Recombinant preparation of calcitonin fragments and use thereof in the preparation of calcitonin and related analogs

#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 In contrast to eel calcitonin, Elcatonin lacks an amino terminal end and the disulfide bridge of eel calcitonin has been replaced by a --(CH.sub.2).sub.5 -- "carbon bridge."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. No. 4,086,221 and U.S. Pat. No. 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.

#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.

#FIG. 2 shows a map of plasmid pAEN. Plasmid pABN includes a nucleic acid sequence(SEQ ID NO:47 and SEQ ID NO:48) coding for a Linker peptide fragment(SEQ ID NO:49) inserted into the plasmid near the carboxy terminal end of the gene sequence of human carbonic anhydrase II ("hCAII").

#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.

#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").

#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.

#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)).