Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-06-28
2004-08-17
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C530S303000, C435S252300, C435S254200, C435S254210, C435S320100, C435S325000, C536S023400, C536S023500
Reexamination Certificate
active
06777207
ABSTRACT:
BACKGROUND
Yeast organisms produce a number of proteins that have a function outside the cell. Such proteins are referred to as secreted proteins. These secreted proteins are expressed initially inside the cell in a precursor or a pre-form containing a pre-peptide sequence ensuring effective direction (translocation) of the expressed product across the membrane of the endoplasmic reticulum (ER). The pre-peptide, normally named a signal peptide, is generally cleaved off from the desired product during translocation. Once entered in the secretory pathway, the protein is transported to the Golgi apparatus. From the Golgi, the protein can follow different routes that lead to compartments such as the cell vacuole or the cell membrane, or it can be routed out of the cell to be secreted to the external medium (Pfeffer et al. (1987) Ann. Rev. Biochem. 56:829-852).
Insulin is a polypeptide hormone secreted by &bgr;-cells of the pancreas and consists of two polypeptide chains, A and B, which are linked by two inter-chain disulphide bridges. Furthermore, the A-chain features one intra-chain disulphide bridge.
The hormone is synthesized as a single-chain precursor proinsulin (preproinsulin) consisting of a prepeptide of 24 amino acid followed by proinsulin containing 86 amino acids, in the configuration: prepeptide-B-Arg Arg-C-Lys Arg-A, in which C is a connecting peptide of 31 amino acids. Arg-Arg and Lys-Arg are cleavage sites for cleavage of the connecting peptide from the A and B chains.
Three major methods have been used for the production of human insulin in microorganisms. Two involve
Escherichia coli
, with either the expression of a large fusion protein in the cytoplasm (Frank et al. (1981) in Peptides: Proceedings of the 7
th
American Peptide Chemistry Symposium (Rich & Gross, eds.), Pierce Chemical Co., Rockford, Ill. pp 729-739), or use a signal peptide to enable secretion into the periplasmic space (Chan et al. (1981) PNAS 78:5401-5404). A third method utilizes
Saccharomyces cerevisiae
to secrete an insulin precursor into the medium (Thim et al. (1986) PNAS 83:6766-6770). The prior art discloses a limited number of insulin precursors which are expressed in either
E. coli
or
Saccharomyces cerevisiae,
vide U.S. Pat. No. 5,962,267, WO 95/16708, EP 0055945, EP 0163529, EP 0347845 and EP 0741188.
SUMMARY OF THE INVENTION
The present invention features novel connecting peptides (mini C-peptides) which confer an increased production yield in insulin precursor molecules and insulin precursor analog molecules when expressed in a transformed microorganism, in particular in yeast. Such insulin precursors or insulin precursor analogs can then be converted into insulin or insulin analogs by one or more suitable, well known conversion steps.
The connecting peptides of the present invention contain at least one Gly and will generally be shorter than the natural human C peptide which, including the flanking dibasic cleavage sites, consists of 35 amino acids. Thus the novel connecting peptides will in general not be of more than 15 amino acid residues in length and preferably not more than 10 amino acid residues. Typically the novel connecting peptides will be of up to 9, 8, 7 or 5 amino acid residues and will preferably not be of more than 4 amino acid residues in length.
As in the natural human insulin molecule, the connecting peptide will contain a cleavage site at its C and N termini enabling in vitro cleavage of the connecting peptide from the A and B chains. Such cleavage sites may be any convenient cleavage sites known in the art, e.g. a Met cleavable by cyanogen bromide; a single basic amino acid residue or a pair of basic amino acid residues (Lys or Arg) cleavable by trypsin or trypsin like proteases;
Acromobactor lyticus
protease or by a carboxypeptidase protease. The cleavage site enabling cleavage of the connecting peptide from the A-chain is preferably a single basic amino acid residue Lys or Arg, preferably Lys.
Alternatively cleavage of the connecting peptide from the B chain may be enabled by cleavage at the natural Lys
B29
amino acid residue in the B chain giving rise to a desB30 insulin precursor or desB30 insulin precursor analog. The desired B30 amino acid residue may then be added by well known in vitro, enzymatic procedures.
In one embodiment the connecting peptide will not contain two adjacent basic amino acid residues (Lys,Arg). In this embodiment, cleavage from the A-chain may be accomplished at a single Lys or Arg located at the N-terminal end of the A-chain and the natural Lys in position B29 in the B-chain.
The connecting peptide may comprise more than one Gly but preferably not more than 5. The connecting peptide will preferably not comprise more than 3 Gly and most preferred it will only comprise a single Gly. The Gly may be immediately N-terminal to the cleavage site adjacent to the A chain.
Furthermore, the B27 (atom CG2) will typically have a proximity to the A1 (atom CA) of less than 5 Å.
Accordingly, in one aspect the invention is related to insulin precursors or insulin precursor analogs comprising a connecting peptide (C-peptide) being cleavable from the A and B chains said connecting peptide comprising at least one Gly, wherein the B27 (atom CG2) has a proximity to the A1 (atom CA) of less than 5 Å.
In another aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising a connecting peptide (C-peptide) being cleavable from the A and B chains said connecting peptide comprising at least one Gly and a cleavage site enabling cleavage of the peptide bond between the A-chain and the connecting peptide, wherein one Gly is immediately N-terminal to said cleavage site.
In another aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising a connecting peptide (C-peptide) being cleavable from the A and B chains said connecting peptide comprising at least one Gly, wherein the connecting peptide is of up to 6 amino acid residues in length.
In a further aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising a sequence of formula:
B(1-27)-X
3
-X
2
-X
1
-Y-A(1-21),
wherein
X
1
comprises 1-5 amino acid residues in length comprising at least one Gly,
X
2
is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain,
X
3
is one of Pro, Asp, Lys, or Ile at position 28 of the B chain, and
Y is Lys or Arg.
In one embodiment X
1
is 1-4, 1-3 or 1-2 amino acid residues in length.
In a further aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising a sequence of formula:
B(1-27)—X
3
—X
2
—X
1
—Y—A(1-21),
wherein
X
1
comprises a Gly immediately N-terminal to Y,
X
2
is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain,
X
3
is one of Pro, Asp, Lys, or Ile at position 28 of the B chain, and
Y is Lys or Arg.
In one embodiment, X
1
is 1-15, 1-10, 1-8, 1-5 or 1-3 amino acid residues in length.
In a further aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising a sequence of formula:
B(1-27)—X
3
—X
2
—X
1
—Y—A(1-21),
wherein
X
1
comprises at least one Gly,
X
2
is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain,
X
3
is one of Pro, Asp, Lys, or Ile at position 28 of the B chain, and
Y is Lys or Arg,
and wherein the B27 (atom CG2) has a proximity to the A1 (atom CA) of less than 5 Å.
In this embodiment X
1
is typically 1-15, 1-10, 1-9, 1-8, 1-5, 1-4 or 1-3 amino length.
In the above formula X
1
will typically contain 1-5 Gly, preferably 1-3 and more preferred only one Gly molecule.
The amino acid residues in X
1
can be any codable amino acid residue and may be the same or different with the only proviso that at least one amino acid residue in X
1
is Gly.
In one embodiment, X
3
is Asp and X
2
is Lys. This embodiment encompasses the insulin precursor analogs containing an Asp in position B28 of the B chain (termed hereinafter “Asp
B28
IP”). In another embodiment X
2
is Lys and X
Kjeldsen Thomas Børglum
Ludvigsen Svend
Began, Esq. Marc A.
Bork, Esq. Richard W.
Green, Esq. Reza
Novo Nordisk A S
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