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AssigneeNovo Nordisk A/S
InventorsThomas Borglum Kjeldsen, Svend Ludvigsen, Niels C. Kaarsholm
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Novel insulin precursors and insulin precursor analogs having a mini C-peptide comprising at least one aromatic amino acid residue have an increased folding stability. The novel insulin precursors and insulin precursor analogs can be expressed in yeast in high yields and are preferably not more 15 amino acid residues in length. Also provided are polynucleotide sequences encoding the claimed precursors or precursor analogs, and vectors and cell lines containing such polynucleotide sequences.
An insulin precursor or insulin precursor analog comprising a connecting peptide (C-peptide) being cleavable from the A and B chains said connecting peptide comprising at least one aromatic amino acid residue and a cleavage site enabling cleavage of the peptide bond between the A-chain and the connecting peptide, wherein one aromatic amino acid residue is immediately N-terminal to said cleavage site.
An insulin precursor or insulin precursor analog comprising a connecting peptide (C-peptide) being cleavable from the A and B chains and being of up to 9 amino acid residues in length, wherein the C-peptide comprises at least one aromatic amino acid residue.
An insulin precursor or insulin precursor analog comprising a connecting peptide (C-peptide) being cleavable from the A and B chains, said connecting peptide comprising one aromatic amino acid residue which is less than 5 Å away from at least one of the residues in position B11, B12, or B26 in the B-chain.
An insulin precursor or an insulin precursor analog comprising the formula:
An insulin precursor or an insulin precursor analog comprising the formula:
An insulin precursor or an insulin precursor analog comprising the formula:
An insulin precursor or insulin precursor analog of claim 75, wherein one aromatic amino acid residue is immediately N-terminal to Y.
Dependencies are inferred from the claim text.
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Hormones: insulins
Peptides
Term end not estimated
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 O-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.
FIG. 1 represents the pAK721 S. cerevisiae expression plasmid expressing the LA19 leader-EEAEAEAEPK(SEQ ID NO:3)-IP(AlaAlaLys) fusion protein.
FIG. 2 is the DNA sequence and inferred amino acid sequence of the encoded fusion protein (α-factor-leader-EEAEAEAPK(SEQ ID NO:4)-AspB28IP portion of pAK1150 (SEQ ID NO: 5 and 6) used as PCR template.
FIG. 3 is the DNA sequence encoding α-factor leader-AspB28IP(GluTrpLys) fusion protein with a synthetic mini C-peptide GluTrpLys generated by randomized optimization (SEQ ID NO:7 and 8). The mini C-peptide (EWK) is indicated by underlining.
Abbreviations and Nomenclature.
By “connecting peptide” or “C-peptide” is meant the connection moiety “C” of the B-C-A polypeptide sequence of a single chain preproinsulin-like molecule. Specifically, in the natural insulin chain, the C-peptide connects position 30 of the B chain and position 1 of the A chain. A “mini C-peptide” or “connecting peptide” such as those described herein, connect B29 or B30 to A1, and differ in sequence and length from that of the natural C-peptide.
By “IP” is meant a single-chain insulin precursor in which a desB30 chain is linked to the A chain of insulin via a connecting peptide. The single-chain insulin precursor will contain correctly positioned disulphide bridges (three) as in human insulin.