Recombinant preparation of calcitonin fragments and use...

Details Recombinant preparation of calcitonin fragments and use... Recombinant preparation of calcitonin fragments and use...

2001-01-02

2002-06-25

Achutamurthy, Ponnathapu (Department: 1652)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S252300, C435S320100, C435S069100, C435S069700, C536S023100, C536S023400, C530S300000, C530S307000, C530S325000, C530S350000

Reexamination Certificate

active

06410707

ABSTRACT:

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).
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 In contrast to eel calcitonin, Elcatonin lacks an amino terminal end and the disulfide bridge of eel calcitonin has been replaced by a —(CH
2
)
5
— “carbon bridge.”
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.
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
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.
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.
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:
A
10
-A
11
-A
12
-A
13
-A
14
-A
15
-A
16
-A
17
-A
18
-A
19
-A
20
-A
21
-A
22
-A
23
-A
24
-A
25
-A
26
-A
27
-Gly-A
29
-A
30
-A
31
-Pro-Xxx   (SEQ ID NO:1)
wherein A
10
is Gly or Ser, A
11
is Lys, Thr or Ala, A
12
is Leu or Tyr, A
13
is Ser, Thr or Trp, A
14
is Gln, Lys or Arg, A
15
is Glu, Asp or Asn, A
16
is Leu or Phe, A
17
is His or Asn, A
18
is Lys or Asn, A
19
is Leu, Tyr or Phe, A
20
is Gln or His, A
21
is Thr or Arg, A
22
is Tyr or Phe, A
23
is Pro or Ser, A
24
is Arg, Gly or Gln, A
25
is Thr or Met, A
26
is Asp, Ala, Gly, or Asn, A
27
is Val, Leu, Ile, Phe, or Thr, A
29
is Ala, Val, Pro or Ser, A
30
is Gly, Val or Glu, A
31
is Thr, Val or Ala. The C-terminal -Xxx group is typically a C-terminal carboxylic acid (“—OH”), a C-terminal carboxamide (“—NH
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
10
to A
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.
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:
wherein A
2
is Gly, Ser or Ala; A
3
is Asn or Ser; A
8
is Val or Met; R
2
is —(CH
2
)
4
— or —CH(NH
2
)CH
2
S—S—; and Y is OH, OR
1
, where —R
1
is a lower alkyl group;
with a recombinantly-formed polypeptide of the formula:
A
10
-A
11
-A
12
-A
13
-A
14
-A
15
-A
16
-A
17
-A
18
-A
19
-A
20
-A
21
-A
22
-A
23
-A
24
-A
25
-A
26
-A
27
-Gly-A
29
-A
30
-A
31
-Pro-Xxx   (SEQ ID NO:1)
wherein a A
10
is Gly or Ser, A
11
is wherein A
10
is Gly or Ser, A
11
is Lys, Thr or Ala, A
12
is Leu or Tyr, A
13
is Ser, Thr or Trp, A
14
is Gln, Lys or Arg, A
15
is Glu, Asp or Asn, Al
16
is Leu or Phe, A
17
is His or Asn, A
18
is Lys or Asn, A
19
is Leu, Tyr or Phe, A
20
is Gln or His, A
21
is Thr or Arg, A
22
is Tyr or Phe, A
23
is Pro or Ser, A
24
is Arg, Gly or Gln, A
25
is Thr or Met, A
26
is Asp, Ala, Gly, or Asn, A
27
is Val, Leu, Ile, Phe, or Thr, A
29
is Ala, Val, Pro or Ser, A
30
is Gly, Val or Glu, A
31
is Thr, Val or Ala, and -Xxx is —OH, —NH
2
, an amino acid residue or a polypeptide group;
in the presence of a non-enzymatic coupling reagent to form a ca

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