Pro-Xxx.
11. The method of claim 10 wherein the minifusion protein has the
formula:
8 Val-Asp-Asn-Trp-Arg-Pro-Ala-Gln-Pro-Leu-Lys-Asn- (SEQ ID NO:24)
Arg-Glu-Ile-Lys-Ala-Phe-Val-Asp-Asp-Asp-Asp-Asn-
Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-
Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro-Ala- Pro-Ala.
12. The method of claim 11 wherein the carbonic anhydrase is human
carbonic anhydrase II and the cleavage agent includes cyanogen
bromide.
13. The method of claim 11 further comprising cleaving the
minifusion protein with hydroxylamine to form a peptide including
an amino acid sequence of the formula:
9 Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr- (SEQ TD NO:6)
Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro-Xxx.
14. The method of claim 10 further comprising reacting the
minifusion protein with a protecting reagent to form a protected
minifusion protein including an amino acid residue having a
protected amino, hydroxyl or carboxy reactive side chain group.
15. The method of claim 1 wherein the carbonic anhydrase is human
carbonic anhydrase II; the target sequence includes an amino acid
sequence of the formula:
Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr--
Asp-Val-Gly-Ala-Gly-Thr-Pro-Ala.sub.c (SEQ ID NO:23), wherein
-Ala.sub.c is a C-terminal residue; and further comprising
transpeptidating the -Ala.sub.c with an o-nitrophenylglycinamide to
form a third peptide; and photolyzing the third peptide to form a
second peptide including an amino acid sequence of the formula:
10 Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr- (SEQ ID NO:6).
Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro-NH.sub.2
16. The method of claim 15 wherein the cleavage site includes
Asn-A.sub.10; and the cleaving step includes contacting the fusion
protein with hydroxylamine.
17. A nucleic acid sequence comprising a sequence of the formula:
GGT AAA CTG TCT CAG GAG CTC CAT AAA CTG CAG ACT TAC CCG CGT ACT GAC
GTT GGT ACC CCG (SEQ ID NO:5)
18. A method for producing a calcitonin carba analog comprising
condensing an N-terminal fragment of the formula: 11wherein A.sub.2
is Gly, Ser or Ala; A.sub.3 is Asn or Ser; A.sub.8 is Val or Met;
R.sup.2 is --(CH.sub.2).sub.4-- or --CH(NH.sub.2)CH.sub.2S--S--;
and Y is OH, OR.sup.1, where --R.sup.1 is a lower alkyl group; with
a recombinantly-formed polypeptide of the formula:
A.sub.10-A.sub.11-A.sub.-
12-A.sub.13-A.sub.14-A.sub.15-A.sub.16-A.sub.17-A.sub.18-A.sub.19-A.sub.20-
-A.sub.21-A.sub.22-A.sub.23-A.sub.24-A.sub.25-A.sub.26-A.sub.27-Gly-A.sub.-
29-A.sub.30-A.sub.31-Pro-Xxx (SEQ ID NO:1) wherein A.sub.10 is Gly
or Ser, A.sub.11 is Lys, Thr or Ala, A.sub.12 is Leu or Tyr,
A.sub.13 is Ser, Thr or Trp, A.sub.14 is Gln, Lys or Arg, A.sub.15
is Glu, Asp or Asn, A.sub.16 is Leu or Phe, A.sub.17 is His or Asn,
A.sub.18 is Lys or Asn, A.sub.19 is Leu, Tyr or Phe, A.sub.20 is
Gln or His, A.sub.21 is Thr or Arg, A.sub.22 is Tyr or Phe,
A.sub.23 is Pro or Ser, A.sub.24 is Arg, Gly or Gln, A.sub.25 is
Thr or Met, A.sub.26 is Asp, Ala, Gly, or Asn, A.sub.27 is Val,
Leu, Ile, Phe, or Thr, A.sub.29 is Ala, Val, Pro or Ser, A.sub.30
is Gly, Val or Glu, A.sub.31 is Thr, Val or Ala, and -Xxx is --OH,
--NH.sub.2, an amino acid residue or a polypeptide group; in the
presence of a non-enzymatic coupling reagent to form a
calcitonin-derivative having the formula: 12
19. The method of claim 18 wherein the non-enzymatic coupling
reagent includes N-hydroxysuccinimide, N-hydroxybenzotriazole or a
carbodiimide.
20. The method of claim 18 wherein the non-enzymatic coupling
reagent includes (i) N-hydroxysuccinimide and
dicyclohexylcarbodiimide; (ii) N-hydroxysuccinimide and
N-ethyl-N'-dimethylaminopropylcarbodiimide; or (iii)
N-hydroxybenzotriazole and dicyclohexylcarbodiimide.
21. A method for producing calcitonin or a related analog
comprising condensing a desaminononapeptide of the frormula:
13wherein A.sub.2 is Gly, Ser or Ala; A.sub.3 is Asn or Ser;
A.sub.8 is Val or Met; Y is OH, OR.sup.1, where --R.sup.1 is a
lower alkyl group; with a recombinantly-formed polypeptide of the
formula: A.sub.10-A.sub.11A.sub.1-
2-A.sub.13-A.sub.14-A.sub.15-A.sub.16-A.sub.17-A.sub.18-A.sub.19-A.sub.20--
A.sub.21-A.sub.22-A.sub.23-A.sub.24-A.sub.25A.sub.26-A.sub.27-Gly-A.sub.29-
-A.sub.30-A.sub.31-Pro-Xxx (SEQ ID NO:1) A.sub.10 wherein A.sub.10
is Gly or Ser, A.sub.11 is Lys, Thr or Ala, A.sub.12 is Leu or Tyr,
A.sub.13 is Ser, Thr or Trp, A.sub.14 is Gln, Lys or Arg, A.sub.15
is Glu, Asp or Asn, A.sub.16 is Leu or Phe, A.sub.17 is His or Asn,
A.sub.18 is Lys or Asn, A.sub.19 is Leu, Tyr or Phe, A.sub.20 is
Gln or His, A.sub.21 s Thr or Arg, A.sub.22 is Tyr or Phe, A.sub.23
is Pro or Ser, A.sub.24 is Arg, Gly or Gln, A.sub.25 is Thr or Met,
A.sub.26 is Asp, Ala, Gly, or Asn, A.sub.27 is Val, Leu, Ile, Phe,
or Thr, A.sub.29 is Ala, Val, Pro or Ser, A.sub.30 is Gly, Val or
Glu, A.sub.31 is Thr, Val or Ala, and -Xxx is --OH, --NH.sub.2, an
amino acid residue or a polypeptide group, in the presence of a
non-enzymatic coupling reagent to form a calcitonin-derivative
having the formula: 14
Description
[0001] Calcitonins and related analogs, such as Elcatonin, are
known polypeptides which can be employed for treating bone atrophy
(see, e.g., U.S. Pat. No. 4,086,221). Naturally occurring
calcitonins, such as eel, salmon or human calcitonin, are
C-terminal amidated polypeptides which consist of 32 amino acids,
the first and the seventh amino acids in each case being
L-cysteines whose mercapto groups are connected to each other by
the formation of a disulfide bridge. The natural calcitonins can be
obtained, for example, by extraction from the mammalian thyroid
gland (see, e.g., U.S. Pat. No. 5,428,129).
[0002] Elcatonin is a modified synthetic "carba" analog of
calcitonin whose activity is comparable with that of eel calcitonin
(Morikawa et al., Experienta, 32, 1004, (1976)). In contrast to eel
calcitonin, Elcatonin lacks an amino terminal end and the disulfide
bridge of eel calcitonin has been replaced by a
--(CH.sub.2).sub.6-- "carbon bridge."
[0003] Currently, a variety of processes are known for the
preparation of Elcatonin using purely chemical methods. These
chemical methods involve condensation of the corresponding amino
acids or peptides (see, e.g., U.S. Pat. Nos. 4,086,221 and
5,428,129). The purely chemical methods, however, all suffer from
the disadvantage that, due to the elaborate purification methods
required, the Elcatonin is obtained in low yield and its
preparation is consequently very expensive.
[0004] It would accordingly be beneficial to be able to avoid the
disadvantages of the purely chemical methods in the preparation of
Elcatonin through the use of a approach which includes the
recombinant preparation of a portion of the molecule. This could be
achieved, for example, if a simple process for the recombinant
preparation of a C-terminal polypeptide fragment was available. The
recombinantly synthesized C-terminal fragment could then be used as
a starting peptide for the preparation of calcitonin or carba
analogs such as Elcatonin. A partially recombinant strategy would
also facilitate the synthesis of peptides/peptide analogs of
calcitonin, Elcatonin and related analogs or derivatives which
could potentially include non-natural amino acids.
SUMMARY OF THE INVENTION
[0005] The present invention is directed to a process for the
recombinant preparation of a calcitonin fragment and the use of the
fragment in the preparation of calcitonin and related analogs
including carba analogs (referred to hereinafter as "calcitonin
carba analogs"), such as Elcatonin. The invention includes
recombinantly forming a fusion protein which includes a target
sequence linked to a carbonic anhydrase through a cleavage site.
The target sequence includes a sequence of at least about 15 amino
acids residues corresponding to a fragment from near the C-terminus
of calcitonin or to a closely related analog of such a fragment.
Typically, the target sequence includes an amino acid sequence
corresponding to amino acids residues 10 through 32 of calcitonin
or closely related analogs (collectively referred to hereinafter as
a "10-32 fragment"). The recombinantly formed fusion protein is
subsequently cleaved with a cleavage reagent to produce a
polypeptide including the target sequence. The cleavage reaction
may be carried out by contacting the fusion protein with either a
chemical cleavage reagent or an enzymatic cleavage reagent. The
choice of a suitable cleavage reagent and the corresponding
cleavage site incorporated into the fusion protein will depend on
the particular target sequence and carbonic anhydrase sequence
present in the fusion protein. Typically, the cleavage reagent and
cleavage site are selected such that the amino acid sequence
constituting the cleavage site does not appear in the amino acid
sequence of either the target sequence or the carbonic anhydrase.
For example, a cyanogen bromide cleavage at methionine would not be
employed with a fusion protein which included the 10-32 fragment
from porcine, bovine or sheep calcitonin.
[0006] The cleavage site is typically present in a linker sequence
which connects the carbonic anhydrase and the target sequence.
Alternatively, the fusion protein may include a construct in which
the C-terminus of the carbonic anhydrase is connected directly to
the N-terminus of the target sequence. This may occur where the
C-terminal residue(s) of the carbonic anhydrase and the N-terminal
residue(s) of the target sequence constitute a cleavage site which
allows cleavage of the peptide bond between the two fragments. In
addition to a cleavage site present in a linker sequence, the
carbonic anhydrase portion of the fusion protein may also include a
different cleavage site which permits the fusion protein to be
cleaved to form a "minifusion protein," i.e., a polypeptide having
a C-terminal portion of the carbonic anhydrase still linked to the
target sequence.
[0007] One embodiment of the invention includes a method for the
recombinant preparation of polypeptides corresponding to amino
acids 10-32 of calcitonin or related analogs ("10-32 fragments").
The method typically includes the recombinant preparation of a
polypeptide fragment ("10-32 fragment-Xxxx") of the formula:
[0008]
A.sub.10-A.sub.11-A.sub.12-A.sub.13-A.sub.14-A.sub.15-A.sub.16-A.su-
b.17-A.sub.18-A.sub.19-A.sub.20-A.sub.21-A.sub.22-A.sub.23-A.sub.24-A.sub.-
25-A.sub.26-A.sub.27-Gly-A.sub.29-A.sub.30-A.sub.31-Pro-Xxx (SEQ ID
NO:1)
[0009] wherein A.sub.10 is Gly or Ser, A.sub.11 is Lys, Thr or Ala,
A.sub.12 is Leu or Tyr, A.sub.13 is Ser, Thr or Trp, A.sub.14 is
Gln, Lys or Arg, A.sub.15 is Glu, Asp or Asn, A.sub.16 is Leu or
Phe, A.sub.17 is His or Asn, A.sub.18 is Lys or Asn, A.sub.19 is
Leu, Tyr or Phe, A.sub.20 is Gln or His, A.sub.21 is Thr or Arg,
A.sub.22 is Tyr or Phe, A.sub.23 is Pro or Ser, A.sub.24 is Arg,
Gly or Gln, A.sub.25 is Thr or Met, A.sub.26 is Asp, Ala, Gly, or
Asn, A.sub.27 is Val, Leu, Ile, Phe, or Thr, A.sub.29 is Ala, Val,
Pro or Ser, A.sub.30 is Gly, Val or Glu, A.sub.31 is Thr, Val or
Ala. The C-terminal -Xxx group is typically a C-terminal carboxylic
acid ("--OH"), a C-terminal carboxamide ("--NH.sub.2"), or group
capable of being converted into a C-terminal carboxamide, such as
an amino acid residue or a polypeptide group (typically having from
2 to about 10 amino acid residues). The 10-32 fragment represented
by residues A.sub.10 to A.sub.32 (SEQ ID NO:2) corresponds to
residues 10 through 32 of the amino acid sequences for eel (SEQ ID
NO:37), salmon I (SEQ ID NO:38), salmon II (SEQ ID NO:39), salmon
III (SEQ ID NO:40), chicken (SEQ ID NO:41), human (SEQ ID No:42),
rabbit (SEQ ID NO:43), porcine (SEQ ID NO:44), bovine (SEQ ID
NO:45) and sheep (SEQ ID NO:46) calcitonin or closely related
analogs (see the calcitonin sequences shown in FIG. 9). The present
method may also be employed to recombinantly produce 10-32
fragments corresponding to modified calcitonin sequences. The
modified calcitonin sequences may include one or more conservative
amino acid substitutions in the natural amino acid sequence.
[0010] The 10-32 fragment may be utilized in the preparation of
calcitonin and related analogs. The preparation typically includes
the condensation of an N-terminal fragment of the formula: 1
[0011] wherein A.sub.2 is Gly, Ser or Ala; A.sub.3 is Asn or Ser;
A.sub.8 is Val or Met; R.sup.2 is --(CH.sub.2).sub.4-- or
--CH(NH.sub.2)CH.sub.2S- --S--; and Y is OH, OR.sup.1, where
--R.sup.1 is a lower alkyl group;
[0012] with a recombinantly-formed polypeptide of the formula:
[0013]
A.sub.10-A.sub.11-A.sub.12-A.sub.13-A.sub.14-A.sub.15-A.sub.16-A.su-
b.17-A.sub.18-A.sub.19-A.sub.20-A.sub.21-A.sub.22-A.sub.23-A.sub.24-A.sub.-
25-A.sub.26-A.sub.27-Gly-A.sub.29-A.sub.30-A.sub.31-Pro-Xxx (SEQ ID
NO:1)
[0014] wherein a A.sub.10 is Gly or Ser, A.sub.11 is wherein
A.sub.10 is Gly or Ser, A.sub.11 is Lys, Thr or Ala, A.sub.12 is
Leu or Tyr, A.sub.13 is Ser, Thr or Trp, A.sub.14 is Gln, Lys or
Arg, A.sub.15 is Glu, Asp or Asn, Al.sub.16 is Leu or Phe, A.sub.17
is His or Asn, A.sub.18 is Lys or Asn, A.sub.19 is Leu, Tyr or Phe,
A.sub.20 is Gln or His, A.sub.21 is Thr or Arg, A.sub.22 is Tyr or
Phe, A.sub.23 is Pro or Ser, A.sub.24 is Arg, Gly or Gln, A.sub.25
is Thr or Met, A.sub.26 is Asp, Ala, Gly, or Asn, A.sub.27 is Val,
Leu, Ile, Phe, or Thr, A.sub.29 is Ala, Val, Pro or Ser, A.sub.30
is Gly, Val or Glu, A.sub.31 is Thr, Val or Ala, and -Xxx is --OH,
--NH.sub.2, an amino acid residue or a polypeptide group;
[0015] in the presence of a non-enzymatic coupling reagent to form
a calcitonin-derivative having the formula: 2
[0016] In one embodiment of the invention, the recombinantly-formed
10-32 fragment is condensed with a desaminononapeptide. The
desaminononapeptide is a carba analog of an N-terminal calcitonin
fragment and typically has the formula: 3
[0017] wherein A.sub.2 is Gly, Ser or Ala; A.sub.3 is Asn or Ser;
A.sub.8 is Val or Met; and Y is OH, OR.sup.1, where --R.sup.1 is a
lower alkyl group (i.e., a C.sub.1-C.sub.6 alkyl group). The
condensation reaction may be carried out using a chemical coupling
reaction such as those described in U.S. Pat. Nos. 4,086,221 and
5,428,129, the disclosures of which are herein incorporated by
reference. Chemical coupling agents are well known to those skilled
in the art. Suitable chemical coupling agents include carbodiimides
and a variety of other non-enzymatic reagents capable of reacting
with the .alpha.-carboxylic acid group of a peptide to form an
activated carboxylic acid derivative and/or capable of catalyzing
the condensation of an activated .alpha.-carboxylic acid derivative
with an N-terminal .alpha.-amino group of another amino acid or
polypeptide. Chemical coupling reactions in which the C-terminal
.alpha.-carboxylic acid of the desaminononapeptide has been
converged to an acid azide, mixed acid anhydride, acid imidazole or
active ester may be employed in the present invention. An
especially effective method of coupling two peptide fragments is
carried out in the presence of a carbodiimide and a reagent capable
of forming an active ester, e.g., a mixture of
dicyclohexylcarbodiimide ("DCC") and either N-hydroxysuccinimide
("HOSu") or 1-hydroxybenzotriazole ("HOBt").
[0018] The recombinantly-formed 10-32 fragment employed in the
condensation preferably has the formula:
[0019]
A.sub.10-A.sub.11-A.sub.12-A.sub.13-A.sub.14-A.sub.15-A.sub.16-A.su-
b.17-A.sub.18-A.sub.19-A.sub.20-A.sub.21-A.sub.22-A.sub.23-A.sub.24-A.sub.-
25-A.sub.26-A.sub.27-Gly-A.sub.29-A.sub.30-A.sub.31-Pro-Xxx (SEQ ID
NO:1)
[0020] wherein A.sub.10 through A.sub.31 and -Xxx are as defined
herein.
[0021] The product of the coupling reaction is typically a
calcitonin carba analog having the formula: 4
[0022] wherein A.sub.10 through A.sub.31 and -Xxx are the same as
defined herein for the desaminononapeptide (SEQ ID NO:3) or the
10-32 fragment-Xxx (SEQ ID NO:1). The coupling of the
desaminononapeptide and the recombinantly-formed peptide is
typically carried out in the presence of a non-enzymatic coupling
reagent.
[0023] A preferred embodiment of the invention provides a method
for the recombinant preparation and amidation of a polypeptide
fragment (referred to herein as the "ECF2-amide") having the
formula:
[0024]
Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-
-Val-Gly-Ala-Gly-Thr-Pro-NH.sub.2 (SEQ ID NO:6)
[0025] and for coupling the ECF2-amide to an amino terminal
fragment of Elcatonin (referred to hereafter as "ECF1"), which has
the formula: 5
[0026] The present invention also provides a nucleic acid sequence
which includes a sequence coding for amino acids 10-32 of
calcitonin or a related analog. The nucleic acid sequence typically
encodes a fusion protein which includes the 10-32 fragment linked
to a carbonic anhydrase through a cleavage site. The portion of the
gene encoding the 10-32 fragment is preferably designed using
optimal codon usage for a targeted host cell, such as E. coli, S.
cerevisiae or P. pastoris.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 shows a map of plasmid pET31F1mhCAII. The plasmid
includes the F1 origin of replication from plasmid pEMBL8
introduced in the opposite orientation of the pSP65 origin of
replication.
[0028] FIG. 2 shows a map of plasmid pABN. Plasmid pABN includes a
nucleic acid sequence coding for a Linker peptide fragment inserted
into the plasmid near the carboxy terminal end of the gene sequence
of human carbonic anhydrase II ("hCAII").
[0029] FIG. 3 shows a map of plasmid PTBN. Plasmid pTBN is a
tetracycline resistant expression vector derived from the insertion
of a 1.5-kb fragment from pABN into plasmid pBR322. The 1.5-kb
fragment includes the T7 promotor, hCAII, Linker and T7 terminator
sequences from pABN.
[0030] FIG. 4A is a schematic illustration of the first step of the
preparation using PCR methodology of a nucleotide sequence encoding
ECF2-Ala and additional restriction sites to permit cloning the
fragment into plasmids. This step was carried out by conducting a
PCR reaction using Taq DNA polymerase on PCR MIX 1.
[0031] FIG. 4B is a schematic illustration of the second step of
the preparation of a DNA sequence encoding ECF2-Ala. The PCR
produces derived from the extension of oligonucleotides 2 and 3 in
PCR MIX 1 (2.sup.Ext (SEQ ID NO:8) and 3.sup.Ext (SEQ ID NO:9)
respectively) have 20 bp of complementary sequence at their 3'
ends. The second PCR reaction using Taq DNA polymerase creates a
double stranded nucleotide fragment which includes a full length
non-interrupted gene sequence encoding ECF2-Ala (SEQ ID NO:10).
[0032] FIG. 5 shows a map of plasmid pTBN26. Plasmid pTBN26
includes a gene sequence for the entire hCAII-Linker-ECF2-Ala
("hCA-ECF2-Ala") fusion protein construct.
[0033] FIG. 6A shows the nucleotide (SEQ ID NO:11) and amino acid
(SEQ ID NO:12) sequences for the N-terminal methionine and hCAII
residues 1-162 of the hCAII-Linker-ECF2-Ala fusion protein
construct.
[0034] FIG. 6B shows the nucleotide (SEQ ID NO:11) and amino acid
(SEQ ID NO:12) sequences for hCAII residues 162-257, and the linker
and ECF2-Ala fragments of the hCAII-Linker-ECF2-Ala fusion protein
construct.
[0035] FIG. 7 shows a nucleotide sequence (SEQ ID NO:5) and the
amino acid (SEQ ID NO:6) sequence for the 23 amino acid C-terminal
fragment ("ECF2") of Elcatonin and eel calcitonin. The nucleotide
sequence shown is the sequence encoding ECF2 present in the gene
for the hCA-ECF2-Ala fusion protein incorporated into plasmid
pTBN26.
[0036] FIG. 8 shows a representative preparative HPLC trace (using
a polysulfolethyl-aspartamide column) of a sample containing
ECF2-amide (SEQ ID NO:6). The peak for ECF2-amide appears between
14.5 and 16.3 minutes.
[0037] FIG. 9A shows the amino acid sequences for residues 1-15 of
calcitonin from a number of species.
[0038] FIG. 9B shows the amino acid sequences for residues 16-32 of
calcitonin from a number of species.
[0039] FIG. 10 shows a double stranded DNA fragment (SEQ ID NO:10)
synthesized from the 5 oligonucleotide described in example 1.1 via
PCR methodology.
[0040] FIG. 11 depicts the nucleotide sequence for oligonucleotide
2.sup.Ext (SEQ ID NO:8) produced during the PCR synthesis of the
gene sequence encoding ECF2.
[0041] FIG. 12 depicts the nucleotide sequence for oligonucleotide
3.sup.Ext (SEQ ID NO:9) produced during the PCR synthesis of the
gene sequence encoding ECF2.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The recombinant preparation of the fusion protein according
to the present invention includes the construction of a nucleic
acid sequence coding for the target sequence linked to a carbonic
anhydrase through a cleavage site ("FP nucleic acid sequence"). The
FP nucleic acid sequence shown in FIG. 6 is one example of a
suitable nucleic acid sequence for use in the present method. The
nucleic acid sequence shown in FIG. 6 includes codons encoding
residues 1-257 of human carbonic anhydrase II ("hCAII").
[0043] Inclusion of this amino acid sequence or other functionally
active fragments of a carbonic anhydrase in the fusion protein
greatly facilitates the isolation of the fusion protein from cell
debris. As used herein, the term "carbonic anhydrase" includes
naturally occurring carbonic anhydrase (and any allelic variants),
functionally active carbonic anhydrase fragments or modified
versions ("mutants") thereof. Herein "functionally active carbonic
anhydrase fragments and mutants" means a fragment or modified
version of a carbonic anhydrase which exhibits the enzyme inhibitor
binding properties of hCAII. Fragments having such properties are
capable of binding carbonic anhydrase inhibitors such as
sulfanilamides extremely tightly. A functionally active fragment
having such binding properties exhibits highly selective affinity
binding to a low molecular weight ligand, e.g. a sulfanilamide, or
a synthetic derivative thereof. The binding is strong so that, in
general, the carbonic anhydrase/ligand conjugate will exhibit a
solution dissociation constant (inverse of the binding constant) of
no more than about 10.sup.-7 M. Generally, the ligand is a
reversible inhibitor for the carbonic anhydrase. Suitable carbonic
anhydrase fragments may lack an N-terminal or C-terminal portion of
the enzyme, so long as the fragments retain the functional
inhibitor binding activity of the enzyme. Similarly, modified
carbonic anhydrases which may be employed as binding proteins in
the present fusion protein may be modified by one or more amino
acid additions, deletions or insertions so long as the modified
enzyme substantially exhibits the inhibitor binding activity
described above. Typically, a carbonic anhydrase is modified by
deleting or altering amino acid residues so that the modified
enzyme does not contain the particular amino acid(s) to be employed
as a cleavage site in the fusion protein. For example, where
cyanogen bromide is to be utilized as a cleavage reagent, a
carbonic anhydrase may be modified to remove or replace any
methionine ("Met") residues normally present.
[0044] The FP nucleic acid sequence includes a sequence encoding a
10-32 fragment, i.e., amino acid residues 10-32 of calcitonin or a
closely related analog. The closely related analogs may be derived
from conservative amino acid substitutions in a calcitonin 10-32
fragment. This portion of the nucleic acid sequence may be designed
to optimize the expression of the fusion protein in a particular
host cell. For example, where the expression of the fusion protein
is to be carried out using an enterobacteria such as E. coli as the
host organism, the nucleic acid sequences encoding the 10-32
fragment and the cleavage site are typically constructed based on
the frequency of codon usage for the targeted host organism (see,
e.g., discussion of frequency of codon usage in Gribskov et al.,
Nucl. Acids Res., 12, 539-549 (1984)).
[0045] The cleavage site may be a chemical cleavage site or an
enzymatic cleavage site. Chemical and enzymatic cleavage sites and
the corresponding agents used to effect cleavage of a peptide bond
close to one of these sites are described in detail in PCT patent
application WO 92/01707, the disclosure of which is herein
incorporated by reference. Examples of peptide sequences (and DNA
gene sequences coding therefor) suitable for use as cleavage sites
in the present invention and the corresponding cleavage enzymes or
chemical cleavage conditions are shown in Table 1. The gene
sequence indicated is one possibility coding for the corresponding
peptide sequence. Other DNA sequences may be constructed to code
for the same peptide sequence. Preferably, the nucleic acid
sequence coding for the cleavage site is designed based on the more
commonly used codons for each amino acid for the host cell to be
employed in the expression of the fusion protein. For example, The
nucleotide sequence may be based on optimum codon usage for an
enterobacteria, such as E. coli (see, e.g., Gribskov et al., Nucl.
Acids Res., 12, 539-549 (1984)).
[0046] The cleavage site may be present as part of a linker peptide
which connects the carbonic anhydrase and the target sequence. For
example, the amino acid sequence (SEQ ID NO:12) for
hCAII-linker-ECF2-Ala ("hCA-ECF2-Ala") depicted in FIG. 6 includes
a seven amino acid linker sequence (-Phe-Val-Asp-Asp-Asp-Asp-Asn-;
(SEQ ID NO:14)) which sets up a chemical cleavage site cleavable by
hydroxylamine ("Asn-Gly") between the C-terminal asparagine residue
of the linker and the N-terminal glycine residue of the target
sequence.
[0047] The fusion protein includes at least one copy and may
include multiple copies of the target sequence. Where multiple
copies of the target sequence are present, the copies may be
tandemly linked together. Alternatively, the copies of the target
sequence may be linked together by an
1 TABLE 1 Peptide Sequence DNA Sequence Enzymes for Cleavage
Enterokinase (Asp).sub.4Lys GACGACGACGATAAA (SEQ ID NO: 16) (SEQ ID
NO: 15) Factor Xa IleGluGlyArg ATTGAAGGAAGA (SEQ ID NO: 18) (SEQ ID
NO: 17) Thrombin GlyProArg or GGACCAAGA or GlyAlaArg GGAGCGAGA
Ubiquitin Cleaving ArgGlyGly AGAGGAGGA Enzyme Renin
HisProPheHisLeu- CATCCTTTTCATC- LeuValTyr TGCTGGTTTAT (SEQ ID NO:
20) (SEQ ID NO: 19) Trypsin Lys or Arg AAA OR CGT Chymotrypsin Phe
or Tyr or Trp TTT or TAT or TGG Clostripain Arg CGT S. aureus VS
Glu GAA Chemical Cleavage (at pH3) AspGly or AspPro GATGGA or
GATCCA (Hydroxylamine) AsnGly AATCCA (CNBr) Methionine ATG
BNPS-skatole Trp TGG 2-Nitro-5- Cys TGT thiocyanatobenzoate
[0048] innerconnecting linker peptide. The innerconnecting linker
peptide may be the same as or different than the intraconnecting
linker peptide which connects the carbonic anhydrase to the first
copy of the target peptide. If the innerconnecting and
intraconnecting linker peptides are the same or include the same
cleavage site, the fusion protein may be cleaved directly to
produce a number of fragments each containing a single copy of the
target peptide. If, however, the innerconnecting and
intraconnecting linker peptides contain different cleavage sites,
it may be possible to initially cleave off the carbonic anhydrase
fragment to form a intermediate polypeptide having more than one
copy of the target peptide.
[0049] Target sequences free of methionine residues may be produced
using the present method from a multicopy construct having
innerconnecting peptides which include a methionine residue. Where
the methionine residue is directly linked to the C-terminus of the
target sequence, the multicopy construct may be cleaved with
cyanogen bromide. The resulting fragments may be transpeptidated
using a carboxypeptidase, e.g., a serine carboxypeptidase such as
carboxypeptidase Y, to replace the C-terminal homoserine residue
with an .alpha.-amidated amino acid. The fragments may be also
transamidated with the carboxypeptidase to replace the C-terminal
homoserine residue with a 2-nitrobenzylamine compound. This
produces a fragment having a C-terminal (2-nitrobenzyl)amido group
which may be photochemically decomposed to produce an
.alpha.-amidated peptide fragment minus the homoserine residue.
[0050] One example of a fusion protein including multiple copies of
a target sequence in a construct which includes
hCA-(MetValAsnAspAspAspAsn-- ECF2).sub.n-Xxx, where hCA, ECF2 and
Xxx are as defined herein and n is an integer (typically 2 to 20).
Such a construct may be treated with CNBr to form
ValAspAspAspAspAsn-ECF2-Hse (SEQ ID NO:49) peptide fragments (where
Hse is a homoserine residue produced by the reaction of CNBr with a
Met residue). The peptide fragments may then be reacted with a
nucleophile such as o-nitrophenylglycine amide ("ONPGA") in the
presence of a peptidase such as carboxypeptidase Y resulting in the
replacement of the Hse residue by ONPGA. Upon photolysis, the
transpeptidation product is converted to a C-terminal carboxamide.
The N-terminal tail sequence, ValAspAspAspAspAsn (SEQ ID NO:50),
may be cleaved off the fragments by treatment with
hydroxylamine.
[0051] A preferred embodiment of the invention is directed to the
preparation of the C-terminal eel calcitonin polypeptide fragment,
ECF2-amide (SEQ ID NO:6). The preparation involves the initial
genetic expression of the following protein construct:
[0052] Met-hCAII'-Met.sub.240-Val.sub.241-hCAII"-Linker-ECF2-aa
("hCA-ECF2-aa")
[0053] where aa is an amino acid residue and Met is the required
N-terminal residue of any E. coli protein. The Met residue is added
to the N-terminus of the first residue of hCAII' (a 239 amino acid
N-terminal polypeptide segment of human carbonic anhydrase II),
Met.sub.240-Val.sub.241 is the only cyanogen bromide labile peptide
bond in human carbonic anhydrase II ("CAII"); hCAII" is a 16 amino
acid fragment (SEQ ID NO:21) from near the C-terminal end of hCAII
(residues 242-257). The C-terminal amino acid residue of ECF2-aa
(SEQ ID NO:22), designated aa, provides an amidation signal to
enable conversion to the Pro-amide that constitutes the C-terminus
of Elcatonin (SEQ ID NO:13). The aa residue is typically an amino
acid residue, such as alanine, which is capable of being exchanged
with a nucleophile via a transamidation reaction.
[0054] The desired product, ECF2-aa (SEQ ID NO:22), can be obtained
from a hCA-ECF2-aa protein construct, in at least two ways. The
first employs a cleavage of hCA-ECF2-aa at the Asn-Gly bond with
hydroxylamine to yield ECF2-aa (SEQ ID NO:22) directly.
Alternatively, cleavage with cyanogen bromide (CNBr) yields a
minifusion protein which includes the hCAII" C-terminal fragment
(SEQ ID NO:21) of hCAII linked to the ECF2-aa polypeptide (SEQ ID
NO:22). The minifusion protein may subsequently be cleaved with
hydroxylamine either before derivatization to provide ECF2-aa (SEQ
ID NC:22) or, after derivatization of the minifusion protein side
chain residues, e.g., where the Lys residues of ECF2-aa (SEQ ID
NO:22) have been derivatized to form Lys residues having
Z-protected side chain amino groups. In the case where
derivatization reagents modify the side chain amino functions of
Lys, then after cleavage of derivatized minifusion protein, the
resulting protected ECF2-aa (SEQ ID NO:22) produced will only
possess a single free amino function at the alpha position of the
N-terminal glycine residue. The free N-terminal a-amino group can
be used for subsequent specific chemical reactions, such as
coupling to the C-terminal residue of ECF1 (SEQ ID NO:7) to provide
Elcatonin (SEQ ID NO:13). Coupling reactions of this type may be
carried out after the ECF2-aa polypeptide (SEQ ID NO:22) has been
converted to an ECF2 derivative having a C-terminal residue
Pro-amide ("ECF2-amide"; (SEQ ID NO:6)), or may be employed to
produce derivatized forms of Elcatonin (e.g., "Elcatonin-aa"; (SEQ
ID NO:30)) for subsequent conversion to Elcatonin (SEQ ID
NO:13).
[0055] A preferred sequence of ECF2-aa is:
2 Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr- ("ECF2-Ala";
SEQ ID NO:23) Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro-Ala
[0056] The ECF2-Ala peptide fragment may be derived from cleavage
of a hCA-ECF2-Ala protein construct, e.g., by treatment with
hydroxylamine. The hCA-ECF2-Ala protein construct may include the
sequence:
3 hCAII'-Met-Val-Asp-Asn-Trp-Arg-Pro-Ala-Gln-Pro-Leu-Lys- (SEQ ID
NO:12) Asn-Arg-Gln-Ile-Lys-Ala-Phe-Val-Asp-Asp-Asp-Asp-As- n-
Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-
Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro-Ala
[0057] Alternatively, the hCA-ECF2-Ala protein construct may be
cleaved at a different bond to produce a pre-ECF2-Ala peptide,
i.e., a minifusion protein. For example, the hCA-ECF2-Ala protein
construct may be cleaved to produce a pre-ECF2-Ala peptide which
includes a C-terminal fragment of hCAII or a modified version
thereof linked to the N-terminus of ECF2-Ala (SEQ ID NO:23). In a
preferred embodiment of the invention, the hCA-ECF2-Ala protein
construct (SEQ ID NO:12) may be cleaved with cyanogen bromide
(CNBr) to yield a minifusion protein ("MFP") having the amino acid
sequence:
4 Val-Asp-Asn-Trp-Arg-Pro-Ala-Gln-Pro-Leu-Lys-Asn-Arg- (SEQ ID
NO:24). Glu-Ile-Lys-Ala-Phe-Va1-Asp-Asp-Asp-Asp-Asn-Gly-L- ys-
Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-
Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro-Ala
[0058] The MFP includes the Val.sub.241-hCAII" fragment from the
C-terminal end of hCAII:
[0059]
Val-Asp-Asn-Trp-Arg-Pro-Ala-Gln-Pro-Leu-Lys-Asn-Arg-Glu-Ile-Lys-Ala
(SEQ ID NO:25)
[0060] and a Linker sequence having the formula:
[0061] Phe-Val-Asp-Asp-Asp-Asp-Asn (SEQ ID NO:14).
[0062] The polypeptide segment
hCAII'-Met.sub.240-Val.sub.241-hCAII" of human carbonic anhydrase
II (hCAII) has been reported in Biochemistry, 9, 2638 (1970) and
can be used as the binding protein segment for purification of a
carbonic anhydrase-EGF2-aa protein construct using procedures such
as those described in (WO 92/01707). In accordance with WO
92/01707, carbonic anhydrase fragments or modified carbonic
anhydrases can also be used as the binding protein segment.
[0063] The amino acid sequences for a number of other carbonic
anhydrases have also been reported (see, e.g., Hewett-Emmett et
al., in The Carbonic Anhydrases, Dodgson et al. eds., Chapt. 2, pp.
15-32 (1991)). If the amino acid sequence of a particular carbonic
anhydrase is known but no gene is available, a cDNA coding for the
carbonic anhydrase may be isolated using procedures well known to
those skilled in the art (see, e.g., Sambrook et al., Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. (1989), and Tanhauser et al., Gene, 117,
113-117 (1992)). This process typically includes constructing
nucleic acids probes (e.g., about 20-30 base pairs in length)
coding for fragments of the carbonic anhydrase in question.
Degenerate nucleotide probes based on the known amino acid sequence
or related DNA sequence can be used to screen a cDNA library (see,
e.g., Wallace et al., Nucleic Acids Res., 6, 3543 (1979) and
Wallace et al., Nucleic Acids Res., 9, 879 (1981)).
[0064] In another embodiment of the invention, the target sequence
may include an amino acid sequence in which at least one of the
C-terminal residues of a 10-32 fragment has been replaced by one or
more amino acid residues ("leaving unit"). Ache C-terminal end of
the target sequence may be modified via a transpeptidation
reaction, either before or after cleavage of the fusion protein to
remove the carbonic anhydrase portion. The transpeptidation
reaction typically results in the removal of the leaving unit from
the C-terminal end of the target sequence and its replacement by
the missing C-terminal residue (or residues) of the "10-32
fragment." For example, a recombinantly produced peptide having
C-terminal leaving unit, such as eel(10-30)-Ala (amino acid
residues 10-30 of eel calcitonin coupled to a C-terminal alanine),
may be transpeptidated using S. aureus V8 to introduce a
Thr-Pro-NH.sub.2 dieptide after residue Glu.sub.30 in place of the
C-terminal Ala residue.
[0065] In addition to being employed in the preparation of carba
analogs of calcitonin, such as Elcatonin, the recombinantly
synthesized 10-32 fragments of the present invention may be used in
the synthesis of calcitonins. For example, a side chain protected
derivative of ECF2-amide, e.g., where Ser, Thr and Glu residues are
protected as benzyl ethers or esters and Lys residues are protected
by a CBZ group, may be coupled using a non-enzymatic coupling
reagent with the cyclic oxidized form of residues 1-9 of eel
calcitonin. Examples of suitable non-enzymatic coupling reagents
which may be used to carry out the coupling reaction include DCC,
N-ethyl-N'-dimethylaminopropyl-carbodiimid- e ("EDAPC"), DCC/HOSu,
DCC/HOBt and EDAPC/HOSu (see, e.g., U.S. Pat. No. 5,429,129, the
disclosure of which is herein incorporated by reference).
[0066] I. Recombinant Formation of the Fusion Protein
[0067] a. Vectors
[0068] Expression vectors incorporating the fusion protein gene are
chosen to be compatible with the host cell. The vectors used have
many features in common. These features include an origin so
replication compatible with the host cell, regulatory DNA sequences
for transcription and regulation of transcription (for inducible
systems), an efficient ribosomal binding site (for prokaryotic
hosts), a poly-A signal (for eukaryotic hosts). In addition,
phenotype genes, regulatory regions and leader sequences may be
included.
[0069] Prokaryotic vectors such as those for expression in E. coli
are characterized by an origin of replication, a genetic marker
(phenotype) for selection of transformed bacteria, and DNA
regulation sequences that will direct the expression of the gene of
interest. The regulation sequences typically will include a
promoter to drive the transcription, an operator to control
transcription (on/off switch), an efficient ribosome binding site
to start translation, and a transcription termination signal. The
start and stop codons are provided by the inserted (fusion protein)
gene.
[0070] Prokaryotic vectors in particular contain a suitable
"expression cassette" which is based upon any of a number of
available promoter/operator systems. Typical promoters for
inclusion in the prokaryotic vector include lactose, tryptophan,
T7, lipoprotein, alkaline phosphatase, lambda leftward or rightward
promoter or a combination of these (hybrid promoters). The lactose
and tryptophan operators, as well as temperature sensitive lambda
promoters are typical on/off switches that can be included in the
prokaryotic vector. Typical phenotypic markers for inclusion in the
prokaryotic vector include genes for development of resistance to
ampicillin, tetracycline, kanamycin, and chloramphenicol.
[0071] Eukaryotic vectors such as those for expression in the
yeast, Saccharomyces cerevisiae, typically are shuttle vectors
which contain an origin of replication for E. coli and one for S.
cerevisiae, a genetic marker for both cell types, and DNA
regulation sequences that will direct the expression in yeast. The
regulation sequences typically include a promoter, a regulatory
sequence, and a transcription termination signal (including a
polyadenylation signal). Optional signal sequences for direction of
cellular secretion can also be inserted into the eukaryotic vector.
Typical markers to be incorporated provide positive selection by
complementation of mutations in the genes necessary for production
of uracil, leucine, histidine, adenine, tryptophan and the like.
Promoter sequences which preferably can be incorporated into the
eukaryotic vector include alcohol dehydrogenase I or II,
glyceraldehyde phosphate dehydrogenase, phosphoglycerokinase,
galactose, tryptophan, mating factor alpha and the like.
[0072] Depending on the nature of the vector selected, the ECF2-aa
gene fragment can be expressed in a variety of organisms. Both
vectors having a specific host range and vectors having a broad
host range are suitable for use in the present invention. Examples
of vectors having a specific host range, e.g. for E. coli, are
pBR322 (Bolivar et al., Gene, 2, 95-113, 1977), pUC18/19 (Yanisch
Perron et al., Gene, 33, 103-119, 1985), pK18/19 (Pridmore, Gene,
56, 309-312, 1987), pRK290X (Alvarez-Morales et al., Nucleic Acids
Res., 14, 4207-4227, 1986) and pRA95 (obtainable from Nycomed Pharm
a AS, Huidove, Denmark).
[0073] Other vectors that can be employed are "broad host range"
vectors which are suitable for use in Gram negative bacteria.
Examples of such "broad host range" vectors are pRK290 (Ditta et
al., Proc. Nat. Acad. Sci., 77, 7347-7351; 1980), pKT240
(Bagdasarian et al., Gene, 26, 273-282, 1983), derivatives of
pRK290, such as pLAFR1 (Long et al., Nature, 289, 485-488, 1982),
derivatives of pKT240, such as pMMB66EH (Furste et al., Gene, 48,
119-131, (1986)) or pGSS33 (Sharpe, Gene, 29, 93-102, (1984)).
[0074] b. Construction of Plasmid pTBN
[0075] An expression vector, pET31F1mhCAII, containing the hCAII
gene was obtained from Dr. P. J. Laipis at the University of
Florida. The ampicillin resistant, T7 expression vector was
constructed by the method described in Tanhauser et al., Gene, 117,
113-117 (1992) from the plasmid pET-3c (Studier et al., Methods
Enzymol., 185, 60-89 (1990)). The T7 expression cassette from the
pET-3c vector was transferred to a truncated pSP65 plasmid. To
enable the production of single stranded plasmid DNA, the F1 origin
from pEMBL8+ (Dente et al., Nucleic Acids Res., 11, 1645-1655
(1983)) was ligated into the Bgl II restriction site of the
positive clones. The resulting plasmid was designated pET31F1m,
where F1m indicates that the F1 origin has the opposite orientation
of the pSP65 origin of replication. Finally, human carbonic
anhydrase II cDNA (hCAII cDNA) was cloned into the pET31F1m plasmid
between the Nde.+-. and BamH I sites to give a plasmid designated
as pET31F1mhCAII (see FIG. 1).
[0076] The Linker region of the plasmid was created by first
synthesizing two complementary oligonucleotides at the DNA
Synthesis Core Facility of the Interdisciplinary Center for
Biotechnology at the University of Florida:
[0077] 5' A GCT TTC GTT GAC GAC GAC GAT ATC TT 3' (SEQ ID
NO:26)
[0078] 5' AGC TAA GAT ATC GTC GCO GTO AAC GAA 3' (SEQ ID NO:27)
[0079] The oligonucleotides were phosphorylated, annealed and
ligated into the Hind III site near the carboxy terminal end of the
gene sequence of hCAII. Plasmids containing the insert have a
unique EcoR V site immediately following the fourth aspartic acid
residue in the polypeptide fragment, Linker (SEQ ID NO:14). The
resulting plasmid is referred to as pABN (see FIG. 2).
[0080] The plasmids pABN and pBR322 (Bolivar et al. Gene, 2, 95-113
(1977) were used to construct a tetracycline resistant expression
vector to be used for the production of ECF2-aa (SEQ ID NO:27),
e.g., where aa is Ala. The 1.5-kb Ssp I/BspE I fragment from pABN
was inserted into the ScaI site of pBR322 resulting in pTBN (see
FIG. 3).
[0081] C. Host Strains and Transformation
[0082] Procedures for methods to restrict, ligate, transform,
select, culture, and lyse according to the invention, generally
follow standard methods known in the art. Literature providing the
details for these methods include, Sambrook et al., Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. (1989), the disclosure of which is incorporated
herein by reference.
[0083] In order to prepare the production strains for the
fermentation, the DNA fragment encoding hCA-ECF2-aa is introduced
into a host strain suitable for the expression of an hCA-ECF2
fusion protein. Examples of microorganisms which are suitable for
expressing this gene typically include strains possessing a high
tolerance of substrate and starting material, are enterobacteria,
such as from the genus Escherichia. Microorganisms of the species
E. coli are particularly preferred. The microorganisms can contain
the hCA-ECF2-aa DNA fragment either on a vector molecule or
integrated in their chromosomes. The selected microorganisms are
transformed using methods well known to those skilled in the art
(see, e.g. Sambrook et al., cited supra) with a vector containing
the hCA-ECF2-aa DNA fragments. Examples of suitable production
strains are E. coli JM109 (DE3) and E. coli BL21 (DE3), in each
case containing a plasmid encoding the fusion protein, e.g.,
pTBN26.
[0084] The eukaryotic cells may include unicellular organisms, such
as yeast cells, as well as immortal cells from higher organisms,
such as plant, insect or mammalian cells. Suitable eukaryotic host
cells include Sacoharomyces cerevisiae, Pichia pastoris,
Aspergillus niger, Spodoptera frupiperda, and corn, tobacco or
soybean plant cells. The higher organisms useful as hosts include
higher order plants and animals having germ cells that are amenable
to transformation. Included are plants such as tobacco, corn,
soybean and fruit bearing plants, and invertebrate and vertebrate
animals such as fish, birds and mammals, such as sheep, goats,
cows, horses and pigs.
[0085] The transformed host strains are typically isolated from a
selective nutrient medium to which an antibiotic has been added
against which the host strains are resistant due to a marker gene
located on the vector.
[0086] d. Fermentation
[0087] The recombinant preparation of the hCA-ECF2-aa fusion
protein is carried out using the microorganisms which contain the
hCA.about.ECF2-aa DNA fragment and/or the vector plasmids
(containing hCA.about.ECF2-aa fragment). The process may be carried
out by methods which are known per se, for example, by the method
described in WO 92/01707. In accordance with this, commercially
available growth media, such as Luria broth, can be used. The
fermentations are batch fed with oxygen and amino acid
supplementation to provide high cell densities. After the
fermentation is complete, she microorganisms may be disrupted as
described in WO 92/01707 and the hCA-ECF2-aa fusion protein
purified, e.g., using sulfanilamide affinity chromatography as
described in WO 92/01707.
[0088] II. Cleavage of the Fusion Protein to Yield ECF2-Ala
[0089] A fusion protein having the sequence shown below may be
cleaved between the amino acids Asn 7 (amino acid 7 of the linker
sequence) and Gly A.sub.10 (amino acid 1 of ECF2-Ala) as indicated
by the "*" below.
5 hCAII'-Met-Val-Asp-Asn-Trp-Arg-Pro-Ala-Gln-Pro-Leu-Lys- (SEQ ID
NO:12) Asn-Arg-Gln-Ile-Lys-Ala-Ser-Phe-Val-Asp-Asp-Asp-As- p-
Asn*-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-
Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro-Ala
[0090] Specific cleavage at this site can be achieved by treatment
with hydroxylamine. The resulting ECF2-Ala fragment (SEQ ID NO:23)
may be purified by conventional procedures.
[0091] III. Amidation of ECF2-aa (SEQ ID NO:22) to Form ECF2-amide
(SEQ ID NO:6)
[0092] The amidation of ECF2-aa (SEQ ID NO:22) may be carried out
in accordance with WO 92/05271, by transamidating an ECF2-aa
peptide such as ECF2-Ala (SEQ ID NO:23) with a nucleophilic amino
compound in the presence of carboxypeptidase Y (see Scheme 1 below)
to form a peptide intermediate capable of being reacted or
decomposed to form ECF2-NH.sub.2 (SEQ ID NO:6). Examples of
suitable nucleophiles include o-nitrobenzylamines, such as
o-nitrophenylglycine amide ("ONPGA"). The transamidation results in
a replacement of the C-terminal -aa residue of ECF2-aa (SEQ ID
NO:22) by the nucleophilic amine. Photolysis of the resulting
photolabile intermediate, e.g., ECF2-ONPGA (SEQ ID NO:28), leads to
cleavage of the nucleophile and the production of an ECF2 fragment
having an amidated proline at the carboxy terminus ("ECF2-amide";
SEQ ID NO:6). The ECF2-amide may be purified by conventional
methods. 6
[0093] IV. Condensation of Elcatonin-Fragment 1 (ECF1) and
ECF2-Amide
[0094] The N-terminal Elcatonin fragment ECF1 (SEQ ID NO:7) and the
Elcatonin related fragment ECF2-Xxx (SEQ ID NO:1) may be coupled
using standard peptide coupling reactions as described herein,
e.g., in the presence of DCC/HOSu, EDAPC/HOSu or DCC/HOBt. If the
coupling reaction is carried out using unprotected forms of ECF1
and ECF2-amide, the C-terminal .alpha.-carboxylic acid of the ECF1
fragment is typically converted into an activated ester prior to
the addition of the ECF2 fragment. The Elcatonin (SEQ ID NO:13)
produced by the coupling reaction may be purified by conventional
techniques, such as preparative HPLC methods.
[0095] Carba-analogs of other calcitonins (e.g. salmon I calcitonin
(SEQ ID NO:38)) and related calcitonin carba analogs may be
prepared in a similar manner using ECF1 (SEQ NO:7) and other 10-32
fragments, e.g., the salmon I calcitonin 10-32 fragment (SEQ ID
NO:29). The present invention also allows the preparation of carba
analogs through the combination of the C-terminal fragment of a
calcitonin from one species with an N-terminal carba fragment
corresponding to a calcitonin from a different species. 7
[0096] V. Cleavage of the Fusion Protein hCA-ECF2-aa to Give the
Minifusion Protein (MFP).
[0097] In another embodiment of the invention which employs a
chemical coupling reaction in the formation of Elcatonin (SEQ ID
NO:13), the
Met-hCAII'-Met.sub.240-Val.sub.241-hCAII"-Linker-ECF2-Ala fusion
protein (SEQ ID NO:12) may be cleaved between the Met.sub.240 and
Val.sub.241 residues via CNBr treatment to yield a 48 amino acid
minifusion protein (Val.sub.241-hCAII"-Linker-ECF2-Ala, hereinafter
"MFP" (SEQ ID NO:24)). The MFP is composed of 3 constituent
fragments. The amino terminal portion of the minifusion protein
essentially constitutes a 24 residue "biological blocking group" on
the N-terminus of the ECF2-Ala fragment (SEQ ID NO:23). The MFP
(SEQ ID NO:24) may be purified by conventional procedures and
subsequently derivatized to protect amino acid side chain
residues.
[0098] VI. Introduction of Protective Groups into a Minifusion
Protein.
[0099] When ECF2-aa (SEQ ID NO:22) is to be isolated in a
chemically blocked form, a minifusion protein may be separated from
the Fusion protein following cleavage, and then subjected to
chemical derivatization to block reactive side chain groups, such
as epsilon amino functions of lysine residues, with various
protecting groups ("R"). The N-terminal peptide segment functions
as a blocking group to protect the .alpha.-amino group of the
N-terminal Gly of ECF2-aa from derivatization by the chemical
protecting reagent.
[0100] The amino protective groups, R, which are customary in
polypeptide chemistry, e.g. those described in Houben-Weyl
(Methoden der organischen Chemie 15/1 and 15/2, Thieme Verlag
Stuttgart 1974), are suitable for use as protective groups.
Preferred amino protective groups include benzyloxycarbonyl- ("Z");
tert-butyloxycarbonyl- ("BOC"); fluorenylmethoxycarbonyl- ("FMOC");
or adamantyloxycarbonyl-("ADOC"). Other reactive side chain groups
may also be protected by a protective group, e.g., hydroxyl and
carboxy groups may be protected as benzyl ethers and benzyl esters
respectively.
[0101] VII. Preparation of a Protected ECF2-Ala Fragment
[0102] A protected 10-32 fragment may be formed by first treating
the minifusion protein with an appropriate derivatizing agent ("R
agent") where R is the amino protective group (where n corresponds
to the number of lysine residues in the minifusion protein). The
resulting protected minifusion protein ("R.sub.n-MFP" (SEQ ID
NO:24)) may then be cleaved to form a protected 10-32 fragment
("R.sub.n-ECF2-Ala" (SEQ ID NO:23)) in which only the free
.epsilon.-amino groups of Lys residues are protected by the "R"
group and the .alpha.-amino group is not protected (see Scheme 3
below). The cleavage can be affected chemically or enzymatically.
For the chemical cleavage, hydroxylamine may be used to cleave the
Asn-Gly bond (indicated by the arrow), e.g., according to the
procedure described by Bornstein, Biochemistry, 12, 2408-2421,
(199) to produce R.sub.n-ECF2-Ala (SEQ ID NO:23). The resulting
R.sub.n-ECF2-Ala fragments (SEQ ID NO:23) can subsequently be
purified by customary chemical methods if desired. In one example
of this embodiment of the invention, a BOC-protected
Val.sub.241-hCAII"-Linker-ECF2-Ala ("BOC.sub.4-MFP" (SEQ ID NO:24))
may be formed by reacting the MFP with BOC-anhydride. The resulting
BOC.sub.4-MFP (SEQ ID NO:24) may be cleaved between the Asn and Gly
amino acids residues with hydroxylamine to yield BOC.sub.2-ECF2-Ala
(SEQ ID NO:23). 8
[0103] IX. Coupling of the Protected R.sub.nECF2-aa to ECF1
Fragment to Produce Non-Amidated Elcatonin.
[0104] The present invention additionally relaxes to the use of
ECF2-aa (SEQ ID NO:22) or modified forms thereof, such as
R.sub.n-ECF2-aa, in the preparation of calcitonin analogs such as
Elcatonin (SEQ ID NO:13). In this embodiment, the free
.alpha.-amino group of R.sub.n-ECF2-aa (SEQ ID NO:22) may first be
condensed using a non-enzymatic coupling reagent, in a manner
customary to the person skilled in the art, onto the free carboxyl
group of a peptide fragment such as ECF1 (SEQ ID NO:7) or a
protected ECF1 to produce an Elcatonin presursor, such as
Elcatonin-Ala. 9
[0105] "Elcatonin-Ala" (SEQ ID NO:31)
[0106] For example, the condensation used to produce the Elcatonin
precursor may be carried out with starting materials in which the
hydroxyl group of the Ser and Thr residues and the side chain amino
groups of the Lys residues are protected as described above. This
results in the formation of a side chain protected, amino acid
extended, non-amidated form of Elcatonin ("R.sub.n-Elcatonin-Ala
(SEQ ID NO:31)).
[0107] The condensation may be carried out, in a known manner, by
the carbodiimide or by the azide method. After the two fragment
have been chemically condensed, the product may be deprotected
using conventional techniques appropriate for the particular
protecting group, e.g., by hydrogenolysis, hydrolytically, by
acids, by reduction, or by hydrazinolysis as described (Houben
Weyl, Meth. der Org. Chemie, 15, 1-2 Thieme Verlag, Stuttgart,
1974).
[0108] IX. Conversion of Elcatonin-Ala to Elcatonin
[0109] The conversion of an amino acid extended Elcatonin precursor
to Elcatonin of the formula: 10
[0110] may be affected, in accordance with WO 92/05271, by reacting
a precursor polypeptide, Elcatonin-aa (SEQ ID NO:30) with a
nucleophilic compound (e.g., ONPGA) in the presence of a
carboxypeptidase to give a cleavable intermediate, which may then
be cleaved by photolysis to give Elcatonin (which exists as a
C-terminal .alpha.-carboxamide).
[0111] The abbreviations used herein for the amino acids are: Gly,
glycine; Lys, L-lysine; Leu, L-leucine; Ser, L-Serine; Gin,
L-glutamine; Glu, L-glutamic acid; His, L-histidine; Thr,
L-threonine; Tyr, L-tyrosine; Pro, L-proline; Arg, L-arginine; Asp,
L-aspartic acid; Val, L-valine; Ala, L-alanine; Met, L-methionine;
Asn, L-asparagine; Trp, L-tryptophan; Ile, L-isoleucine; and Phe,
L-phenylalanine.
EXAMPLES
[0112] 1.1 Preparation of the Plasmid pTBN26 Containing DNA
Encoding the hCA-ECF2-Ala Fusion Protein
[0113] A gene encoding amino acids 10-32 of ECF2 was constructed
using PCR methodology. The following five oligonucleotides were
synthesized at the University of Florida:
6 1. 5' GGA TCC AAG CTT GTT A 3' (SEQ ID NO:32) 2. 5' GTC GAC GAA
TTC GAT A 3' (SEQ ID NO:33) 3. 5' GGA TCC AAG CTT GTT AAC GGT AAA
CTG TCT CAG GAG CTC CAT AAA CTG 3' (SEQ ID NO:34) 4. 5' CTG ACG TTG
GTG CTG GTA CCC CGG CTT AAG ATA TCG AAT TCG TCG AC 3' (SEQ ID
NO:35) 5. 5' GGT ACC AGC ACC AAC GTC AGT ACG CGG GTA AGT CTG CAG
TTT ATG GAG CTC CTG AGA 3' (SEQ ID NO:36)
[0114] Oligonucleotides 2-5 -were combined in one PCR reaction
mixture, PCR MIX 1. The 3' end of oligonucleotide 3 is
complementary to the 3' end of oligonucleotide 5, while
oligonucleotide 2 is complementary to the 3' end of oligonucleotide
4. These four nucleotides annealed together as shown in FIG. 4A.
During PCR, Taq DNA polymerase was used to extend oligonucleotide 3
to the 5' end of oligonucleotide 5, oligonucleotide 5 was extended
to the 5' end of oligonucleotide 3, and oligonucleotide 2 was
extended to the 5' end of oligonucleotide 4, as indicated by the
dotted lines in diagram A.
[0115] A second PCR reaction was used to join the PCR products from
PCR MIX 1 resulting in a double stranded nucleotide fragment (SEQ
ID NO:10) containing the complete, non-interrupted Asn-ECF2-Ala
gene sequence as well as restriction sites to facilitate cloning.
The PCR extended products derived from oligonucleotides 2
(2.sup.Ext (SEQ ID NO:8) and 3.sup.Ext respectively (SEQ ID NO:9))
have 20 bp of complementary sequence at their 3' ends (see
schematic illustration in FIG. 4B). During the first few cycles of
the reaction with PCR MIX 2, oligonucleotides 2.sup.Ext (SEQ ID
NO:8) and 3.sup.Ext (SEQ ID NO:9) annealed where their sequences
were complementary and extended to create the double stranded
fragment including the full length non-interrupted gene sequence
for ECF2 (SEQ ID NO:10). Finally, oligonucleotides 1 and 2 were
used to amplify the full length gene sequence. FIGS. 11 and 12 show
the nucleic acid sequences for the complete, non-interrupted ECF2
gene sequence (SEQ ID NO:10) and for oligonucleotides 2.sup.Ext
(SEQ ID NO:8) and 3.sup.Ext (SEQ ID NO:9) respectively.
[0116] The amplified PCR produce was digested with Hpa I and EcoR
V. This blunt-ended DNA fragment, that codes for the C-terminal
amino acid (Asn) of the polypeptide fragment Linker as well as the
entire colypeptide fragment ECF2, was inserted into the pABN
plasmid at the unique EcoR V site. The plasmids were screened to
insure the Asn-ECF2 gene was inserted in the correct orientation.
The resultant plasmid was designated pABN26. Finally, pABN26 was
digested with Xba I and BspE I and the entire
Met-hCAII'-Met.sub.240-Val.sub.241-hCAII"-Linker-ECF2-aa sequence
was transferred to pTBN plasmid that had been digested with the
same enzymes to create production plasmid pTBN26 (FIG. 5).
[0117] The DNA sequence of the final construct with the Met
cyanogen bromide cleavage site underlined and the Asn-Gly (SEQ ID
NO:11) and corresponding amino acid sequence (SEQ ID NO:12)
cleavage site indicated by the arrow is shown in FIG. 6.
[0118] 1.2 Transformation of the Plasmid pTBN26 (Vector Containing
the hCA-ECF2-aa DNA Fragment)
[0119] The transformation was carried out in accordance with the
procedures described in WO 92/01707. The host microorganisms used
were E. coli HB 101 and E. coli BL21(DE3), both being described in
van Heeke et al., Protein Expression and Purification, 4, 265-275,
(1993).
[0120] 1.3 Selection of the Vectors Containing hCA-ECF2-Ala DNA
[0121] The selection was carried out using standard procedures such
as those described in WO 92/01707. The selection was based on the
introduction of tetracycline resistance into the host organism.
[0122] 2. Fermentation of E. coli Containing Plasmid pTBN26
[0123] 2.1 Growth of a Preculture for Inoculating the Fermentor
[0124] The E. coli strain BL21(DE3) containing plasmid pTBN26 was
grown in Luria broth (LB) medium containing tetracycline (15 mg/L)
and glucose solution (50 mg/L) in a shaking flask at 37.degree. C.
until an optical density at 550 nm (OD550) of about 4 had been
reached, about 14 hours. The composition of the Luria broth medium
(LB medium) is: 1.0 g/L Tryptone, 1.0 g/L NaCl and 0.5 /L yeast
extract.
[0125] 2.2 Fermentation
[0126] Fermentation was performed in a New Brunswick MPP-80
fermentor. The fermentation media, containing 300 g yeast extract,
30 g NaCl and 1200 g casamino acids dissolved in 15 L of H.sub.2O,
was added to the fermentor followed by 30 L of distilled water. The
fermentor was sterilized in place at 121.degree. C. for 25 minutes.
After the contents of the fermentor had cooled to 37.degree. C.,
the following sterile solutions were added sequentially: 480 g
glucose in 800 mL H.sub.2O, 120 g MgSO.sub.4.H.sub.2O in 250 mL
H.sub.2O, 495 g K.sub.2PO.sub.4 and 465 g KH.sub.2PO.sub.4 in 3.0 L
of H.sub.2O, and 0.9 g tetracycline hydrochloride in a mixture of
30 mL of 95% EtOH and 20 mL H.sub.2O. Also, a sterile mineral mix
containing 3.6 g FeSO.sub.4.7 H.sub.2O, 3.6 g CaCl.sub.2.2
H.sub.2O, 0.90 g MnSO.sub.4, 0.90 g AlCl.sub.3.6 H.sub.2O, 0.09 g
CuCl.sub.2 .2 H.sub.2O, 0.18 g molybdic acid, and 0.36 g
COCl.sub.2.6 H.sub.2O dissolved in 490.0 mL H.sub.2O and 10.0 mL
concentrated HCl was added.
[0127] Reagent grade NH.sub.4OH (28%) was attached to the automatic
pH feed pump of the fermentor and the pH was adjusted to 6.8. The
pH of the fermentation liquid was monitored continuously using a
calibrated electrode and maintained at pH 6.8 by the intermittent
addition of NH.sub.4OH. Additionally, dissolved oxygen was
monitored continuously using a calibrated oxygen monitor. Oxygen
concentration was maintained by adjusting the stirring rate.
Aeration was maintained at 40 L/min. When all systems were
operating normally, inoculation was performed by adding 600 mL of
inoculum by sterile procedures.
[0128] Turbidity, dissolved oxygen, and glucose levels were
monitored throughout fermentation. When the turbidity reached an
OD(550) of 15 to 20, the medium was supplemented with 300 g of
yeast extract and 1,200 g of casamino acids. When the agitation
rate reached 500 rpm, pure oxygen was supplemented into the air
feed at a rate of 5 L/min and the aeration rate was reduced to 20
L/min to maintain dissolved oxygen at 40%.
[0129] When the turbidity reached 30 OD.sub.550 units, the
fermentation medium was rendered 2 mM with respect to
isopropylthiolgalactoside to induce the expression of the plasmid.
In addition, enough ZnCl.sub.2 was added to render the final
concentration 100 .mu.M, and a sterile amino acid solution
containing 225 g of L-Ser and 75 g each of L-Tyr, L-Trp, L-Phe,
L-Pro, and L-His was added to the medium. Two hours after
induction, cell samples were removed for analysis. Dry weights and
SDS-PAGE protein analysis were performed on samples taken at
induction and every 30 minutes thereafter.
[0130] At the end of the fermentation, the medium was transferred
under sterile conditions to a sterile holding tank and cooled to
8.degree. C. The spent medium was separated from the cells by
crossflow filtration using a Millipore Pro Stack equipped with a
200 kD molecular weight cut-off membrane. The cells were
concentrated to 5 L, then either processed immediately or packaged
in 1 L aliquots in plastic freezer bags and stored at -20.degree.
C. for later processing.
[0131] Concentrated cells (5 L) were diluted in 32 L of lysis
buffer (50 mM Tris, 1 mM EDTA, 0.5% Triton-X100, pH 7.8, containing
0.05 mM phenylmethanesulfonyl fluoride) and then passed two times
through a Gaulin APV pilot scale homogenizer operated at 12,000 psi
at 4-15.degree. C. The solution was then made 1.3 .mu.M with
respect to lysozyme, incubated for 15 minutes (4-15.degree. C.)
then passed through a homogenizer a second time.
[0132] Approximately 50% of the soluble E. coli protein represents
hCA-ECF2-aa, assessed by measuring the enzymatic activity of the
human carbonic anhydrase portion of the fusion protein in
accordance with Verpoorte et al., J. Biol. Chem. 242, 4221-4229,
1967.
[0133] 2.3 Purification of the hCA-ECF2-Ala Fusion Protein
[0134] The lysate (32 L) obtained from Example 2.2 was diluted 1:1
with the lysis buffer without added phenylmetfanesulfonyl fluoride
and polyethylenimine was added to a final concentration of 0.35%.
The whole was stirred for 20 min. and then centrifuged at
10,000.times.g to remove precipitated DNA, RNA, nonessential
proteins, and membrane vesicles and then filtered using a Pall
Profile Filter (1.0 .mu.m).
[0135] The soluble fusion protein was subsequently purified by
affinity chromatography. The pH of the filtered protein solution
was adjusted to 8.7 by adding Tris base and loaded onto a 1 L
column of p-aminomethylbenzenesulphonamide affinity resin (van
Heeke et al., Methods. Molec. Biol., 36, 245-260, 1994) at a flow
rate of 200 mL/min. After loading, the column was washed with 5
column volumes of 0.1 M Tris-sulphate buffer pH 9.0, containing 0.2
M K.sub.2SO.sub.4 and 0.5 mM EDTA. The resin was then washed with 5
column volumes of 0.1 M Tris-sulphate buffer, pH 7.0, containing 1
M NaCl. The recombinantly produced hCA.about.ECF2-Ala fusion
protein was eluted from the affinity material with 5 column volumes
of 0.1 M Tris-sulphate buffer, pH 6.8, containing 0.4 mM potassium
thiocyanate and 0.5 mM EDTA. Fractions containing the
hCA.about.ECF2-Ala fusion protein were combined and treated with
acetic acid to pH 4.0 to precipitate the product, which was
subsequently collected by centrifugation. The resulting paste was
frozen and lyophilized The yield for this step was 85%.
[0136] 3. Cleavage of the hCA-ECF2-Ala Fusion Protein to Give
ECF2-Ala
[0137] 3.1 Cleavage to the hCA-ECF2-Ala Fusion Protein Using
Hydroxylamine
[0138] The fusion protein was cleaved with hydroxylamine between
Asn and Gly into an hCAII-containing fragment and the polypeptide
fragment ECF2-Ala (SEQ ID NO:23). Cleavage was achieved by
incubating 40 g of hCA-ECF2-Ala dissolved in 1 L of hydroxylamine
buffer (2 M hydroxylamine hydrochloride, 5 M guanidine
hydrochloride, 50 mM 3-(cyclohexylamino)
-2-hydroxy-1-propanesulphonic acid, adjusted to pH 10 with lithium
hydroxide) for 4 h. An aliquot was removed every hour, and the
extent to which the fusion protein has been cleaved to ECF2-Ala was
determined by HPLC analysis (C18 Vydac, 4.6.times.300 mm), buffer
A: 0.1% trifluoroacetic acid, 5% acetonitrile by volume, 95% by
volume water; buffer B: 0.1% trifluoroacetic acid, 5% by volume
water; 95% by volume acetonitrile; linear gradient of 5% buffer A
to 68% buffer B; flow rate: 1 mL/min). The reaction was then
diluted to 4 L with 15% acetic acid whereupon the resultant
precipitated hCAII-containing fragment was removed by
centrifugation at 10000.times.G.
[0139] The resulting supernatant from the centrifugation containing
ECF2-Ala (SEQ ID NO:23) was then desalted by loading onto a
preparative C8 column (5.times.5.1 cm) equilibrated with 10 mM
acetic acid in 5% acetonitrile at a flow rate of 50 mL/min.
Following loading, the column was washed with 10 mM acetic acid in
10% acetonitrile, and the ECF2-Ala eluted with 10 mM acetic acid in
45% acetonitrile. The fractions containing ECF2-Ala, identified by
analytical HPLC as above, were pooled and lyophilized. In all 1.14
g of ECF2-Ala (SEQ ID NO:23) was obtained, corresponding to a yield
of 82%.
[0140] 3.2 Purification of ECF2-Ala
[0141] For the further purification of ECF2-Ala (SEQ ID NO:23), a
semipreparative polysulphoethylaspartamide HPLC was used as
described in detail in Section 5.3 with a yield of 85%.
[0142] 4. Conversion of ECF2-Ala to ECF2-Amide
[0143] The amidation of ECF2-Ala (SEQ ID NO:23) was carried out
according to procedures described in WO 92/05271. ECF2-Ala (12.8
mg) was dissolved in 1 mL of 5 mM EDTA, 25 mM
morpholinoethane-sulfonic acid, pH 7.0, and to this was added 72 mg
of o-nitrophenylglycine amide (ONPGA). The pH was adjusted to 6.0
with 5 M NaOH and carboxypeptidase-Y (120 .mu.g) was added. After
stirring in the dark for about 48 h, acetonitrile (1 mL) was added
and the ECF2-ONPGA (SEQ ID NO:28) product purified by C18 reverse
phase HPLC as in section 5.2 and the product lyophilized. The
course of the amidation reaction was followed by extracting samples
for analysis after 10, 60, 120 and 180 min by analytical C18 HPLC
as in WO 92/05271.
[0144] For the subsequent photolysis, the freezed-dried ECF2-ONPGA
(12 mg) was dissolved in 5 mL 50% ethanol. To this solution was
added NaHSO.sub.3 (26 mg) and sodium benzoate (7.2 mg) and the pH
adjusted to 9.5 with 5 M NaOH. Nitrogen was then passed through the
reaction mixture for 15 min. The subsequent photolysis, analysis of
the course of the reaction, and identification of the resulting
ECF2-amide (SEQ ID NO:6), were carried out in accordance with the
procedures described in WO 92/05271.
[0145] 5. Cleavage of the hCA-ECF2-Ala Fusion Protein to Produce
the Minifusion Protein
[0146] 5.1 Chemical Cleavage of the hCA-ECF2-Ala Fusion Protein
Using Cyanogen Bromide
[0147] When chemical rather than enzymatic fragment coupling was
employed, the MFP (SEQ ID NO:24) was first cleaved from the fusion
protein (SEQ ID NO:12). To cleave the Met.sub.240-Val.sub.241
linkage (24 amino acids in the sequence prior to the beginning of
ECF2-Ala), 40 mg/mL of the hCA-ECF2-Ala fusion protein (SEQ ID
NO:12) was treated with 0.02 M cyanogen bromide in 70% formic acid
at room temperature for 6 h under an argon atmosphere in the dark.
Methionine (0.03 M) was then added to a final concentration of 0.03
M to terminate the cleavage reaction and the resulting solution was
stirred for 30 min. Twice the reaction volume of a solution
containing 10% acetic acid and 112 g/L of sodium sulphate was added
to the reaction mixture to precipitate the hCAII"-containing
fragment and the mixture was stirred for 20 min. The precipitated
material was removed by centrifugation (10 min at 10,000.times.G).
The minifusion protein (SEQ ID NO:24) remained soluble in the
supernatant. A 58% recovery of MFP (SEQ ID NQ:24) in relation to
hCA-ECF2-Ala fusion protein (SEQ ID N0:12, employed, was
obtained.
[0148] 5.2 Desalting the Minifusion Protein
[0149] The supernatant from the acid precipitation (4.5 g of mini
fusion protein) was loaded onto a C8 Vydac semi-preparative column
(22.times.250 mm), which had been equilibrated n 0.1%
trifluoracetic acid and 5% acetonitrile. The protein was eluted
using 0.1% trifluoracetic acid in 25% acetonitrile, and
subsequently lyophilized. In all, 4.05 g of 85% pure mini fusion
protein was obtained, corresponding to a yield of 90%.
[0150] 5.3 Purification of the Minifusion Protein Using
Polysulphoethylaspartamide HPLC
[0151] Polysulphoethylaspartamide chromatography was used for
purification of the minifusion protein (SEQ ID NO:24) , ECF2-Ala
(SEQ ID NO:23) and ECF2-amide (SEQ ID NO:6). The peptide was taken
up in buffer A (25 mM acetic acid, 35% acetonitrile) such that the
final concentration was 5 mg/mL and then loaded onto a
polysulphoethylaspartamide HPLC column (2.2.times.25 cm) at a flow
rate of 20 mL/min. It was subsequently possible to elute the
protein, over a period of 30 min, using a linear gradient of 10%
buffer B (25 mM acetic acid, 400 mM sodium acetate, 35%
acetonitrile) to 47% buffer B. Yields typically were 50% or
greater. FIG. 8 shows a representative HLPC trace of a sample
containing ECF2-amide (SEQ ID NO:6). The peak for ECF2-amide
appears between 14.5 and 16.3 min.
[0152] 5.4 Desalting Following Polysulphoethylaspartamide HPLC
[0153] Following lyophilization, the protein was loaded, for
desalting, onto a C8 column (5.times.20 cm, equilibrated with 95%
ethanol). The column was then washed with 4 column volumes of water
and with 4 column volumes of 5% ethanol containing 1% acetic acid.
The peptide was then eluted with 2 column volumes of 90% ethanol.
The fractions were analyzed by HPLC. Yields typically were 80% or
greater.
[0154] 6. Incorporation of Protective Groups into the Minifusion
Protein
[0155] General Strategy:
[0156] The .alpha.-amino group of the ECF2 fragment (SEQ ID NO:6)
within the recombinant MFP (SEQ ID NO:24) is biologically
protected, i.e. protected by the presence of the N-terminal hCAII"
fragment. The .epsilon.-amino groups of the Lys groups in MFP were
protected with acyl donors, such as Z, BOC, ADOC or FMOC. The
reaction of the arginine-guanido function with Z-OSU was prevented
by salt formation with HCl. The reaction of the His nitrogen with
Z-OSU was suppressed by adding agents such as
N-hydroxysuccinimide.
Experimental Procedure
[0157] MFP (SEQ ID NO:24) (270 mg) was dissolved in water (20 mL),
and dioxane (4 mL) and 680 .mu.L of 0.1 N HCl (to ensure salt
formation at the two arginine-guanidino functions) was added. Prior
to reaction, 78 mg of N-hydroxysuccinimide and 104.7 .mu.L of
triethylamine were added to protect the His nitrogen, and either
169 mg of Z-OSU, 146 mg of BOC-OSU, 135 mg of ADOC fluoride or 229
mg of FMOC-OSU were then added to the whole, with cooling.
[0158] The reaction mixture was then stirred at room temperature
for 24 h and subsequently dried in vacuo. The resulting residue was
carefully triturated with dichloromethane (twice) and then with
acetonitrile (twice). The product was collected by filtration and
dried in vacuo. The yields of the Z protected mini fusion protein
was 300 mg and of BOC-protected protein 295 mg, and that of
ADOC-protected and FMOC-protected protein was 310 mg.
[0159] Checking for complete reaction of mini fusion protein with
the respective protective groups was carried out by means of thin
layer chromatography using protected and unprotected peptides.
Complete reaction was verified on thin layer silica plates (eluent:
phenol:H2O, 775:225). The yield for the Z-OSU reaction when 540 mg
of MFP (SEQ ID NO:24) was used was 75% and was typical of that
found for the other blocking agents.
[0160] 7. Cleavage of a Protected Mini Fusion Protein to Produce a
Protected ECF2-Ala Fragment
[0161] 7.1 Cleavage Using Hydroxylamine
[0162] In order to liberate the .alpha.-amino group which was
biologically protected by amino acid sequence, the protected mini
fusion protein was cleaved with hydroxylamine between Asn and Gly
into a Z-protected ECF2-Ala fragment (SEQ ID NO:23) and the 24
amino acid-long Trp-containing fragment. The composition of the
hydroxylamine buffer was the same as in Section 3.1.
[0163] Hydroxylamine buffer (26 mL) was added to 260 mg of
Z-protected minifusion protein (SEQ ID NO:24), and the whole was
then ultrasonicated (20 sec) and the pH adjusted to pH 10.0 with 4
M lithium hydroxide. The resulting mixture was incubated at
30.degree. C. and pH 10.0 for up to 5 h. The pH was then adjusted
to 6.0 using concentrated acetic acid.
[0164] An aliquot was removed every hour, and the extent to which
the Z-protected minifusion protein (SEQ ID NO:24) has been cleaved
to the Z-protected ECF2-Ala fragment (SEQ ID NO:23) was determined
by HPLC analysis (C18 Vydac column, 5.times.300 mm) , buffer A:
0.1% trifluoroacetic acid; buffer B 0.1% trifluoroacetic acid, 5%
by volume H2O, 95% acetonitrile; eluent: linear gradient of 5%
buffer A to 68% buffer B; 1 mL/min). Termination of the
hydroxylamine cleavage was as in 3.1.
[0165] Fractions obtained from the C8 column were analyzed by HPLC
using a Vydac C18 column. The buffers for the HPLC were those
previously described. The flow rate was 1 mL/min. The proteins were
eluted by a linear gradient from 41% buffer A to 71% buffer B.
Detection was at 210 nm. In all, 156 mg of Z-protected ECF2-Ala
("Z-ECF2-Ala" (SEQ ID NO:23)) were obtained, corresponding to a
yield of 60%.
[0166] 7.2 Purification of Z-ECF2-Ala
[0167] Z-ECF2-Ala (SEQ ID NO:23) was purified by use of a
semi-preparative C8 HPLC. Z-ECF2-Ala (160 mg) was dissolved in 5 M
acetic acid (5 mL), and buffer A (100 mM acetic acid, 5%
acetonitrile ( 5 mL) was subsequently added. The sample was then
loaded onto a semi-preparative HPLC C8 column (details as in 3.2.).
Following the hydroxylamine cleavage and the purification, 120 mg
of Z-ECF2-Ala (SEQ ID NO:23) was obtained, corresponding to a yield
of 75% for the C8 purification step.
[0168] For the further purification of Z-ECF2-Ala (SEQ ID NO:23), a
semi-preparative polysulphoethylaspartamide HPLC was used as
described in Section 5.3 below. Using this method, 59 mg of 95-98%
pure Z-ECF2-Ala was obtained, corresponding to a yield of
85.5%.
[0169] 8. Condensation of ECF2-amide and ECF1-OMe
[0170] Amidated ECF2 (SEQ ID NO:6) produced according to Example 4
and the cyclic Elcatonin-fragment ECF1 (SEQ ID NO:3) may be coupled
in the presence of a non-enzymatic coupling reagent. For example,
dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (HOSu) may
be added to a solution of ECF1 (SEQ ID NO:3) having the side
hydroxyl groups of Ser and Thr protected as a benzyl ether and
ECF2-amide (SEQ ID NO:2) having the reactive side chain groups of
Lys, Ser, Thr and/or Glu residues protected. The reaction is
stirred at about 0.degree. C. for 8 hours. Fragment condensation
during this time was followed by HPLC analysis. After termination
of the reaction, the mixture may be diluted by adding 1%
trifluoracetic acid. The product Elcatonin (SEQ ID NO:13) may be
purified by preparative HPLC. Unreacted ECF2-amide (SEQ ID NO:2)
may be isolated and purified by HPLC and recycled.
[0171] 9. Chemical Coupling of Z-ECF2 to ECF1
[0172] The coupling of the free .alpha.-amino group of the
Z-ECF2-Ala fragment (i.e., EFC2-Ala having the side chain amino
groups protected with a carbobenzyloxy group) to the free
C-terminal .alpha.-carboxyl group of the peptide fragment ECF1 may
carried out either by the carbodiimide or the azide methods (see,
e.g., Greenstein et al., in Chemistry of the Amino Acids, Vol. 2,
John Wiley, New York, pp 804ff, 1016ff, (1961)). The coupling
reaction is typically carried out by adding the Z-ECF2-Ala fragment
to an activated ester formed from the C-terminal .alpha.-carboxylic
acid of the ECF1 fragment. Removal of the Cbz protecting groups
from the coupling product may be carried out via hydrogenolysis and
the resulting product purified by preparative HPLC to yield
Elcatonin-Ala (i.e., "ECF1-ECF2-Ala"; SEQ ID NO:31)
[0173] 10. Conversion of Elcatonin-Ala to Elcatonin-Amide
[0174] The Elcatonin-Ala (SEQ ID NO:31) peptide produced according
to Section 9 may be amidated using the procedure described in
Section 4. The amidated Elcatonin (SEQ ID NO:13) may be purified
and subsequently desalted using the procedures described in
Sections 5.3 and 5.4.
[0175] The invention has been described with reference to various
specific and preferred embodiments and techniques. However, it
should be understood that many variations and modifications may be
made while remaining within the spirit and scope of the
invention.
[0176] The publications referred to in this specification are
indicative of the level of ordinary skill in the art to which this
invention pertains and are herein incorporated by reference to the
same extent as if each individual publication was specifically and
individually indicated by reference.
Sequence CWU 1
1
* * * * *