Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Insulin; related peptides
Reexamination Certificate
2000-12-14
2003-02-18
Mosher, Mary E. (Department: 1647)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Insulin; related peptides
C435S069100, C435S069700, C435S320100, C435S325000, C435S252300, C435S254200, C435S254210, C536S023400, C536S023500
Reexamination Certificate
active
06521738
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.
Circular Dichroism (CD) is used to determine protein stability and relative stabilities of molecules. CD observed below 240 nm is due to the peptide amide chromophore and may be used to estimate protein secondary structure (Johnson (1988) Ann. Rev. Biophys.Chem. 17:145-166). The spectrum of insulin is characterized by minima at 220 and 209 nm, a negative to positive crossover near 203 nm, and a maximum at 195 nm. Upon denaturation, the negative CD in the 240-218-nm range gradually diminishes, consistent with the loss of ordered secondary structure that accompanies protein unfolding. Consequently, the folding stability of an insulin precursor may be quantitated by measuring the loss of secondary structure as a function of added denaturant, e.g., guanidinium hydrochloride (GuHCl) (see e.g., Pace (1975) CRC Crit. Rev. Biochem. 3:1-43).
SUMMARY OF THE INVENTION
The present invention features novel connecting peptides (C-peptides) which confer an increased production yield and/or increased stability in insulin precursor molecules and insulin precursor analog molecules when expressed in a transformed microorganism, in particular yeast. Such insulin precursors or insulin precursor analogs can then be converted into insulin or an insulin analog by one or more suitable, well known conversion steps.
The connecting peptides of the present invention contain at least one aromatic amino acid residue Phe, Trp, or Tyr 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 9 amino acid residues. Typically the novel connecting peptides will be of up to 7 or up to 5 amino acid residues and preferably not more than 4 amino acid residues.
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 aromatic amino acid residue but preferably not more than 5. The aromatic amino acid residues may be the same or different. The connecting peptide will preferably not comprise more than 3 aromatic amino acid residues and most preferred it will only comprise a single aromatic amino acid residue.
In one embodiment of the present invention one of the aromatic amino acid residues in the connecting peptide is immediately N-terminal to the cleavage site adjacent to the A chain. Furthermore, one of the aromatic amino acid residues will preferably be positioned less than 5 Å away from at least one of the residues in positions B11, B12 or B26 in the B chain. In one embodiment, the aromatic amino acid immediately N-terminal to the cleavage site adjacent to the A chain is less than 5 Å away from at least one of the residues in positions B11, B12 or B26 in the B chain.
The insulin precursors or insulin precursor analogs are characterized by having a high folding stability in solution. The precursors according to the present invention will have an increased Cmid stability compared to insulin or insulin analogs, which do not comprise an aromatic amino acid residue in the connecting peptide. The Cmid stability is thus higher than about 5.5 M GuHCl, typically higher than about 6.0 M GuHCl and more typically higher than about 6.5 M GuHCl.
Accordingly, in one aspect the present invention relates to insulin precursors or insulin precursor analogs comprising a connecting peptide (C-peptide) being cleavable from the A and B chains and 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.
In another aspect the present invention relates to insulin precursors or insulin precursor analogs comprising a connecting peptide (C-peptide) being cleavable from the A and B chains and consisting of up to 9 amino acid residues of which at least one is an aromatic amino acid residue.
In still a further aspect the present invention relates to an insulin precursor or an insulin precursor analog comprising a connecting peptide (C-peptide) being cleavable from the A and B chains, wherein the connecting peptide contains one aromatic amino acid residue which is less than 5 Å away from at least one of the residues in positions B11, B12 or B26 in the B chain.
In still a further aspect the present invention is related to insulin precursors or insulin precursor an
Kaarsholm Niels C.
Kjeldsen Thomas Borglum
Ludvigsen Svend
Green Reza
Mosher Mary E.
Novo Nordisk A S
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