METHOD FOR MAKING INSULIN PRECURSORS AND INSULIN PRECURSOR ANALOGS

Abstract: 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.

Description: METHOD FOR MAKING INSULIN PRECURSORS AND INSULIN PRECURSOR ANALOGS 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 β-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 icroor- ganisms. 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, IL. 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 insu- lin 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 US 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 or- dered 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 (GuHCI) (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; Acro- mobactor 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 A 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 A away from at least one of the residues in posi- tions BH , 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 GuHCI, typically higher than about 6.0 M GuHCI and more typically higher than about 6.5 M GuHCI. 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 A 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 analogs comprising a connecting peptide (C-peptide) comprising at least one aromatic amino acid residue and being cleavable from the A and B chains Said insulin precursors or insulin precursor analogs having an increased Cmid stability relative to insulin pre- cursor or insulin precursor analogs which do not comprise an aromatic amino acid residue a the connecting peptide. The increased activity is determined by a variety of methods known to one of skill in the art, and described below. In one embodiment, increased stability is measured by CD determination of the concentration of guanidine hydrochloride (GuHCI) needed to achieve half- maximum unfolding of an insulin precursor molecule (Cmid). In a further aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising the formula: B(1-27) - X 2 - X 3 - Xi - Y - A(1-21) wherein X T is a peptide sequence of 1 - 15 amino acid residues comprising one aromatic ami- no acid residue immediately N-terminal to Y, X 2 is one of Pro, Asp, Lys, or lie at position 28 of the B chain, X 3 is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain, and Y is Lys or Arg. In one embodiment, the total number of amino acid residues in X^ will be from 1 - 10, 1-9, 1-8, 1-7, 1-6, 1-5 or 1-4 amino acid residues in length. In another specific embodiment Xi is 1-3 amino acid residues and preferably 1-2 amino acid residues. The amino acid residues in Xi can be any codable amino acid residue and may be the same or different with the only proviso that one is an aromatic amino acid residue immediately N-terminal to Y. In a further aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising the formula: B(1-27) - X 2 - X 3 - Xi - Y - A(1-21) wherein X is a peptide sequence of 1 - 15 amino acid residues of which one is an aromatic amino acid residue which is less than 5 A away from at least one of the amino acid residues in position B11 , B12 or B26 in the B chain, X 2 is one of Pro, Asp, Lys, or lie at position 28 of the B chain, X 3 is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain, and Y is Lys or Arg. In one embodiment the number of amino acid residues in Xi is 1-9, 1-5 or 1-4. In another embodiment the number of amino acid residues is 1-3 or 1-2. In another aspect, the present invention is related to insulin precursors or insulin precursor analogs comprising the formula: B(1-27) - X 2 - X 3 - X 1 - Y - A(1-21) wherein Xi is a peptide sequence of 1 - 8 amino acid residues of which at least one is an aromatic amino acid residue, X 2 is one of Pro, Asp, Lys, or lie at position 28 of the B chain, X 3 is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain, and Y is Lys or Arg. The total number of amino acid residues in X-i will be from 1-7, 1-6, 1-5 or 1-4 amino acid residues. In a more specific embodiment X-i is 1-3 amino acid residues and preferably 1- 2 amino acid residues. The amino acid residues in Xi 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 Xi is an aromatic amino acid residue. In the above formulas Xi may comprise up to 5 aromatic amino acid residues which may be the same or different. In a specific embodiment, Xi comprises up to 3 aromatic amino acid residues which may be the same or different and Xi will preferably contain only one aromatic amino acid residue. The aromatic amino acid residues are Trp, Phe or Tyr, preferably Phe or Trp. In one embodiment, X 2 is Asp and X 3 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 3 is Pro. In a further embodiment the se- quence X r Y is selected from the group of: (a) Met-Trp-Lys; (b) Ala-Trp-Lys; (c) Val-Trp-Lys; (d) lle-Trp-Lys; (e) Leu-Trp-Lys; (f) Glu-Glu- Phe-Lys (SEQ ID NO: 15); (g) Glu-Phe-Lys; (h) Glu-Trp-Lys; (i) Ser-Trp-Lys; (j) Thr-Trp-Lys; (k) Arg-Trp-Lys; (I) Glu-Met-Trp-Lys (SEQ ID NO:1); (m) Gln-Met-Trp-Lys (SEQ ID NO:2); and (n) Asp-Trp-Lys. In another embodiment X 2 is Pro, X 3 is Lys and Xi is 1-2 amino acid residues of which one is Trp or Phe. In another embodiment X 2 is Lys, X 3 is Pro-Thr and Xi consists of up to 15 amino acid residues of which one is Trp, Tyr or Phe. In this embodiment X-i will contain a cleavage site at the C-terminal end, e.g a mono basic or dibasic (Lys, Arg) cleavage site. The present invention is also related to polynucleotide sequences which code for the claimed insulin precursors or insulin precursor analogs. In a further aspect the present invention is related to vectors containing such polynucleotide sequences and host cell containing such polynucleotide sequences or vectors. In another aspect, the invention relates to a process for producing the insulin precursors or insulin precursor analogs in a host cell, said method comprising (i) culturing a host cell comprising a polynucleotide sequence encoding the insulin precursors or insulin precursor analogs of the invention under suitable conditions for expression of said precursor or precursor analog; and (ii) isolating the precursor or precursor analog from the culture me- dium. In still a further aspect, the invention relates to a process for producing insulin or insulin analogs in a host cell said method comprising (i) culturing a host cell comprising a polynucleotide sequence encoding an insulin precursor or insulin precursor analogs of the invention; (ii) isolating the precursor or precursor analog from the culture medium and (iii) convert- ing the precursor or precursor analog into insulin or an insulin analog by in vitro enzymatic conversion. In one embodiment of the present invention the host cell is a yeast host cell and in a further embodiment the yeast host cell is selected from the genus Saccharomyces. In a further embodiment the yeast host cell is selected from the species Saccharomyces cerevisiae. BRIEF DESCRIPTION OF THE DRAWINGS 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)-Asp B28 IP portion of pAK1150 (SEQ ID NO: 5 and 6) used as PCR template. Fig. 3 is the DNA sequence encoding α-factor leader-Asp B28 IP(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. Fig. 4 shows folding stability of the insulin analog Asp B28 IP(MetTrpLys) relative to Asp B28 IP. Fig. 5 shows the solution structures of Asp B28 IP(MetTrpLys) as backbone lines of ensemble of 20 converged structures. Fig. 6 shows a ribbon presentation of Asp B28 IP(MetTrpLys). The figure is produced us- ing MOLSCRIPT (Kraulis (1991) J. Appl. Crystallog. 24:946-950). Amino acid residue anno- tations are derived as follows: B1-B29 (B chain) are numbered 1-29, residues C1-C3 (connecting peptide) are numbered 30-32, and residues A1-A21 (A chain) are numbered 33-53. Fig. 7 is the ID proton NMR spectrum for Asp B28 IP(MetTrpLys) recorded at 27°C at 1.0 mM concentration in 10%/90% D 2 0/H 2 0 with 10 mM phosphate buffer at pH 8.0. Fig. 8 is the DNA and inferred amino acid sequence of the expression cassette expressing the YAP3-TA39-EEGEPK(SEQ ID NO:17)-Asp B28 IP fusion protein with a synthetic mini C-peptide GluTrpLys (SEQ ID NO:9 and 10) and Fig. 9 is the DNA and inferred amino acid sequence of the expression cassette expressing plasmid expressing the YAP3-TA57-EEGEPK(SEQ ID NO:17)-Asp B28 IP fusion pro- tein with a synthetic mini C-peptide GluTrpLys (SEQ ID NO:11 and 12). DETAILED DESCRIPTION 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. With "desB30" or "B(1-29)" is meant a natural insulin B chain lacking the B30 amino acid residue, "A(1-21)" means the natural insulin A chain, "B(1-27)" means the natural B chain lacking the B28, B29, and B30 amino acid residues; "Asp B28 IP" means a single-chain insulin precursor with aspartic acid at position 28 of the B-chain and no C-peptide (B29 is linked to A1). The mini C-peptide and its amino acid sequence is indicated in the three letter amino acid code in parenthesis following the IP; Thus "Asp B28 IP(MetTrpLys)" means a single- chain insulin precursor with aspartic acid at position 28 of the B-chain and a mini C-peptide with the sequence Met-Trp-Lys connecting B29 to A1. By "insulin precursor" is meant a single-chain polypeptide which by one or more subsequent chemical and/or enzymatic processes can be converted into human insulin. By "insulin precursor analog" is meant an insulin precursor molecule having one or more mutations, substitutions, deletions and or additions of the A and/or B amino acid chains relative to the human insulin molecule. The insulin analogs are preferably such wherein one or more of the naturally occurring amino acid residues, preferably one, two, or three of them, have been substituted by another codable amino acid residue. In one embodiment, the instant invention comprises analog molecules having position 28 of the B chain altered relative to the natural human insulin molecule. In this embodiment, position 28 is modified from the natural Pro residue to one of Asp, Lys, or lie. In a preferred embodiment, the natural Pro residue at position B28 is modified to an Asp residue. In another embodiment Lys at position B29 is modified to Pro; Also, Asn at position A21 may be modified to Ala, Gin, Glu, Gly, His, He, Leu, Met, Ser, Thr, Trp, Tyr or Val, in particular to Gly, Ala, Ser, or Thr and preferably to Gly. Furthermore, Asn at position B3 may be modified to Lys. Further examples of insulin precursor analogs are des(B30) human insulin, insulin analogs wherein Phe B1 has been de- leted; insulin analogs wherein the A-chain and/or the B-chain have an N-terminal extension and insulin analogs wherein the A-chain and/or the B-chain have a C-terminal extension. Thus one or two Arg may be added to position B1. The term "immediately N-terminal to" is meant to illustrate the situation where an amino acid residue or a peptide sequence is directly linked at its C-terminal end to the N- terminal end of another amino acid residue or amino acid sequence by means of a peptide bond. In the present context, the term "functional analog of insulin" and the like, is meant to indicate a polypeptide with a similar biological action as the native human insulin protein. By a distance shorter than 5 A between two amino acid residues is meant the short- est inter-atomic distance less than 5 A between any atom in the first amino acid and any atom in the second amino acid. Atomic distances are measured from three-dimensional structures of the molecule determined either by NMR (Wϋthrich, K., 1986, NMR of Proteins and Nucleic Acids, Wiley, New York) or by X-ray crystallography (Drenth, J., 1994, Principles of Protein X-ray crystallography, Springer Verlag Berlin). A distance from one amino acid to another is measured as the shortest inter-atomic distance between any atom in the first amino acid and any atom in the second amino acid if not stated differently. The present invention features novel mini C-peptides connecting position 29 of the insulin B chain and position 1 of the insulin A chain which significantly increased production yields in a yeast host cell. By the term "significantly increased production," "increased fermentation yield," and the like, is meant an increase in secreted amount of the insulin precursor molecule or insulin precursor analog molecule present in the culture supernatant compared to the yield of an insulin precursor or insulin precursor analog with no aromatic amino acid residue in the mini C peptide. An "increased" fermentation yield is an absolute number larger than the control; preferably, the increase is 50% or more larger than the con- trol (AspB 28 IP) level; even more preferably, the increase is 100% or more larger than control levels. By the term "increase in stability" is meant, for example, an increased value of Cmid in solution relative to that obtained for an insulin analog precursor (e.g., Asp B28 IP) without an aromatic amino acid residue in the mini C-peptide. By the term "Cmid" is meant the concentration of GuHCI necessary to unfold one-half of the protein population in an assay measuring the far-UV circular dichroism of the insulin molecule as a function of increasing concentrations of denaturant. "POT is the Schizosaccharomyces pombe triose phosphate isomerase gene, and "TPI1" is the S. cerevisiae triose phosphate isomerase gene. By a "leader" is meant an amino acid sequence consisting of a pre-peptide (the signal peptide) and a pro-peptide. The term "signal peptide" is understood to mean a pre-peptide which is present as an N-terminal sequence on the precursor form of a protein. The function of the signal peptide is to allow the heterologous protein to facilitate translocation into the endoplasmic reticulum. The signal peptide is normally cleaved off in the course of this process. The signal peptide may be heterologous or homologous to the yeast organism producing the protein. A number of signal peptides which may be used with the DNA construct of the invention including yeast aspartic protease 3 (YAP3) signal peptide or any functional analog (Egel-Mitani et al. (1990) YEAST 6:127-137 and US 5,726,038) and the α-factor signal of the MFα1 gene (Thorner (1981) in The Molecular Biology of the Yeast Saccharomyces cerevisiae, Strathem et al., eds., pp 143-180, Cold Spring Harbor Laboratory, NY and US 4,870,00. The term "pro-peptide" means a polypeptide sequence whose function is to allow the expressed polypeptide to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory vesicle for secretion into the culture medium (i.e. exportation of the polypeptide across the cell wall or at least through the cellular membrane into the periplasmic space of the yeast cell). The pro-peptide may be the yeast α-factor pro-peptide, vide US 4,546,082 and 4,870,008. Alternatively, the pro-peptide may be a synthetic pro-peptide, which is to say a pro-peptide not found in nature. Suitable synthetic pro-peptides are those disclosed in US 5,395,922; 5,795,746; 5,162,498 and WO 98/32867. The pro- peptide will preferably contain an endopeptidase processing site at the C-terminal end, such as a Lys- Arg sequence or any functional analog thereof. The polynucleotide sequence of the invention may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage et al. (1981) Tetrahedron Letters 22:1859-1869, or the method described by Matthes et al. (1984) EMBO Journal 3:801-805. According to the phosphoamidite method, oligonucleotides are synthesized, for example, in an automatic DNA synthesizer, purified, duplexed and ligated to form the synthetic DNA construct. A currently preferred way of preparing the DNA construct is by polymerase chain reaction (PCR). The polynucleotide sequence of the invention may also be of mixed genomic, cDNA, and synthetic origin. For example, a genomic or cDNA sequence encoding a leader peptide may be joined to a genomic or cDNA sequence encoding the A and B chains, after which the DNA sequence may be modified at a site by inserting synthetic oligonucleotides encoding the desired amino acid sequence for homologous recombination in accordance with well-known procedures or preferably generating the desired sequence by PCR using suitable oligonucleotides. The invention encompasses a vector which is capable of replicating in the selected microorganism or host cell and which carries a polynucleotide sequence encoding the insulin precursors or insulin precursor analogs of the invention. The recombinant vector may be an autonomously replicating vector, i.e., a vector which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra- chromosomal element, a mini-chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used. The vector may be linear or closed circular plasmids and will preferably contain an element(s) that permits stable integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome. In a preferred embodiment, the recombinant expression vector is capable of replicating in yeast Examples of sequences which enable the vector to replicate in yeast are the yeast plasmid 2 μm replication genes REP 1-3 and origin of replication. The vectors of the present invention preferably contain one or more selectable markers which permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Selectable markers for use in a filamentous fungal host cell include amdS (acetamidase), argB (ornithine carbamoyl- transferase), pyrG (orotidine-5'-phosphate decarboxylase) and trpC (anthranilate synthase. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 , and URA3. A preferred selectable marker for yeast is the Schizosaccharomyces pompe TPI gene (Russell (1985) Gene 40: 125-130). In the vector, the polynucleotide sequence is operably connected to a suitable promoter sequence. The promoter may be any nucleic acid sequence which shows transcrip- tional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extra-cellular or intra-cellular polypeptides either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription in a bacterial host cell, are the promoters obtained from the £. coli lac operon, Streptomyces coelicolor agarase gene {dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amylo- liquefaciens alpha-amylase gene (amyQ), and Bacillus licheniformis penicillinase gene {penP). Examples of suitable promoters for directing the transcription in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhi- zomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, and Aspergillus niger acid stable alpha-amylase. In a yeast host, useful promoters are the Saccharomyces cerevisiae Ma1 , TPI, ADH or PGK promoters. The polynucleotide construct of the invention will also typically be operably connected to a suitable terminator. In yeast a suitable terminator is the TPI terminator (Alber et al. (1982) J. Mol. Appl. Genet. 1 :419-434). The procedures used to ligate the polynucleotide sequence of the invention, the promoter and the terminator, respectively, and to insert them into suitable yeast vectors contain- ing the information necessary for yeast replication, are well known to persons skilled in the art. It will be understood that the vector may be constructed either by first preparing a DNA construct containing the entire DNA sequence encoding the insulin precursors or insulin precursor analogs of the invention, and subsequently inserting this fragment into a suitable expression vector, or by sequentially inserting DNA fragments containing genetic information for the individual elements (such as the signal, pro-peptide, mini C-peptide, A and B chains) followed by ligation. The present invention also relates to recombinant host cells, comprising a polynucleotide sequence encoding the insulin precursors or the insulin precursor analogs of the invention. A vector comprising such polynucleotide sequence is introduced into the host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra- chromosomal vector as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. The host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote. Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, Streptomyces cell, or gram negative bacteria such as E. coli and Pseudomonas sp. Eukaryote cells may be mammalian, insect, plant, or fungal cells. In a preferred embodiment, the host cell is a yeast cell. The yeast organism used in the process of the invention may be any suitable yeast organism which, on cultivation, produces large amounts of the insulin precursor and insulin precursor analogs of the invention. Examples of suitable yeast organisms are strains selected from the yeast species Saccharomyces cerevisiae, Saccharomyces kluyveri, Schi- zosaccharomyces pombe, Sacchoromyces uvarum, Kluyveromyces lactis, Hansenula poly- morpha, Pichia pastoris, Pichia methanolica, Pichia kluyveri, Yarrowia lipolytica, Candida sp., Candida utilis, Candida cacaoi, Geotrichum sp., and Geotrichum fermentans. The transformation of the yeast cells may for instance be effected by protoplast formation followed by transformation in a manner known per se. The medium used to cultivate the cells may be any conventional medium suitable for growing yeast organisms. The secreted insulin precursor or insulin precursor analogs of the invention, a significant proportion of which will be present in the medium in correctly processed form, may be recovered from the medium by conventional procedures including separating the yeast cells from the medium by centrifugation, filtration or catching the insulin precursor or insulin precursor analog by an ion exchange matrix or by a reverse phase absorption matrix, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, fol- lowed by purification by a variety of chromatographic procedures, e.g. ion exchange chroma- tography, affinity chromatography, or the like. The insulin precursors and insulin precursor analogs of the invention may be expressed with an N-terminal amino acid residue extension, as described in U.S. Patent No. 5,395,922, and European Patent No. 765,395A, both of which patents are herein specifically incorporated by reference. The extension is found to be stably attached to the insulin precursor or insulin precursor analogs of the invention during fermentation, protecting the N- terminal end of the insulin precursor or insulin precursor analog against the proteolytic activity of yeast proteases such as DPAP. The presence of an N-terminal extension on the insulin precursor or insulin precursor analog may also serve as a protection of the N-terminal amino group during chemical processing of the protein, i.e. it may serve as a substitute for a BOC (t-butyl-oxycarbonyl) or similar protecting group. The N-terminal extension may be removed from the recovered insulin precursor or insulin precursor analog by means of a proteolytic enzyme which is specific for a basic amino acid (e.g., Lys) so that the terminal extension is cleaved off at the Lys residue. Examples of such proteolytic enzymes are trypsin or Achro- mobacter lyticus protease . After secretion to the culture medium and recovery, the insulin precursor or insulin precursor analogs of the invention will be subjected to various in vitro procedures to remove the possible N-terminal extension sequence and the mini C-peptide to give insulin or the desired insulin analog. Such methods include enzymatic conversion by means of trypsin or an Achromobacter lyticus protease in the presence of an L-threonine ester followed by conversion of the threonine ester of the insulin or insulin analog into insulin or the insulin analog by basic or acid hydrolysis as described in US patent specification No. 4,343,898 or 4,916,212 or Research Disclosure, September 1994/487 the disclosures of which are incorporated by reference hereinto. As described below, insulin precursors or insulin precursor analogs with synthetic C- peptides were constructed featuring at least one aromatic amino acid (Example 1). Saccharomyces cerevisiae expression plasmids containing a polynucleotide sequence encoding the claimed insulin precursors or insulin precursor analogs were constructed by PCR and used to transform a S. cerevisiae host cell. The amount of expressed product, e.g. an insulin analog was measured as a percentage of the relevant control level, e.g. amount of expressed Asp B28 IP lacking mini C-peptide (Table 1) and Asp B28 IP(AlaAlaLys) with a mini C- peptide without an aromatic amino acid residue (Table 2). The novel C-peptides of the invention gave increased yields by up to 7-fold levels. The present invention is described in further detain in the following examples which are not in any way intended to limit the scope of the invention as claimed. The attached Figures are meant to be considered as integral parts of the specification and description of the invention. All references cited are herein specifically incorporated by reference for all that is described therein. EXAMPLES General Procedures All expressions plasmids are of the C-POT type, similar to those described in EP 171 , 142, which are characterized by containing the Schizosaccharomyces pombe triose phosphate isomerase gene (POT) for the purpose of plasmid selection and stabilization in S. cer- evisiae. The plasmids also contain the S. cerevisiae triose phosphate isomerase promoter and terminator. These sequences are similar to the corresponding sequences in plasmid pKFN1003 (described in WO 90/100075) as are all sequences except the sequence of the EcoRI-Xoal fragment encoding the fusion protein of the leader and the insulin precursor product. In order to express different fusion proteins, the EcoRl-Xba\ fragment of pKFN1003 is simply replaced by an EcoRI-Xbal fragment encoding the leader-insulin precursor-fusion of interest. Such EcoRI-Xbal fragments may be synthesized using synthetic oligonucleotides and PCR according to standard techniques. Yeast transformants were prepared by transformation of the host strain S. cerevisiae strain MT663 (MATaiMATc pep4-3/pep4-3 HIS4/his4 tpi::LEU2/tpi::LEU2 Cir + ). The yeast strain MT663 was deposited in the Deutsche Sammlung von Mikroorganismen und Zellkul- turen in connection with filing WO 92/11378 and was given the deposit number DSM 6278. MT663 was grown on YPGaL (1 % Bacto yeast extract, 2% Bacto peptone, 2% galac- tose, 1% lactate) to an O.D. at 600 nm of 0.6. 100 ml of culture was harvested by centrifuga- tion, washed with 10 ml of water, recentrifuged and resuspended in 10 ml of a solution con- taining 1.2 M sorbitol, 25 mM Na 2 EDTA pH = 8.0 and 6.7 mg/ml dithiotreitol. The suspension was incubated at 30°C for 15 minutes, centrifuged and the cells resuspended in 10 ml of a solution containing 1.2 M sorbitol, 10 mM Na 2 EDTA, 0.1 M sodium citrate, pH 0 5.8, and 2 mg Novozym®234. The suspension was incubated at 30°C for 30 minutes, the cells collected by centrifugation, washed in 10 ml of 1.2 M sorbitol and 10 ml of CAS (1.2 M sorbitol, 10 mM CaCI 2 , 10 mM Tris HCl (Tris = Tris(hydroxymethyl)aminomethane) pH = 7.5) and resuspended in 2 ml of CAS. For transformation, 1 ml of CAS-suspended cells was mixed with approx. 0.1 mg of plasmid DNA and left at room temperature for 15 minutes. 1 ml of (20% polyethylene glycol 4000, 10 mM CaCI 2 , 10 mM Tris HCl, pH = 7.5) was added and the mixture left for a further 30 minutes at room temperature. The mixture was centrifuged and the pellet resuspended in 0.1 ml of SOS (1.2 M sorbitol, 33% v/v YPD, 6.7 mM CaCI 2 ) and incubated at 30°C for 2 hours. The suspension was then centrifuged and the pellet resuspended in 0.5 ml of 1.2 M sorbitol. Then, 6 ml of top agar (the SC medium of Sherman et al. (1982) Methods in Yeast Genetics, Cold Spring Harbor Laboratory) containing 1.2 M sorbitol plus 2.5% agar) at 52°C was added and the suspension poured on top of plates containing the same agar-solidified, sorbitol containing medium. S. cerevisiae strain MT663 transformed with expression plasmids was grown in YPD for 72 h at 30°C. Quantitation of the insulin-precursor yield in the culture supernatants was performed by reverse-phase HPLC analysis with human insulin as an external standard (Snel & Damgaard (1988) Proinsulin heterogenity in pigs. Horm. Metabol. Res. 20:476-488). Example 1. Construction of Synthetic C-peptides With Aromatic Amino Acid(s). Synthetic genes encoding fusion proteins, consisting of Asp B28 IP associated with a leader sequence consisting of a pre-peptide (signal peptide) and a pro-peptide, were constructed using PCR under standard conditions (Sambrook et al. (1989) Molecular Cloning, Cold Spring Harbor Laboratory Press) and E.H.F. polymerase (Boehringer Mannheim GmbH, Sandhoefer Strasse 116, Mannheim, Germany). The resulting DNA fragments were isolated and digested with endonucleases and purified using the Gene Clean kit (Bio101 Inc., La Jolla, CA, USA). Standard methods were used for DNA ligation and transformation of E. coli cells were performed by the CaCI 2 method (Sambrook et al. (1989) supra). Plasmids were purified from transformed E. coli cells using QIAGEN columns (QIAGEN, Hilden, Germany). Nucleotide sequences were determined using the ALF Pharmacia Biotech DNA sequencing system with purified double-stranded plasmid DNA as template. Oligonucleotide primers for PCR were obtained from DNA technology (Arhus, Denmark). Secretory expression of the Asp B28 IP in S. cerevisiae was performed using the S. cerevisiae strain MT663 and the 2 μm based yeast expression vector CPOT (see Fig. 1) as described in Thim, L. et al. (1986) Proc. Natl. Acad. Sci.USA 83:6766-6770. The yeast expression vector contains the Schizosaccharomyces pombe triose phosphate isomerase gene (POT) for plasmid selection and stabilization in S. cerevisiae. Furthermore, the S. cerevisiae triose phosphate isomerase gene (TPI1) promoter and terminator are used for transcription initiation and termination of the recombinant gene encoding the leader-Asp B28 IP fusion protein. Secretion of the Asp B28 IP was facilitated by the α-factor leader, although a variety of known yeast leader sequences may be used. As shown in Fig. 1 , the pAK721 S. cerevisiae expression plasmid expressing the LA19 leader-EEAEAEAEPK(SEQ ID NO:3)-IP fusion protein was constructed based on the S. cerevisiae-E. coli shuttle POT plasmid (U.S. patent 5,871 ,957). In Fig. 1 L-IP indicates the fusion protein expression cassette encoding the leader-IP fusion protein; TPI-PROMOTER is the S. cerevisiae TPI1 promoter, TPI-TERMINATOR is the S. cerevisiae TPI1 terminator; TPI-POMBE indicates the S. pombe POT gene used for selection in S. cerevisiae; ORIGIN indicates a S. cerevisiae origin of replication derived from the 2μm plasmid; AMP-R indicates the β-lactamase gene conferring resistance toward ampicillin, facilitating selection in E. coli; and ORIGIN-PBR322 indicates an E. co// origin of replication. DNA encoding a number of fusions proteins of leader sequences and Asp B28 IP with different mini C-peptides was generated by PCR using appropriate oligonucleotides as primers, as described below. Standard methods were used to subclone DNA fragments encoding the leader-Asp B 8 IP fusion proteins into the CPOT expression vector in the following configuration: leader-Lys-Arg-spacer-Asp B28 IP, where Lys-Arg is a potential dibasic endoprotease processing site and spacer is an N-terminal extension. To optimize processing of the fusion protein by the S. cerevisiae Kex2 endoprotease, DNA encoding a spacer peptide (N-terminal extension), e.g. EEAEAEAPK (SEQ ID NO:4), was inserted between the DNA encoding the leader and the Asp B28 IP (Kjeldsen et al. (1996) Gene 170, 107-112.). However, the present of the spacer peptide is not mandatory. The mature Asp B28 IP was secreted as a single-chain N- terminally extended insulin precursor with a mini C-peptide, connecting Lys B29 and Gly A1 . After purification of the Asp B28 IP and proteolytic removal of the N-terminal extension and the mini C-peptide, the amino acid Thr B30 can be added to Lys B29 by enzyme-mediated transpep- tidation, to generate Asp B28 human insulin (Markussen, et al. (1987) in "Peptides 1986" (Theodoropoulos, D., Ed.), pp. 189-194, Walter de Gruyter & Co., Berlin.). Development of synthetic mini C-peptides was performed by randomization of one or more codon(s) encoding the amino acids in the mini C-peptide. All synthetic mini C- peptides feature an enzymatic processing site (Lys) at the C-terminus which allows enzymatic removal of the synthetic mini C-peptide (U.S. Patent No. 4,916,212, herein specifically incorporated by reference). Randomization was performed using doped oligonucleotides which introduced codon(s) variations at one or more positions of the synthetic mini C- peptides. Typically one of the two primers (oligonucleotides) used for PCR was doped. An example of an oligonucleotides pair used for PCR generation of leader-Asp B28 IP with randomized synthetic mini C-peptides used to generated synthetic mini C-peptides with the general formula: Xaa-Trp-Lys (XWK) are as follows: Primer A: 5'-TAAATCTATAACTACAAAAAACACATA-3' (SEQ ID NO:13) and Primer B: 3'-CCAAAGAAGATGTGACTGTTCNNMACCTTCCCATAGCAACTTGTTACAAC- ATGAAGATAGACAAGAAACATGGTTAACCTTTTGATGACATTGATCAGATCTTTGA-TTC-5' (SEQ ID NO:14), where N is A, C, G, or T and M is C or A. Polymerase chain reaction. PCR was typically performed as indicated below: 5 μl Primer A (20 pmol), 5 μl Primer B (20 pmol), 10 μl 10X PCR buffer, 8 μl dNTP mix, 0.75 μl E.H.F. enzyme, 1 μl pAK1150 plasmid as template (approximately 0.2 μg DNA) and 70.25 μl distilled water. Typically between 10 and 15 cycles were performed, one cycle typically was 94° C for 45 sec; 55° C for 1 min; 72° C for 1.5 min. The PCR mixture was subsequently loaded onto a 2 % agarose gel and electrophoresis was performed using standard techniques. The resulting DNA fragment was cut out of the agarose gel and isolated by the Gene Clean kit. Fig. 2 shows the sequence of pAK1150 DNA used as template for PCR and inferred amino acids of the encoded fusion protein (α-factor-leader-(EEAEAEAPK)(SEQ ID NO:4)- Asp B28 IP of pAK1150 (SEQ ID NO:5 and 6). The pAK1150 plasmid is similar to pAK721 shown in Fig. 1. The α-factor-leader's C-terminus was modified to introduced a Nco I restriction endonuclease site, which changes the inferred amino acid sequences from SerLeuAsp to SerMetAla. Moreover, the encoded Asp B28 IP does not feature a mini C-peptide but Lys B29 is directly connected to Gly* 1 . The purified PCR DNA fragment was dissolved in water and restriction endonucleases buffer and digested with suitable restriction endonucleases (e.g. Bgl II and Xba I) according to standard techniques. The Bglll-Xbal DNA fragments were subjected to agarose electrophoresis and purified using The Gene Clean Kit. The expression plasmid pAK1150 or a similar plasmid of the CPOT type (see Fig. 1) was digested with the restriction endonucleases Bgl II and Xba I and the vector fragment of 10765 nucleotide basepairs isolated using The Gene Clean Kit. The two digested and isolated DNA fragments (the vector fragment and the PCR fragment) were ligated together using T4 DNA ligase and standard conditions. The ligation mix was subsequently transformed into a competent E. coli strain (R-, M+) followed by selection with ampicillin resistance. Plasmids from the resulting E. coli's were isolated using QIAGEN columns. The plasmids were subsequently used for transformation of a suitable S. cerevisiae host strain, e.g., MT663 (MATalMAT pep4-3/pep4-3 HIS4/his4 tpi::LEU2/tpi::LEU2 Cir + ). Individual transformed S. cerevisiae clones were grown in liquid culture, and the quantity of Asp B28 IP secreted to the culture supematants were determined by RP-HPLC. The DNA sequence encoding the synthetic mini C-peptide of the expression plasmids from S. cerevisiae clones secreting increased quantity of the Asp B28 IP were then determined. Subsequently, the identified synthetic mini C-peptide sequence might be subjected to another round of randomization optimization. An example on a DNA sequence encoding a leader-Asp B28 IP(GluTrpLys) fusion protein featuring a synthetic mini C-peptide (GluTrpLys) resulting from the randomized optimization process described is shown in Fig. 3 (SEQ ID NO:7 and 8). Table 1 and 2 show the insulin precursors analogs generated by the above method and production yield expressed as a percent of control. Fermentation was conducted at 30°C for 72 h in 5 ml YPD. Yield of the insulin precursor was determined by RP-HPLC of the culture supernatant, and is expressed relative to the yield of a control strain expressing either a leader-Asp B28 IP fusion protein in which the B29 residue is linked to the A1 residue by a peptide bond; or leader-Asp B28 IP fusion protein in which the B29 residue is linked to the A1 residue by a mini C-peptide, respectively. In the tables, "α*" indicates an α-factor leader in which the C- terminus up to the LysArg has been modified from "SLD (SerLeuAsp)" to "SMA (SerMet Ala)" and "ex4" is an N-terminal extension with the amino acid sequence EEAEAEAPK(SEQ ID NO:4). YAP3 is the YAP3 signal sequence TA39 is a synthetic pro-sequence QPIDD- TESNTTSVNLMADDTESRFATNTTLAGGLDWNLISMAKR (SEQ ID NO: 16). The sequence EEGEPK (SEQ ID NO:17) is an N-terminal extension to the B-chain of the insulin analogue. TA57 is a synthetic pro-sequence QPIDDTESQTTSVNLMADDTESAFATQTNSG- GLDWGLISMAKR (SEQ ID NO: 18). TABLE 1 TABLE 2 Example 2. Structure Determination of Asp B28 IP(MetTrpLys) in Aqueous Solution by NMR Spectroscopy. NMR spectroscopy. Samples for NMR were prepared by dissolving the lyophilized protein powder in 10/90 D 2 0/H 2 0 with a 10 mM phosphate buffer and adjusting the pH as desired by addition of small volumes of 1 M DCI or NaOD. All pH meter readings are without correction for isotope effects. Samples of Asp B28 IP(MetTrpLys) for NMR were prepared at concentrations ranging from 25μM to 1mM at pH 8.0. Two-dimensional 1 H- 1 H NMR spectra of 1mM samples, DQF-COSY (Piantini et al. (1982) J. Am. Chem. Soc. 104:6800-6801 , Ranee et ai. (1983) Biochem. Biophys. Res. Commun. 117:479-485), TOCSY (Braunschweiler et al. (1983) J. Magn. Reson. 53:521-528, Bax et al. (1985) J. Magn. Reson. 65:355-360) and NOESY (Jee- ner et al. (1979) J. Chem. Phys. 71 :4546-4553) were recorded at 600 MHz on a Varian Unity Inova NMR spectrometer equipped with a 1 H/ 13 C/ 15 N triple resonance probe with a self-shielded triple-axis gradient coil using standard pulse sequences from the Varian user library. The operating temperature was set to 27°C. For each phase sensitive two-dimensional NMR spectrum 512 ti increments were acquired each with 2048 or 4096 real data points according to the TPPI- States method (Marion et al. (1989) J. Magn. Reson. 85:393-399). Spectral widths of 6983 Hz in both dimensions were used, with the carrier placed exactly on the water resonance which was attenuated by using either saturation between scans for 1.5 seconds or selective excitation by a gradient-tailored excitation pulse sequence (WATERGATE, Piotto et al. (1992) J. Biomol. NMR 2:661-665). DQFCOSY spectra were recorded using a gradient enhanced version applying magic-angle gradients (Mattiello et al. (1996) J. Am. Chem. Soc. 118:3253-3261). For TOCSY spectra mixing times between 30 and 80 ms were used and for NOESY mixing times between 50 and 200 ms. The processing of the two-dimensional NMR spectra was performed using the software package Xwinnmr (version 2.5, NMR processing software from Bruker Analytische Messtech- nik GmbH, D-76275 Ettlingen, Germany). Each dimension was processed with shifted sine-bell apodization and zero-filling performed once in each dimension. Baseline corrections were ap- plied if necessary using Xwinnmr standard procedures. The spectral assignment, cross peak integration, sequence specific assignment, stereo specific assignment, and all other bookkeeping were performed using the program PRONTO (PRONTO Software Development and Distribution, Copenhagen Denmark) (Kjaer et al. (1991) NATO ASI Series (Hoch, J. C, Redfield C, & Poulsen, F. M., Eds.) Plenum, New York). Che- mical shifts are measured in ppm and the water resonance set to 4.75 ppm. Structure calculations. Distance restraints for the subsequent structure calculation were obtained from integrated NOESY cross peaks classified as either weak, medium or strong corresponding to upper distance restraints of 5.5, 3.3, and 2.7 A, respectively. For distance restraints involving methyl groups, an additional 0.5 A was added to the upper limit (Wagner et al. (1985) J. Mol. Biol. 196:611-639). Structure calculations were performed using the hybrid method combining distance geometry (Crippen et al. (1988) Distance Geometry and Molecular Conformation, Research Studies Press, Taunton, Somerset, England; Kuszewski et al. (1992) J.Biomol NMR 2:33-56) and simulated annealing based on the ideas of Nilges et al. (1988) FEBS Lett. 229:317-324 using X-PLOR 3.0 (Brϋnger (1992) X-PLOR Version 3.1: A System for X-ray Crystallography and NMR ,Yale University Press, New Haven) according to the examples given by the X-PLOR manual (dg_sub_embed.inp, dgsa.inp, refine.inp). Residue numbers are derived from standard insulin residue numbering, residues in the B-chain are numbered B1- B29, residues in the C-peptide (e. g. MetTrpLys) are numbered C1-C3 and residues in the A- chain are numbered A1-A21. Spectral assignment of the NMR spectra followed for most resonances the standard sequential assignment procedure described by Wϋthrich (1986 NMR of Proteins and Nucleic Acids, Wiley, New York). The standard assignment procedure fails when the amid proton of a particular amino acid residue exchanges to rapidly with protons in the water. At pH 8.0 this occurs for several amino acid residues, however, comparison with earlier mutant insulin NMR spectral assignments and identification of neighbouring (in space) amino acid residues through NOEs allow an almost total spectral assignment. Analysis of the NOESY spectra showed that several amino acid residues had a NOE network to the surrounding residues similar to what has previously been determined for other insulin molecules, i.e., human insulin His 816 mutant (Ludvigsen et al. (1994) Biochemistry 33:7998-8006) and these similar con- nections are found for residues B1-B10, B13-B14, B17-B24 and A4-A21. Additionally the di- hedral angle restraints for the above listed residues were adopted from those used previously (Ludvigsen et al. (1994) supra). Several amino acids in particular B27-B29, C1-C3, A1-A3 have cross peaks patterns which are consistent with peptide chains that are less well ordered than commonly well- defined secondary structural elements. Thus additional NOEs were converted into distance restraints without any further classification than upper limits of 5.5 A or 6.0 A if a methyl group were included. An ensemble of 20 converged structures (Fig. 5) was calculated and the relevant parameters listed in Table 3 for the converged structures. Each NOE here identical to a distance restraint is only counted once even though it might occur several times in the NOESY spectrum. Ramachandran plot quality assessment is standard quality parameters to evaluate local geometry quality. In general the described quality parameters are comparable to 2.5 A resolution of X-ray based protein structures (Laskowski et al. (1996) J. Biol- mol.NMR 8:477-486). Table 3 Description of the calculated structure. A representative structure most resembling the average of the ensemble is displayed in Fig 6. Asp B28 IP(MetTrpLys) is structurally similar to the native insulin structure for regions comprising residues B1-B10, B14-B23, A4-A21. The differences are mostly pronounced for regions in the vicinity of the connecting peptide in positions B26-B29, C1-C3, A1-A3 and less pronounced for residues B11-B13. The structure of Asp B28 IP(MetTrpLys) near the C-peptide is strikingly different from the native like structure (Ludvigsen (1994) supra). The methionine and tryptophan side-chains in Asp B28 IP(MetTrpLys) opens the traditional insulin core structure by moving on one side the side-chains of particular Tyr B26 and Phe B25 away and leaving the otherwise usual neighboring hydrophobic patch comprised by the side-chains of Leu 811 , Val B12 , lle A2 and Tyr* 19 intact. This pocket created by moving Phe B25 , Tyr B26 and the peptide chain comprised by residues B25 to B29 is apparently well suited to accommodate the packing of the side-chains Met 01 and Trp C2 from the C-peptide. Several NOEs from these two side-chains to structurally neighboring residues verify this very new arrangement of side- chains not previously observed in any insulin structure. Met 01 is placed in a pocket composed by the residues Leu B15 , Phe B24 , Tyr B26 , Trp c1 , lie* 2 and Tyr* 19 which all have NOEs to Met 01 . Trp C2 has an even more extensive NOE network, but due to fast exchange of the indole amid proton only four resonances belonging to the aromatic ring system of Trp 02 can be assigned. Despite this 21 inter-residue NOEs between Trp 02 and its neighbors spanned by Leu 811 , Val B12 , Leu 815 , Tyr 826 , Met 01 and lie* 2 have been observed in the NOESY spectrum of Asp B28 IP(Met Trp Lys). The presence of a tryptophan side-chain in the pocket also has extensive impact on the chemical shifts observed in the spectra of Asp B28 IP(Met Trp Lys). Under the conditions used for NMR the spectra of Asp 828 lP(Met Trp Lys) are influenced by some degree of self- association (Fig. 7) but the exchange between monomer and dimer is on the timescale of NMR only observed as an average between the two states. Between concentrations of 25 μM and 0.2 mM the degree of self-association does not change as seen by NMR. Table 4 shows chemical shifts of Asp B28 IP(MetTrpLys) at 27° Celcius obtained at 600 MHz, pH 8 in 10%/90% D 2 0/H 2 0 with 10 mM phosphate buffer. Chemical shifts are ref- erenced by setting the residual water signal to 4.75 ppm. N/A means no assignment. Asp B28 IP(MetTrpLys) assignments (1-29 = B1-B29; 30-32 = C1-C3 and 33-53 = A1-A21) and Table 5 provides the atomic coordinates of Asp B28 IP(MetTrpLys) in PDB format. Table 4: NMR spectral assignments for Asp . 28i IP(MetTrpLys) Spin system HN HA Other: Phe-1 4.52 HB#a: 2.976, HB#b: 3.040, HD#: 7.104, HE#: 7.214, HZ: 7 177 Val-2 N/A N/A HB: N/A, HG#a: N/A, HG#b: N/A Asn-3 N/A HB#a: N/A, HB#b: N/A, HD2#a: N/A, HD2#b: N/A Glu-4 N/A HB#a: N/A, HB#b: N/A His-5 4.30 HB#a: 3.311, HB#b: 2.992, HD2: 6.850, HE1 : 7.605 Leu-6 4.47 HB#a: 1.635, HB#b: 0.807, HG: 1.506, HD#a: 0.753, HD#b: 0.675 Cys-7 8.30 4.83 HB#a: 2.901, HBftb: 3.173 Gly-8 N/A, N/A Ser-9 N/A HB#: N/A His-10 N/A 4.41 HB#a: 3.140, HB#b: 3.351, HD2: 7.112, HE1: 7.729 Leu-11 N/A 3.93 HB#a: N/A, HB#b: 1.143, HG: 1.257, HD#a: 0.375, HD#b: 0 .559 Val-12 7.25 3.37 HB: 2.000, HG#a: 0.849, HG#b: N/A Glu-13 N/A N/A HB#a: N/A, HG#a: N/A, HG#b: N/A Ala-14 7.71 3.98 HB#: 1.307 Leu-15 7.83 3.74 HB#a: 0.671, HB#b: 1.254, HG: 1.157, HD#a: N/A, HD#b: 0 .295 Tyr-16 8.15 4.31 HB#a: 3.115, HD# : 7.204, HE#: 6.734 Leu-17 7.99 4.05 HB#a: 2.004, HB#b: 1.849, HG: 1.732, HD#a: 0.900, HD#b: 0.879 Val-18 8.49 3.71 HB: 1.996, HG#a: 0.994, HG#b: 0.834 Cys-19 8.57 4.81 HB#a: 2.820, HB#b: 3.250 Gly-20 7.75 3.96, N/A Arg-22 N/A N/A HB#a: N/A, HB#b: N/A, HG#a: N/A, HG#b: N/A, HD#a: N/A, HD#b: N/A Gly-23 7.12 4.06, 3.76 Phe-24 7.56 4.99 HB#a: 2.971, HB#b: 3.206, HD#: 6.746, HE#: 6.877, HZ: N/A Phe-25 N/A 4.78 HB#a: 3.051, HB#b: N/A, HD#: 7.172, HE#: 7.249 Tyr-26 N/A 4.63 HBita: 2.770, HB#b: N/A, HD#: 6.694, HE#: 6.291 Thr-27 N/A N/A HB: N/A, HG2#: N/A Asp-28 N/A N/A HB#a: N/A, HB#b: N/A ys-29 Met-30 7.84 4.18 HBff: 2.627, HG#: 2.904, HE#: 2.110 Trp-31 4.19 HB#a: 3.182, HE3 : 7.037, HH2 : 6.763, HZ2: 7.147, HZ3 : 6. 437 ys-32 Gly-33 N/A, N/A Ile-34 7.98 3.53 HB: 0.953, HGlita: 0.382, HGl#b: 0.562, HG2#: 0.234, HD# : 0.029 Val-35 7.75 N/A, 4.00 HB: N/A, HB: 1.925, HG#a: N/A, HG#b: N/A, HG#b: 0.807 Glu-36 N/A N/A HB#a: N/A, HG#a: N/A Gln-37 7.73 3.96 HB#: 2.033, HG#: 2.313 Cys-38 8.24 4.89 HB#a: 3.103, HB#b: 2.700 Cys-39 N/A N/A HB#a: N/A, HB#b: N/A Thr-40 3.95 HB: 4.411, HG2#: 1.167 Ser-41 6.97 4.59 HB#a: 3.698, HB#b: 3.867 Ile-42 7.59 4.32 HB: 1.399, HGl#a: N/A, HG2#: 0.573, HD#: 0.389 Cys-43 N/A N/A HB#a: N/A Ser-44 N/A N/A HB#a: N/A, HB#b: N/A Leu-45 3.86 HB#a: 1.412, HB#b: 1.489, HG: 1.565, HD#a: 0.808, HD#b: 0.733 Tyr-46 7.59 4.27 HB#a: 2.938, HD#: 7.049, HE#: 6.784 Gln-47 7.49 3.87 HB#a: 2.235, HB#b: 1.948, HG#a: 2.305, HG#b: 2.076 Leu-48 7.66 4.11 HB#a: 1.930, HB#b: 1.409, HG: 1.683, HD#a: 0.677, HD#b: N/A Glu-49 7.75 4.12 HB#a: N/A, HB#b: 1.935, HG#a: 2.291, HG#b: 2.191 Asn-50 7.21 4.34 HB#a: 2.363, HB#b: N/A Tyr-51 7.76 4.50 HB#a: 3.269, HB#b: 2.774, HD#: 7.250, HE#: 6.670 Cys-52 7.35 5.01 HB#a: 2.785, HB#b: 3.322 Asn-53 8.18 4.40 HB#a: 2.555, HB#b: 2.716, HD2#a: 7.426, HD2#b: N/A Table 5 Atomic coordinates of Asp IP(MetTrpLys) in PDB format ATOM 1 CA PHE 1 -6.075 -6.762 -0.761 1.00 0.00 ATOM 2 HA PHE 1 -5.526 -6.571 0.150 1.00 0.00 ATOM 3 CB PHE 1 -5.359 -6.093 -1.942 1.00 0.00 ATOM 4 HB1 PHE 1 -5.934 -6.258 -2.842 1.00 0.00 ATOM 5 HB2 PHE 1 -4.382 -6.535 -2.061 1.00 0.00 ATOM 6 CG PHE 1 -5.208 -4.597 -1.715 1.00 0.00 ATOM 7 CD1 PHE 1 -4.925 -4.081 -0.436 1.00 0.00 ATOM 8 HD1 PHE 1 -4.817 -4.749 0.405 1.00 0.00 ATOM 9 CD2 PHE 1 -5.346 -3.722 -2.799 1.00 0.00 ATOM 10 HD2 PHE 1 -5.563 -4.112 -3.783 1.00 0.00 ATOM 11 CE1 PHE 1 -4.787 -2.702 -0.251 1.00 0.00 ATOM 12 HE1 PHE 1 -4.573 -2.308 0.732 1.00 0.00 ATOM 13 CE2 PHE 1 -5.204 -2.341 -2.612 1.00 0.00 ATOM 14 HE2 PHE 1 -5.310 -1.668 -3.451 1.00 0.00 ATOM 15 CZ PHE 1 -4.926 -1.831 -1.338 1.00 0.00 ATOM 16 HZ PHE 1 -4.818 -0.768 -1.195 1.00 0.00 ATOM 17 C PHE 1 -7.491 -6.201 -0.628 1.00 0.00 ATOM 18 O PHE 1 -8.361 -6.497 -1.425 1.00 0.00 ATOM 19 N PHE 1 -6.144 -8.234 -0.995 1.00 0.00 ATOM 20 HT1 PHE 1 -6.303 -8.723 -0.091 1.00 0.00 ATOM 21 HT2 PHE 1 -5.250 -8.560 -1.415 1.00 0.00 ATOM 22 HT3 PHE 1 -6.930 -8.446 -1.642 1.00 0.00 ATOM 23 N VAL 2 -7.730 -5.399 0.380 1.00 0.00 ATOM 24 HN VAL 2 -7.013 -5.181 1.011 1.00 0.00 ATOM 25 CA VAL 2 -9.095 -4.819 0.578 1.00 0.00 ATOM 26 HA VAL 2 -9.838 -5.571 0.354 1.00 0.00 ATOM 27 CB VAL 2 -9.270 -4.345 2.030 1.00 0.00 ATOM 28 HB VAL 2 -8.787 -3.387 2.154 1.00 0.00 ATOM 29 CGI VAL 2 -10.760 -4.206 2.339 1.00 0.00 ATOM 30 HG11 VAL 2 -11.281 -3.863 1.457 1.00 0.00 ATOM 31 HG12 VAL 2 -10.897 -3.493 3.139 1.00 0.00 ATOM 32 HG13 VAL 2 -11.156 -5.164 2.641 1.00 0.00 ATOM 33 CG2 VAL 2 -8.653 -5.362 3.001 1.00 0.00 ATOM 34 HG21 VAL 2 -8.836 -6.363 2.637 1.00 0.00 ATOM 35 HG22 VAL 2 -9.103 -5.245 3.976 1.00 0.00 ATOM 36 HG23 VAL 2 -7.589 -5.193 3.071 1.00 0.00 ATOM 37 C VAL 2 -9.279 -3.628 -0.369 1.00 0.00 ATOM 38 O VAL 2 -8.349 -2.892 -0.637 1.00 0.00 ATOM 39 N ASN 3 -10.473 -3.437 -0.874 1.00 0.00 ATOM 40 HN ASN 3 -11.204 -4.045 -0.641 1.00 0.00 ATOM 41 CA ASN 3 -10.725 -2.296 -1.806 1.00 0.00 ATOM 42 HA ASN 3 -9.782 -1.927 -2.184 1.00 0.00 ATOM 43 CB ASN 3 -11.594 -2.772 -2.975 1.00 0.00 ATOM 44 HB1 ASN 3 -12.168 -1.940 -3.357 1.00 0.00 ATOM 45 HB2 ASN 3 -12.265 -3.545 -2.633 1.00 0.00 ATOM 46 CG ASN 3 -10.702 -3.324 -4.087 1.00 0.00 ATOM 47 OD1 ASN 3 -9.628 -3.832 -3.824 1.00 0.00 ATOM 48 ND2 ASN 3 -11.103 -3.251 -5.327 1.00 0.00 ATOM 49 HD21 ASN 3 -11.968 -2.842 -5.538 1.00 0.00 ATOM 50 HD22 ASN 3 -10.539 -3.603 -6.046 1.00 0.00 ATOM 51 C ASN 3 -11.448 -1.171 -1.055 1.00 0.00 ATOM 52 O ASN 3 -12.648 -1.002 -1.182 1.00 0.00 ATOM 53 N GLN 4 -10.727 -0.404 -0.276 1.00 0.00 ATOM 54 HN GLN 4 -9.763 -0.562 -0.193 1.00 0.00 > o ι-3 ι >-3 > > > > > > > -3 > > H ι-3 ι-3 > > > -3 > > > > ι-3 > > > >-3 ι-3 ι-3 -3 ι ι-3 ι-3 >-3 ι-3 H H ι-3 H o o o o o o o o O O o o o o o o o o O O O O O O O O O O O O O 0 O O O O O O 0 O O 0 0 0 O O O O O O O O O 0 O O O O 2 s 2 2 2 2 2 2 IS 2 2 s s s s 2 s s S S S S s S S S S S s S S S S s S 2 S s s 2 s S 2 S 2 2 2 2 2 2 2 2 2 2 2 (-> en en cn en cn cn cn cn cn cn Cπ n cπ to en co to en to cn cπ to cπ EC tc EC tn n O O EG EC £E o tn 2 o O D o D o o D D o tn o tn tn O tn 0 tn -2 O O 2; tn Ω tn Ω tn 2 Ω tn tn Ω tn Ω tc 2 0 Ω ei Ed 2 O Ω tn tc Ω tc tn Ω tC Ω Go ¬ ω 2 t t to 2 M M M 0 D > > to t σ α cn ω ω ω o O cn W ω O 2 to ra M D Ω Ω Ω to tn CD t to t- 1 to to to to to to to to Ω Ω o o o o o o t-< t- 1 t" 1 tf t-< t→ t- 1 tr 1 if tr 1 tr 1 IT 1 tr 1 tr 1 tr 1 t" 1 t- 1 tn tn tn c tn tn tn tn tn tn W tn tn tc tn tc tc «: M M H M H IS <r> Ώ Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω H M H M a t?a M H H M H H H tr" t→ tr 1 tr 1 tr* tr 1 t" 1 i→ t" 1 t" 1 t→ t" 1 t- 1 t- 1 ω CΛ ω oα ω CΛ α σ G G G G G G G G c G α σ G G G α G ω 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 -J -J en en en cn cn cn cn en en en en en cn en cn cπ cπ cπ cπ Cπ cπ cπ Cπ cn cπ i ji ji ji ji j ji ji ji j ji ji Ji 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I 1 1 1 I I I 1 1 ( 1 1 t- 1 (- 1 t- I- 1 (- 1 en Cπ π n cn cπ cπ cn en cπ cn en to •~o to to to t en en σ o cπ en cn t *» cn cn to en cn π cn o to o to o cn en t t co to cn to o π en to cn o ιo cπ *> en cn en en ω on cπ t to to cn to t-> to 0 1 1 1 1 Cπ en cn cn cπ o o o o o t to to t to en cπ en Cπ cπ cn π t to t- 1 to to σ 1- 1 l- i to en Cn o o cn cn cπ cπ o o >e> ~o cn cn to to cn to cπ to to t t cπ cπ us cπ en cπ o t cπ t ω en cπ cπ t to cn o o ID to cn cπ to cπ en t to cπ cn en t cπ cπ < > cn I 1 Cπ en cπ Cπ Cπ ω to t o o o o o to t t 0 to O t to o co Cπ to co C en to to en cπ cn to en cn t en cn cπ cπ to cn o o to t cn to cn en to cπ en cn en cπ cn en cn cn to cn o t cπ cn cn t to to en t cπ t σ cπ n (-» t-> t- 1 t- 1 (-> o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o o o o o σ > (-3 > > •-3 > > > > > > > H > > > > > > > > >-3 > > > > > > > > ι-3 > > > >-3 > > ι-3 ι-3 > > > ι-3 ι-3 > O O o o o o o O O o O o o O o o o o o O O O O o O O o o o o O o O O O O o O O o O o O O O o o O o O o O O o O O o o O 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ι-> cn cn en en en en en en en cπ cπ cπ cπ cn cπ cπ cπ cπ co to to t to to to to to to t -j en Cπ to o cn Cπ t o en cπ t o cn cπ to en oo to cn Ω ES 2 o Ω 2 < t- 1 tr 1 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ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 O o O O o O O O o o O o O o o O o o o O O O o o o o O o O O O O o o O o o O o O O O O o O O o O o o O o O o O o o o O 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 to to t to to to to t to to to to to to t to to to t t t to to to to to to to to to t to to to t to to to to to to to to to to to to to to to to to to on en en cn cn on en cn on en cn cn cπ cπ cn Cπ Cπ cn cπ cπ o co en cπ to o -J on n to o on cπ co o on cπ to o oo en cn co to o en t tc EC tn EC EC EC EG EC EC EC EC tn En tn Ω EC 2 Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω EC Ω EC Ω EC 2 O Ω α O D Ω D D D Ω EC Ω EC EC Ω EC Ω EC 2 O Ω EC O Ω EC Ω EC Ω EC Ω tc Ω Ω tc EC Ω EC Ω OS > 2 to to to Ω Ω t to > > 2 to to to α D Ω Ω tB to ω > 2 EC EC ei 171 M ei o O o D Ω tn to ω py ω to to to to to to to to to Ω Ω Ω Ω Ω < < < < < < < < < < < < < < < < tf if f if if if if tf tf tf if tf if tf if if t- if if ι-3 ι-3 ι-3 ι-3 ι-3 H ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 K K | K! > > > > > > > if > > > > ei ei ei ei ei ei ei ei ei ei ei ei ei ei es ei ei ei ei K ι ι-< K K K CΛ if if if if tf if t→ if if tf if if if if c a α c G a α a a G o G G a a G G a G 50 50 50 50 50 iϋ 50 50 50 50 50 50 50 V V 50 50 50 50 -o -J -J -J on on en on cn cn en cn en on cn en en en on on on en on 1 I I cn Cπ o o t to to to t o to o o o to o o o o o to to o to o oo on o n on on cπ cπ to en -J co on Cπ α cn on t t on en en on o en ω cn o cn cn t to on cn On on en on -J en o on cπ o o o cn o cπ to t t n to to en oo cπ en to o to on 1 1 1 1 I 1 I I I 1 I I 1 1 I 1 1 1 1 1 I I I Cπ en en cπ en en t Ji Cn Ji i en Cπ cπ on -j cπ on en cπ on cn Ji cn -j en on en On -J en cπ Ji Cπ i i en cπ o cπ cn cn cn cn o o o to oo t o o en t o o o on cn to to co cn o cπ en on en o to cn to o Cπ en to cn on on en o cn o en on o cn cn cπ cn cn to o to o Cπ en en cn o o on on on en 1 I 1 1 I 1 1 1 I 1 I t o to t to Ji Ji to to o o o o o o to o o to o t on On on on en on Cπ Cπ to to on cn on en tn to Cπ to t en en ω co on Cπ en to to en o t cπ o o t cn on Cn cn o to on cn to to t to en cn to on o cn Cn on o t on cπ cn to o to o on cn o cn on cn cn cn en to en o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o σ o o σ o o o o o o o σ o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o > > 5 y >> 3 > > > > > > > > > > > > > > > > > > IP t- > ι-3 <-3 > > > ι >-3 > ι-3 > >ι-3 > ι >-3 > O o O O o o O O O O O O o O O O o o O O O O O O O o O o O o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 co oo co oo oo co oo co oo oo co oo oo oo t t t ro to to to co oo co ro o o o o o o o o o o en cn o CD CO cn On ω t o on cπ ω o on to o en cπ ro o en cπ EC EC En EC 2 o Ω EC tn Ω EC 2 O Ω EC tn 2 En tn 2 Ω EC 2 tc EC Ω EC tn Ω EC EC Ω EC Ω tc 2 o Ω o o Ω tc tc Ω tc tn Ω EC Ω EC 2 o Ω EC tn Ω EC 2 CΛ Ω o En o Ω EC > > 2 to t tn ei ei D σ O Ω Ω Ω CO ω CO > 2 to to D Ω Ω Ω to to o 2 > 2 Ω ω to ω to to to to to to to to ro Ω Ω Ω Ω Ω Ω Ω C > > > > > > > > > > Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω E if tf f tf if 50 50 50 50 50 50 50 50 50 50 50 > i if if 5 50 50 0 50 50 > Ω Ω Ω Ω Ω Ω Ω Ω Ω iϋ 50 50 50 5 0 > 50 50 tf if tf if if if if tf tf tf tf tf tf tf if tf tf tf if tf tf tf | K DO K K << Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω a G a α σ G a G G G a G G a σ K K «; CΛ to to to to to to to to t to to to to t t t to to to to to to t t to to to t to t to to to to to to to to to t ro t ro to to ro to to 00 to to to to to to t to to to to t IO to to to to to t to to to to to o o o o o o o h- l h- 1 h- l o o cn ω t t o o o o o o o o CJI cπ cπ on cn on cπ on cn on on cn o to co cn co on cn to en on o on on cπ to cn on cn co o o cn to Cπ cπ ro cπ cn o o -j CD O to 00 Ji 00 to CD o on On en C Ji -J cn CO CO on on cπ On cn n 00 o to on Ji CO on en to o CD CD cπ CD on CO CO on GO cn o 00 1 1 1 1 1 1 1 1 1 I 1 1 1 1 1 1 1 I I 1 1 1 1 I 1 1 h- l 1 1 1 1 1 1 1 1 1 1 1 1 1 to on cπ cn on cn oo CO to co to o on cn o t to en o to en en o o t cπ to to en en cπ o ω on o on cπ en to on to to on o o Cπ to to on o on o cπ cn to cn ro on o to on on on on Cπ to on to cπ o π to on o ro on 1 1 I 1 1 I 1 1 1 1 1 1 1 1 1 1 I 1 1 [ o to o 00 h- 1 to Cπ Cπ Ji 00 to to i Ji 00 to t O o o o to Cn to to to to t t to to o t o o o on o cn o to 00 cπ o o to on on ro to on cn cn o ro on o to cn cπ Cπ to t o en Cπ o o cn to o o cπ o o cn on o cπ o o to on Cπ o o cπ to cπ on CJI o cn ro on o o o o o o o o o o o o o o o o o o o o σ o o o o o o o σ o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o α o o o o o o o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o σ σ o o o o o o o o o o o o o o > > 3 > > > ι-3 ι-3 ι-3 ι-3 > > > > > > > > > > > > > > > > > > > ι-3 ι-3 ι-3 ι-3 >-3 1-3 1-3 ι-3 ι-3 h ι-3 1-3 ι-3 1-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 i-3 ι-3 ι-3 h3 >-3 > 1-3 >ι-3 ι-3 > > ι-3 > ι-3 ι-3 >i-3 ι-3 >h3 U > > > ι-3 h 1-3 1- ι-3 h3 h3 ι-3 1-3 h3 3 h3 ι-3 O O O o o o O O o o o o O O O o O O o O O O o o O o O O O o o o o o O o O o o o O o o o O O O O o O O o O o o o o o o 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 22 ji j j=» i Ji Ji i j Ji 00 00 00 00 CO 00 00 co 00 GO 00 CO CO 00 GO 00 00 00 00 00 00 00 00 00 ω 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 CO 00 00 00 00 00 00 GO o o o o o o o o o CD CD CD CD CD CD CD CO CD CD 00 CO 00 CO CO 00 CO CO CO CO -J -J -J -J -J -J -J -J -J -J on cn cn on cn cn cn cn on cn On cn cπ On cπ cn cπ Cπ CJl Cπ CO -j en CJl Ji 00 ro h- i o CD CO -J cn n Ji 00 CO l-> o CD CO -J cn cπ Ji 00 to h- i o CD CO -J on cπ Ji 00 to h- o CD CO -j on On i oo ro h-> o CD CO ~J cn Cπ Ji 00 to h- i O Ω EC O o EC Ω tn Ω EC Ω EC Ω Ω EC EC Ω EC Ω EC 2 O Ω tn Ω EC Ω EC Ω EC Ω EC Ω Ω EC tc Ω EC Ω EC 2 O Ω EC Ω EC Ω EC Ω EC Ω EC Ω Ω EC tc Ω EC Ω tn EC tn tSl to CO to H α D D D Ω ω to ω h- CO CO h- i h- CO h- i > > 2 IN ESI CO to to CO D to to h- i CO α D to DO tn 2 N N M O CO O D O D D Ω to to to 2 ro to h- i ro h- 1 α Ω h- i to h- i to ro h- l h-> CO to h- i h- i to h- i h3 h3 H ■-3 ι-3 H ι-3 ι-3 ι-3 t-3 h3 h3 ι-3 h3 ι-3 ι-3 ι-3 H h3 ι-3 Tf TJ o TJ TJ Tf Tf T ) TJ TJ TJ Tf TJ TJ TJ TJ TJ Tf TJ TJ TJ T) TJ TJ TJ TJ TJ TJ TJ T) TJ TJ TJ TJ TJ TJ TJ TJ TJ K| < K h K K < EC EC EC EC EC EC EC EC EC EC EC EC EC EC tc EC EC tc EC EC EC EC EC tc EC EC EC tn tn EC EC EC EC EC EC tc EC EC EC 50 50 50 50 50 50 50 50 50 • 0 ia 0 50 io 50 V> 5 50 50 O CO to O CO CO CO CO O CO to CO CO O CO CO O CO CO CO CO CO O CO CO to to CO O to CO CO CO O CO CO O O CO IO O to to t CO to CO CO CO to to CO ro CO to CO to to to to CO to to ro ro to to to IO CO ro to ro to ro to ro to ro IO ro CO to to to CO to CO CO CO ro to ro ro t to co ro en en cn cn on on on cn en cn on on cn cn on on on on en on On On On cn On π on On cπ cπ n CJl Cπ Cπ Cπ on On cπ on on ji ji Ji Ji ji ji i Ji Ji Ji Ji ji ji ji Ji Ji Ji Ji Ji h- i h- i h- l - i h- h- i h- i h- i h- l h- i h- i h- i h- h- i h- i h- - i h- l h- i h- h- i h- l h- h- i h- i h- i h- i CO cn on - CD 00 en -j o CD CO CO CD h- l o o o o h- i h- l O O i ji 00 00 Ji Ji to 00 Ji 00 00 00 00 ro h- i h- i h- i σ CD CO Ji CJl cπ On Cπ CJl cn cn cn on cn -j -J -J CD CO CD h- l cn on cπ o CO ω 00 -J CO o to cn j ro on h- i CD 00 GO 00 CD en GO on -J 00 Ji CO to o CD Ji h- i ji CD h-> Ji - -j O on CO Ji cπ -J Cn -J on cn ji -j ro o Ji 00 CD ro Ji h- i h- i CO 00 to ji h- i -J o -j 00 00 -j h- i CD o Cn to to -J CO j O 00 00 cn CO Ji oo on -J o on h- i CO 00 o Ji O O cn -J cn 00 -J 00 -J i o cn o to o o o Ji l- i h- l GO j on h- i 00 CO CO h- i ro CD CO CO D Cπ oo Ji CO on 00 π ji h- i CO to CD ro i CO CJl Ji CD CΛ -J 00 h- i Ji 00 CO on CO Ji -J cn h- i h- i cn -j CO CO cn to CO GO 00 h- i o h- i CD Ji 1 1 I 1 1 1 1 1 1 1 1 en GO ji cπ cn cπ Ji On cn en on cπ cn on -j on cn On Ji Ji 00 00 o o o to to h- i o O o h- i CO h- i to h-> o o O O o o 00 to h- i o to to o o o o (- ■ h- i -j cn h- i j cn CD to GO o CO -j D On -4 ji on Ji 00 h- l to On CD en 00 to to h- i ro on 00 CO C --J -J O CD to -J CD 00 00 CO CD o CO 00 to to CD h- i CO GO 00 en CD CO CO On o cn on ji CD CO 00 ji CO CD -j CO CD h- CO cn h- i CO cn CO -J O o CO o cπ Cn o cπ CO Ji CO Ji CD -J -J -j CO CD CO -J to ro -j h- i h- i -J -j on to to -J Ji ro CO oo cn O -J h- i h- i CO o Ji -J CO en h- i i 00 o CO CD cn D Ji on cπ O 00 O on to Ji cπ Ji on -J h- i 00 to CO h- i Ji CO CO o to on O O on -j CO CO ro to CO CO CO to h- i CD Ji 1 1 1 o CJl cπ i on Ji to to Ji ji h- i to to ro h- 1 o o to h- l o o on Ji n i ro 00 Ji 00 h- i CO to o h- i h- i to h- i o h- i to CJl Ji j ji Ji 00 to to h- l CO ro o o o o o to h- h- i to 00 CD CO on CD 00 o o h- i cn O CO -J o on h- i 00 to oo GO CJl CD oo D h- i h- i 00 h-> to cπ Cπ cπ Ji on cπ to o 00 Ji Ji cn -j o o -J cπ CO CO Ji o 00 o -J h- i co en - ro h- i on to on o CD CD o ~ 00 CO -j o o on CO ji t ro h- i Ji 00 CD to CD on CD on o cπ o CO -J 00 CD 00 H> -J -J on h- o CO ω O ro ji j -J ji cπ o o cπ ro i t- i CD to to CO cn ji 00 to to on h- i cn o cπ 00 CD CO cn o on cπ Cπ *-j on -j -J CO 00 h- l o h- i CO h- i Ji h- i o 00 o on to ji 00 cn GO en en o CO CO ro 00 o Ji 00 on co cn h- i h- i h- i h- i h- i h- i h- i h- i - i h- i h- i h- i h- h- i h- i h- h- i h- i h- l h- i h- i h- i h- i h- i h- i h- i h- h- i h- i h- i h- i h- i h- i h- i h- i h- i I- 1 h- i h- i h- h- i h- i h- i - i h- h- l h- i h- i h- i h- i h- i h- i h- l h- l O O O o o o o o o O o O O σ o O O o O σ O o σ O o o O O o o o o o O o O o o o O o o o O o σ o o o O o o O o o o o o o O o O o o o o o o o o O o o o O O o O o O o o O o o O O o o o o o O o O o o o O o o o O o o o o o O o o O o o o o o o O o O o o o o o o o o O o o o O O o O o O o o o o o O O o o o o o O o O o o o O o o o O o o o o o o o o o o o o o o o O o O o o o o o o o o O o o o O O o O σ O o o o o σ O O o o o o o O o O o o o O o o o O o o o o o o o o o o o o o o o O o O o o o o o o o o O o o o O O o o o o o o o o o O O o o o o o O o O o O o O o o o O o o o o o o o o o o o o o o o > > > > > > > > > > > D > > > > > > > > > •-3 H > > > > > > > > > > > > > > > > > > > > O o o O o o o O O o O o O O O O O O O O o o o O o O O o O o O o O o O O o o o o o o o o o o o o o o o o o o o o o o o 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 on on en on cn en on on cn cπ cπ on cπ n oo to to ro to ro ro to to to o cn cn to o on o cn cπ to o oo cn o on cn to o oo cn on ro o EC EC EC EC Ω tn EC Ω EC Ω EC 2 O Ω EC EC EC 2 EC EC Ω EC tn Ω EC EC Ω EC EC Ω EC Ω En 2 O Ω o O Ω EC EC Ω EC Ω EC 2 o Ω Ω Ω Ω Ω tn o EC Ω tc Ω EC 2 o Ω Ω Ω DO ω to > 2 CO O to α D D Ω Ω Ω to ω DO 2 α O Ω to ω DO ro t to ro > > 2 to to Ω Ω Ω DO CO 2 to to (-> ro t to to to 2 2 2 2 2 2 2 2 2 2 if if tf if t-ι tf if if if if tr" tf tf tf tf tf if if tf O CO CO CO CO CO > > > > > > > > CO CO > K X X X K κl K X K K; K K EC EC tc tn EC EC EC EC EC EC EC EC tc «l to ia V 0 V 50 50 50 50 50 • 0 iϋ io 50 iϋ to ro to to to to to to to to to t to ro to to to to ro to to ro to to to to to to to to to to to to to to to to o o o o o o o on cn cn oo on on cn cn on cn to ro ro en to co cπ on cn on to to cπ on σ o o o to cn on cπ cπ ro cn cπ to cπ o cπ ro on n n ro on cn en o to on on on to ro to cn cn o on o on cn en cπ cπ on on to to to ro t o ro o to o cπ on cn on to cn o cn o ro to o on on cn on cn on o on o on o cn o to on en o cn o on o o on to on cπ cπ to o on on en o to n on cn to on o to o o ro o to en cπ on o o cn to o to on o cn 1 1 1 1 1 1 1 1 1 1 I t to t o to en on cn cπ cn cπ cπ to to to to to to o o o ro on on o on to cn on ro cπ n cn o to o o on on o on o to o en on to cn cn to to o o o o ro on o en on o o cn ro o ro o on o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o o o o o o o o > > > >-3 > ι-3 > >• > ι-3 ι-3 > > > > > > > > > ι-3 ι-3 ι-3 3 > > ι-3 ι-3 ι-3 ι-3 ι-3 > ι-3 > ι-3 > ι-3 > > > > > > > > > > > > > > > ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 > > ι n-3* ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 > ι-3 ι-3 ι- > O O O Ω o o o Ω o o o o o o o Ω o Ω Ω Ω Ω Ω Ω o Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω 2 2 2 2 2 o Ω o Ω Ω Ω Ω Ω o o Ω o o Ω Ω Ω Ω Ω Ω Ω o o Ω Ω Ω 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 On on on On cπ cπ cn Cπ cn cπ cπ Cπ cπ cn cπ cn to to to ro to o o o o o o o o o o co oo oo -J en on on co o on on to o on oo to o cn n to o cn cπ co to o on cπ to σ Ω EC EC Ω EC 2 o Ω EC EC EC 2 EC tn Ω tn EC Ω EC EC Ω EC EC Ω EC Ω EC 2 o Ω EC Ω EC Ω EC Ω EC Ω Ω EC 2 Ω EC Ω Ω EC EC Ω EC Ω tn 2 Ω Ω EC EC EC Ω CO > 2 N CO O O σ α o Ω Ω Ω DO DO DO > > 2 tn EC CO GO CO CO CO D o o Ω DO DO DO ro to to o > fr 2 CO to CO to D to to to to ro to to Ω Ω Ω Ω Ω Ω tf tf tf if tf if if tf tf tf tf tf tf if if tf tf tf if tf if If ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 >-9 ι-3 ι-3 ι-3 ι-3 ι-3 ι-3 ι ι-3 ι-3 ι-3 ι-3 2 2 2 2 2 2 2 tf if if if tf if X X X X X X X X X X X X X X X X X X X 50 50 50 50 0 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 iΛ 50 CO CO O O to to to X X x X X CΛ to CΛ co CΛ CO CΛ TJ TJ Tf TJ Tf TJ TJ TJ TJ TJ TJ TJ TJ TJ T) TJ TJ T) T) TJ TJ TJ Tf TJ ι-3 ι-3 ι-3 ι-3 ι-3 ω 00 00 co to t t ro t ro to ro to to to ro ro to to to to to ro to ro o o σ o o o o o o t to o cn to ro to to to ro to ro oo on cn Cπ on cn cn cn en o to cn on cn on on cn on ro cn en to to ro oo o to to to o On en cπ to cn cπ to on o cπ o to to cn o o o o n to cπ cπ Cπ cn cn o on cn cn cn on cπ ro o to o oo t o cπ cn cπ o on cn o to cπ to Cπ ro o o On o o o cπ on on cn on cπ to o o o o on en on on o o -J ro t on en ω o to on to o o o o o cn oo on o o o ro o ro cπ cπ to (-> on on On o cn to oo cπ on o cπ cn on on o o o on ro o on cπ cn cπ cπ o on ro o Cπ t 1 1 1 I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I 1 1 1 1 oo Ji 00 to to 00 00 ro to o o o o o ro o o o o o o cn to on to on cπ to to o o h- l o o to cπ on on on on co on o oo cn on to cn on o o cn n o o o to on cπ Cπ cn ro to on o o o ro on on o o on o to ro On cn on cn cπ on cn on to cπ on to cπ on Cπ en cn o en cn cn o o o o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o > D > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > Ω Ω Ω Ω Ω Ω o Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 cn cπ on cπ on cπ cπ cπ cn cπ cπ cn cn cπ on cπ cπ cπ cπ cπ on cπ on π cn on cn cπ cπ cn cπ cπ cπ cπ cπ on cπ cn cn cπ cn on on on on on on cn cn cn cn cn cn cπ cπ cπ cn t t cπ to σ oo en on o on cπ oo o en ji on ji o on o EC EC tn EC tn EC tn EC tc tc tn EC Ω tc EC Ω EC Ω EC 2 Ω Ω Ω O Ω EC Ω EG EC Ω EC Ω EC 2 Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω EC Ω EC Ω EC 2 Ω Ω D D Ω Ω Ω Ω Ω Ω Ω Ω EC Ω EC Ω EC 2 Ω Ω DO DO to 2 CO CO Ω O CO to to to Ω Ω DO DO to to α Ω 2 to ro to > 2 to σ ro to t to -> Ω (-> Ω ω to ω > > 2 Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω < < < < < < < < < < < < < < < < Ω tf tf if tf tf tf If tf if rr 1 if tf tf tf tf tf if tf tf t f tr 1 if tf tf if tf tf tf tf tf if tf if 2 2 2 2 2 2 2 3 G a α G a a a a G G G G G > > tr 1 > > > tf > > > > > f tf t tf tf tf tf > tf tf CO O ei ei ei CO e a ei ei ei O CO to O CO ei CO to X ω ω en cn on en cn cn on cn cn on on en on cn cn cπ cn cπ cπ cπ on cn cπ cπ 1 1 1 ( 1 1 to to Ji Ji to cπ ro ro to o o o o to ro o o O o to o o to o o o cn to cn o o ω to ro to on t on en ro o cn cπ cn cn to to ro cn on on o o to on to o en o on en ro cn on to cn on cn o to to o o en cn on on on en cn cn on o cn cπ cπ on cn cπ to ro o to cn cn to cπ cn o on ro to o o cn cn en on to to o on to on on o o o to cn o cn o to t cn π cn cπ ro o to cπ o on I 1 1 1 1 I 1 ! 1 1 1 1 1 cn cn cπ cπ Ji Ji to o o ro to to t to to o o o O o o o o o o o o o to on o on o en o o o o o ro on o t ω o on on ro cπ o to to on o on o o o ro o to to o cn o o o cπ cn t o on o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o o σ o σ o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o o o o >3 > >-3 >t-3 > ι-3 t-3 > t-3 > > > > > > > > i > > > > > f-3 f-3 ι f-3 f-3 f-3 ι-3 ι-3 ι-3 h3 t-3 ι-3 t-3 t-3 ι-3 f-3 t-3 3 ι-3 f-3 f-3 f-3 f-3 f-3 t-3 > f -3 t-3 t-3 > > > ; h3 f- > > ι-3 h3 f-3 1-3 > > t-3 i-3 >f-3 >f-3 1-3 > > f -3 f-3 f-3 f-3 f-3 ι-3 f-3 t-3 f-3 ι-3 f-3 t-3 f-3 ι-3 Ω Ω Ω Ω Ω Ω o Ω o Ω Ω Ω Ω O Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω O Ω Ω Ω Ω Ω Ω Ω Ω Ω O Ω Ω Ω Ω Ω o Ω Ω Ω Ω Ω Ω 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 cn on on cn cn on cn cn on cn en cn cn on cn cn en cn cn cn cn cn on cn cn cn on on on en on en on en on on on on cn cn on en on on on Cπ On on cn cn cπ CJl Cn cn cn cn on cπ CJl 00 00 00 00 co 00 00 CO Co 00 to to ro t ro ro ro to to O f- h- i h- i h- l l- i h- i o o o o o o o o o o CD CD CD CD CO D CO CD CD CD 00 CO CO CO CO CO o CD CO cn cn 00 to o CO CO cn n oo to o CD CO on cn ω CO o CO 00 cn on 00 to h- l o CD CO -J on cn 00 to o CD 00 -4 on EC tn tn EC EC Ω EC Ω EC 2 Ω Ω tn Ω EC tc Ω EC Ω tn 2 Ω Ω Ω Ω Ω Ω EC Ω EC Ω EC Ω EC 2 Co Ω Ω Ω EC tc EC Ω EC 2 O Ω Ω Ω EC EC EC Ω EC 2 Ω Ω to CO 2 Ω Ω EC EC DO > 2 Ω Ω to to 00 > > 2 t ro to Ω Ω Ω to to > 2 Ω DO DO DO CO to O Ω Ω to 00 ro ro h- l h- i to > 2 Ω DO DO to 2 CO ro to h- l t ro h- 1 ro h- i H H h-l h-l h-l to CΛ CΛ Λ CΛ O CO CΛ CΛ CΛ CO t-3 t-3 t-3 3 t-3 f-3 t-3 ι-3 t-3 t-3 ι-3 ι-3 f -3 t-3 Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω tf if tf tf tf GO to CO GO CO GO GO to CO CO GO tn EC EC EC EC EC EC EC EC EC tc EC EC EC X X f κl K X X h h X f X f X X if if tf tf f if if tf If CO O O ei GO iύ 50 50 50 50 0 50 50 50 0 50 io 0 50 50 50 50 ia 50 50 50 50 50 CΛ CΛ CΛ CΛ CΛ CΛ CΛ CΛ CΛ CΛ CO to CΛ CΛ CΛ CΛ CO CΛ CΛ CO 2 2 2 2 2 2 2 2 2 Ji Ji Ji Ji Ji Ji Ji Ji ω ω 00 00 CO 00 GO 00 ω 00 00 00 00 00 00 CO 00 00 GO GO CO 00 CO CO 00 00 00 00 00 to to to CO to h- l l- h- 1 h- i h- i h- i h- i h- i o O o o σ o o o o o o σ o o O CD CD CD CD CD CD CD D CO CO CO CO 00 CO 00 00 00 J ^J -4 -4 -4 1 1 1 1 1 1 1 1 1 1 1 1 I 1 1 1 1 1 I I 1 1 1 1 1 1 I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 h- CO CD O O O CD CO CO 00 o CD o CO CD O CO -J cn cn -J on -J CO CO CD oo on -J on CD CO Cπ CJl -J on cn to -J on 00 Ji cπ cπ 00 Ji cπ Ji ro 00 to ro to Ji 00 h- l on CO to O -J CD CD -J CO on CO CO h- l 00 CO CD ~4 en 00 h- CO Ji CO h- i ro ~j -j on 00 h- i h- i o 00 -J CO CO CD 00 CO h- l Ji ro en on on cn Ji CO h- l o cn 00 00 on -4 OO o cn cn cn Ji oo ro -J t cn CD h- l 00 CJl cn D CO on to On h- l Ji CD on D on CO on on CD CD -J O on -J 00 o 00 -4 00 00 to 00 cπ cn CD on CO n CD on 00 cn h- i cn O cn cn CO CO CO CD on cπ o 00 on to 00 00 σ σ on on CD O CO CO -J on o h- i on o CD CD oo oo to h- to cn ro to Jl -4 ω On ro 00 00 on π Cn CD CO en on -J -J OO CO CD O ro to to CD o h- i h- i o o CD D CO CO CO CO CO on -J Ji on on on cπ Ji π on -J -J -4 Cn cn on -4 CO CD cn Ji o CD on OO CO CD CO en CO o to Ji ro cn Ji -J o CO Ji 1 h- i on CO on 00 00 CO en cπ ro h- i -J ro 00 cn -J 00 cn cn 00 CJl D cn o to -4 Ji o 00 CO CD D o on CO ~J Ji CO CD on ro 00 -4 00 O cπ cπ 00 on 00 00 Ji 00 00 CO CO o Ji to CO h- i σ cn CO CO On on on CO CO ω on D on cn cn to o on CD on on CD 00 h- l h- i CD Ji to -J D CO CD cn 00 h- i oo to Ji to CO CO -J CO Cπ o CO to n cn CO o CO ro cn to o -J -o CO on o o Ji CO 00 CD 1 1 1 1 1 1 I 1 1 1 1 1 1 1 1 I 1 1 1 1 1 1 1 1 I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I 1 ro 00 Ji 00 to Ji cπ n cn 00 ro to o o to h- 1 to 00 to o o o 00 ro o o to o o o ro o 00 to to ro 00 cn CO -4 cn Cn ro 00 -J -J -J CD CO CO o CO to 00 cn cn CD CD cn CD o Ji en h- i h- i CO cn 00 o cπ to CJl 00 CO o h- i CJl On to -J h- i o to 00 CO cn t h- l on cπ on to on 00 00 h- i -J ro O CD CO cn -J h- 1 CO h- i ro 00 On h- i CO o CD o on O to CO to o CO -J o CD h- i 00 00 Ji on h- i -J Ji --J CO on Ji CO CO CO Cn cn 00 on CD h- l CD ro GO cπ h- i to CO O cn to CO o -J h- 1 CJl CD o 00 Ji CD 00 -j CO 00 CO cn Cn -J h-» cπ Ji o CD Ji on to on D to -J on 00 00 to CO Ji cn cπ on -j o 00 to CO 00 00 to -4 00 h- i h- l h- i h- i h- i h- i h- i h- i h-i h- i h- l h- i h- i h-» h- i h- l h- i h- i h- l h- l h- i h- l h- i h- i h- i h-> H» M h- i h- i h- i h- i h- l h- i h- h- i h- i h- i h- i h- 1 h- i o o o O o o O o O o o o O o o o O o O O o o O O o o O o O o o o o O o O o o O O o o O o σ σ o o O o o o o o O o O O o o o O o o σ o o o o o O o o o O o O o o o O O o o O o O o o o o O o O o o O O o o O o o o o o O o o o o o o o o O O o o o o o o o o o o o o O o o o O o O o o o O O o o O o o o o o o O o O o o O O o o O o o o o o o o o o o o o o o O O o o o o o o o o o o o o O o o o O o O o o o O O o o O o o o o o o O o O o o O O o o O o o o o o o o o o o o o o o O O o o o o o o o o o o o o O o o o O o O o o o O O o o O σ o o o o o O o O o o O O o o O o o o o o o o o o o o o o o O O > > 3s > > > > > > > > > > > ^ > > > > > > > > > > > > > > > > > > > > > > Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω o Ω Ω Ω Ω Ω Ω Ω o Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 on en cn on on on on cn cn on on cn cn on on on cn on on on cn cn cn on en on on on on en on on on on cn on on cn on cn cn on on on on on on cn cn on on on cn on on o o o oo on on on cn cn en cn on on en cπ cn cn cπ on on on cn n to o on cπ i to o en cπ oo ro o on cπ ro o on cn to o cn cn ji to o on EC EC tn EC EC EC EC EC EC EC tn tn EC EC Ω EC Ω EC 2 Ω Ω D D D Ω α D D Ω EC Ω EC EC Ω EC Ω EC 2 Ω Ω EC Ω EC EC Ω EC Ω EC 2 Ω Ω Ω EC EC EC Ω EC 2 Ω Ω O D D Ω Ω Ω Ω Ω Ω Ω Ω EC to > 2 to to to O o Ω Ω DO DO DO > 2 Ω Ω DO DO DO > > 2 Ω to DO DO 2 o ro t ro Ω Ω to to to ro to ro to if tf if tf If tf tf tf tf tf tf if if tr 1 tf tf if to Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω X X X X ei CO ei GO ei CO ei GO CO i ei ei ei CO i CO ei ei CO CO CO CO GO CO to GO CO GO GO X X X X X X tr 1 if if tf tf tf If If tf tf tf tf tf 50 50 50 50 50 a α G σ a G G G α G G a G σ c G a a 50 50 50 50 50 50 50 50 50 50 50 t GO GO ei ei CO CO ei CO i CO ei CO on on on on on n cπ n on π cn cn cn cπ π j ω to to ro to to ro to 1 I I 1 1 1 1 1 to o to ro cn cn on on on cn cn cn cn on on o o o to t o o on to o to cπ o to o cπ t to cn on cn cn ro o o cn o on oo o on on co co cn to on o cn on on o to on cπ on o cπ cπ cn cn o oo co t σ o cπ o to to o 1 1 1 1 ro -4 cπ to o o o o to t to to to o o o o o o cn o o o o co o σ cπ cπ on ro cn cn to to ro on to o cn on o ro cn cn o o to en to to to cn ro on o cn to to to on cn cπ to to t to o o cπ on n t o cπ o 1 1 1 1 ( 1 1 1 1 1 1 1 1 o en cn Ji ro cπ ji on cπ on cπ on cπ on on cn cn Ji to Ji to cn on on on on on cn cn o to ro cn to cn to ro on o cn o o cn cπ to to cn on to ro ro to o to oo to cn cπ on to to on o ro to to to on on o o ro on on o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > > H ι-3 > Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω O o Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω O Ω Ω Ω Ω Ω o o Ω Ω Ω Ω Ω Ω Ω Ω 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 oo oo oo oo oo oo to o o o o o o o o o oo en en on on cn cn on o oo on ro en cπ to o cn on cπ t o on ro o on cπ EC EC EC Ω tn Ω EC Z Ω Ω Ω EC EC tn Ω EC 2 Ω Ω EC Ω Ω tn Ω EC Ω EC Ω EC Ω Ω EC EC Ω EC Ω EC 2 Ω Ω O D 2 Ω Ω EC EC Ω EC Ω tn 2 Ω Ω Ω Ω Ω tn EC Ω DO to 2 Ω to DO DO Ω Ω to > 2 EC tn CO GO et CO D O α D Ω DO DO DO > α Ω DO DO DO to to ro to to > 2 D to to > 2 GO to σ Ω to ro > > > > > > Ω Ω Ω Ω Ω Ω Ω Ω Ω Ω IP > Ω Ω Ω Ω in to K X X X X X X | X X X X X X X X X X X X X X X X X X X co ω > > It- > ω D tn > > Ω Ω tn tn > Ω Ω ω tf tf tr 1 If tf tf tf tf 2 2 2 2 2 2 to tn tn 50 50 50 50 50 50 50 50 < 50 50 50 50 50 50 50 50 50 50 50 50 2 2 2 2 2 2 2 2 2 2 2 2 2 2 σ G G α C a G C on cn cπ cπ on cπ cπ on Cn cn cn cπ cn cn cn cπ cn cπ cn cn cn cπ on cπ on cπ cn on cπ to ro ro to ro ro ro o o o o o o o o ro ro o o on o on t to on on cn cn cπ cn cπ cπ cπ to on o σ to on o cn on on ro to o o t on to o to on en to on Cn o on cn cn cn o o on cn cπ oo on on t o to cn to en oo on o o cn o o on ro o co o cπ on o en to on cπ 1 1 1 I σ ro ro to to σ Cn to to o o o o o o o o o o o o o t to cn on cπ to cn on o on cπ cn on to o o o co ro Cn to on to to on oo cn o cn on on ro ro cn cn ro cn o cπ on o o Ji to o co cn o cπ i on on to oo to on on cn to 1 1 1 1 1 1 1 1 1 t to to to to cn to ro on ro cn ro Cπ on on ro o on cn o oo o on n cn on o o π o on cn en o cπ to to cn cn cπ on o on cn o en cn cn to cπ (-> on o on o to ro cn on ro to o on oo en oo o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o ATOM 822 HB2 ASN 53 12 384 -0 933 -1 828 1.00 0.00 ATOM 823 CG ASN 53 13 918 0 105 -2 919 1.00 0.00 ATOM 824 OD1 ASN 53 15 130 0 023 -2 861 1.00 0.00 ATOM 825 ND2 ASN 53 13 354 1 248 -3 198 1.00 0.00 ATOM 826 HD21 ASN 53 12 378 1 316 -3 245 1.00 0.00 ATOM 827 HD22 ASN 53 13 908 2 041 -3 361 1.00 0.00 ATOM 828 C ASN 53 12 920 -2 464 -4 773 1.00 0.00 ATOM 829 OT1 ASN 53 13 826 -2 084 -5 497 1.00 0.00 ATOM 830 OT2 ASN 53 12 553 -3 625 -4 690 1.00 0.00 END Example 3. Relative Folding Stability of Asp B28 IP Analogs. For evaluation of folding stability for insulin precursor analogs, denaturation samples were prepared by combining different ratios of insulin precursor analog and GuHCI stock solutions with 10 mM Tris/CI0 4 " , pH 8.0. Protein stock solutions were typically 0.06 mM in 10 mM Tris/CI0 4 " , pH 8.0. GuHCI stock solutions were 8.25 M in 10 mM Tris/CIOV, pH 8.0. CD spectra were recorded with a Jasco J-715 Spectropolarimeter calibrated with (+)-10- camphorsulfonic acid. All spectra were recorded at 20 °C. The denaturation samples were scanned from 250 to 218 nm. Typical cell path length and protein concentration were 0.5 cm and 3 μM, respectively. All spectra were smoothed before subtraction of appropriate solvent blanks. The circular dichroism is expressed as, Δε, based on the molar concentration of peptide bond. For presentation purpose each curve is normalized to a 0-1 scale by dividing the observed change at each point by the total change observed in the experiment. Data analysis. GuHCI denaturation curves were analyzed by assuming that the folding/unfolding transition is two-state as described by Santoro & Bolen (1988) Biochemistry 27:8063-8068 and Kaarsholm et al. (1993) Biochemistry 32:10773-10778, both of which publications are specifically incorporated herein by reference for teaching methods of calculating stability by GuHCI denaturation. This analysis yields a number of parameters including the GuHCI concentration at the midpoint of the denaturation curve, Cmid reflecting the concentration of denaturant necessary to unfold one-half of the protein population. An increase in folding stability is thus manifest by an increased value of Cmid. Equilibrium constants can be obtained at each denaturant concentration using K = (Δε N - Δε)/(Δε - Δεu), where Δε is the observed value of the CD, and Δε N and Δεu represent the CD values for native and unfolded forms, respectively, at the given GuHCI concentration (Pace, 1975). Values for Δε N and Δεu at GuHCI concentrations in the transition region are obtained by linear extrapolation of the pre- and post-transition baselines into the transition region, i.e. Δε N = Δε° N + m N [GuHCI], and Δε υ = Δε°u + mu.GuHCI], where Δε° N and Δε°u are intercepts, and m N and mu are slopes of the pre- and post-transition baselines, respectively. The free energy of unfolding at a given denaturant concentration in the transition zone is given by ΔG = -RTInK. Assuming a linear dependence of ΔG on denaturant concentration: ΔG = ΔG H Q - m[GuHCI], where ΔG H Q is the value of ΔG in the absence of denaturant, and m is a measure of the dependence of ΔG on denaturant concentration. Hence, ΔG values derived from K in the transition zone may be extrapolated back to 0 M denaturant to give ΔGu Q. The relationship between Δε and [GuHCI] for the complete unfolding curve is shown in Eq. 1 (Santoro & Bolen, 1988): A£ = (A Λ + m N [GuHCI]) + (A ε ° υ + mu [GuHCI]) ex f-f Δ G H2 „ -m[GuHO])/RT) l + exp(-(AG H2o -m[GuHCl])/RT) With Δε as the response and [GuHCI] as the independent variable, eq. (1) is subject to nonli- near least squares analysis using the NLIN procedure of PC SAS (SAS Inc. Cary, North Carolina). Six parameters then describe the denaturation curve: Δε° N , Δε°u, m N , u, m, and ΔG l_l Q. In addition, the GuHCI concentration at the midpoint of the denaturation curve, Cmid, is given by ΔG H 0 /m. Evaluation of the relative folding stability of Asp B28 IP derivative molecules with C- peptide Met Trp Lys (Asp B 8 IP(MetTrpLys)) was evaluated relative to Asp B28 IP. The results show that the Asp B28 IP(MetTrpLys) molecule was much more stable than Asp B28 IP (Fig. 5), as evidenced by the change in Cmid. While Cmid for Asp B28 IP is approximately about 5.5 M GuHCI, that of Asp B28 IP(MetTrpLys) is increased to at least about 6.5 M GuHCI, an increase of approximately 18%. Example 4 The insulin analogue precursor Asp B28 IP(EWK) was produced culturing yeast strain MT663 transformed with an expression plasmid expressing a YAP3-TA39-EEGEPK(SEQ ID NO: 17)- Asp B28 IP(EWK) fusion protein or a YAP3-TA57-EEGEPK(SEQ ID NO:17)-Asp B28 IP(EWK) fusion protein. cDNA encoding the leader sequences YAP3-TA39 and YAP3-TA57 and cDNA encoding the Asp B28 IP(EWK) and the N-terminal extension were cloned into an expression vector of the C-POT type using standard techniques (Sambrook J, Fritsch EF and Maniatis T, Mo- lecular cloning, Cold spring Harbour laboratory press, 1989). The DNA and inferred amino acids sequences are shown in Fig 8 and 9. Table 6 shows the yields. Fermentation was conducted at 30°C for 72 h in 5 ml YPD. IP yield was determined by RP-HPLC of the culture supernatant and is expressed relative to the IP yield of a control strain. In Table 6, "α*" indicates an α-factor leader in which the C-terminus up to the LysArg has been modified from "SLD (SerLeuAsp)" to "SMA (SerMet Ala)" and "ex4" is an N-terminal extension with the amino acid sequence EEAEAEAPK(SEQ ID NO:4). YAP3 is the YAP3 signal sequence. TA39 is a synthetic pro-sequence QPIDDTESNTTSVNLMADD- TESRFATNTTLAGGLDWNLISMAKR (SEQ ID NO: 16). The sequence EEGEPK(SEQ ID NO: 17) is an N-terminal extension to the B-chain of the insulin analogue. TA57 is a synthetic pro-sequence QPIDDTESQTTSVNLMADDTESAFATQTNSGGLDWGLISMAKR (SEQ ID NO: 18). TABLE 6

Claims: What we claim is: 1. 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. 2. An insulin precursor or insulin precursor analog according to claim 1 , wherein the connecting peptide is of up to 15 amino acid residues in length. 3. An insulin precursor or insulin precursor analog according to claim 1 , wherein the connecting peptide is of up to 9 amino acid residues. 4. An insulin precursor or insulin precursor analog according to claim 1 , wherein the connecting peptide is of up to 5 amino acid residues, preferably up to 3 amino acid residues. 5. An insulin precursor or insulin precursor analog according to claim 1 , wherein the cleavage site enabling cleavage of the peptide bond between the A-chain and the connecting peptide is Lys or Arg. 6. An insulin precursor or insulin precursor analog according to claim 1 , wherein the connecting peptide comprises up to 5 aromatic amino acid residues 7. An insulin precursor or insulin precursor analog according to claim 1 , wherein the connect- ing peptide comprises up to 3 aromatic amino acid residues, preferably only one aromatic amino acid residue. 8. An insulin precursor or insulin precursor analog according to claim 7, wherein one of the aromatic amino acid residues is less than 5 A away from at least one of the amino acid residues in position B11 , B12 or B26 in the B chain. 9. An insulin precursor or insulin precursor analog according to claim 1 , wherein the aromatic amino acid residue immediately N-terminal to the cleavage site is less than 5 A away from at least one of the amino acid residues in position B11 , B12 or B26 in the B chain. 10. An insulin precursor or insulin precursor analog according to claim 1 , wherein the insulin precursor or insulin precursor analog exhibits an increased Cmid stability in solution relative to an insulin precursor or insulin precursor analog which does not comprise an aromatic ami- no acid residue in the connecting peptide. 11. An insulin precursor or insulin precursor analog of claim 10, wherein Cmid is higher than about 5.5 M GuHCI. 12. An insulin precursor or insulin precursor analog of claim 10, wherein Cmid is higher than about 6.0 M GuHCI. 13. An insulin precursor or insulin precursor analog of claim 10, wherein Cmid is at least about 6.5 M GuHCI. 14. 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. 15. An insulin precursor or insulin precursor analog according to claim 14, wherein the connecting peptide is of up to 5 amino acid residues. 16. An insulin precursor or insulin precursor analog according to claim 14, wherein the connecting peptide is of up to 3 amino acid residues in length. 17. An insulin precursor or insulin precursor analog according to claim 14, wherein the connecting peptide comprises up to 5 aromatic amino acid residues. 18. An insulin precursor or insulin precursor analog according to claim 14, wherein the connecting peptide comprises up to 3 aromatic amino acid residues, preferably one aromatic amino acid residue. 19. An insulin precursor or insulin precursor analog according to claim 14, wherein the connecting peptide has a Lys or Arg immediately N-terminal to the A chain. 20. An insulin precursor or insulin precursor analog according to claim 19, wherein one aromatic amino acid residue is immediately N-terminal to the Lys or Arg. 21. An insulin precursor or insulin precursor analog according to claim 14, wherein one of the aromatic amino acid residues in the connecting peptide is less than 5 A away from at least one of the residues in positions B11, B12, or B26 in the B-chain. 22. An insulin precursor or insulin precursor analog according to claim 20, wherein the aromatic amino acid residues positioned immediately N-terminal to the Lys or Arg is less than 5 A away from at least one of the residues in positions B11 , B12, or B26 in the B-chain. 23. An insulin precursor or insulin precursor analog according to claim 14, wherein the insulin precursor or insulin precursor analog exhibits an increased Cmid stability in solution relative to an insulin precursor or insulin precursor analog which does not comprise an aromatic ami- no acid residue in the connecting peptide. 24. An insulin precursor or insulin precursor analog of claim 23, wherein Cmid is higher than about 5.5 M GuHCI. 25. An insulin precursor or insulin precursor analog of claim 23, wherein Cmid is higher than about 6.0 M GuHCI. 26. An insulin precursor or insulin precursor analog of claim 23, wherein Cmid is at least about 6.5 M GuHCI. 27. 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 A away from at least one of the residues in position B11 , B12, or B26 in the B-chain. 28. An insulin precursor or insulin precursor analog according to claim 27, wherein the connecting peptide is of up to 15 amino acid residues in length. 29. An insulin precursor or insulin precursor analog according to claim 27, wherein the connecting peptide is of up to 9 amino acid residues. 30. An insulin precursor or Insulin precursor analog according to claim 27, wherein the con- necting peptide is of up to 5 amino acid residues. 31. An insulin precursor or insulin precursor analog according to claim 27, wherein the connecting peptide is of up to 3 amino acid residues. 32. An insulin precursor or insulin precursor analog according to claim 27, wherein the connecting peptide comprises up to 5 aromatic amino acid residues. 33. An insulin precursor or insulin precursor analog according to claim 27, wherein the connecting peptide comprises up to 3 aromatic amino acid residues, preferably only one aromatic amino acid residue. 34. An insulin precursor or insulin precursor analog according to claim 27, wherein the connecting peptide has a Lys or Arg immediately N-terminal to the A chain. 35. An insulin precursor or insulin precursor analog according to claim 34, wherein one aromatic amino acid residue is immediately N-terminal to the Lys or Arg. 36. An insulin precursor or insulin precursor analog according to claim 35, wherein the aromatic amino acid residues positioned immediately N-terminal to the Lys or Arg is less than 5 A away from at least one of the residues in positions B11 , B12, or B26 in the B-chain. 37. An insulin precursor or insulin precursor analog according to claim 27, wherein the insulin precursor or insulin precursor analog exhibits an increased Cmid stability in solution relative to an insulin precursor or insulin precursor analog which does not comprise an aromatic ami- no acid residue in the connecting peptide. 38. An insulin precursor or insulin precursor analog of claim 37, wherein Cmid is higher than about 5.5 M GuHCI. 39. An insulin precursor or insulin precursor analog of claim 37, wherein Cmid is higher than about 6.0 M GuHCI. 40. An insulin precursor or insulin precursor analog of claim 37, wherein Cmid is at least about 6.5 M GuHCI. 41. An insulin precursor or an insulin precursor analog comprising the formula : B(1-27) - X 2 - X 3 - Xi - Y - A(1-21) wherein Xi is a peptide sequence of 1 - 8 amino acid residues of which at least one is an aromatic amino acid residue, X 2 is one of Pro, Asp, Lys, or lie at position 28 of the B chain, X 3 is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain, and Y is Lys or Arg. 42. An insulin precursor or insulin precursor analog of claim 41 , wherein X^ is 1-6 amino acid residues. 43. An insulin precursor or insulin precursor analog of claim 41 , wherein X^ is 1-4 amino acid residues. 44. An insulin precursor or insulin precursor analog of claim 41 , wherein X-i is 1-3 amino acid residues. 45. An insulin precursor or insulin precursor analog of claim 41 , wherein X^ is 1-2 amino acid residues. 46. An insulin precursor or insulin precursor analog of claim 41 , wherein X^ comprises up to 5 aromatic amino acid residues. 47. An insulin precursor or insulin precursor analog of claim 41 , wherein X 1 comprises up to 3 aromatic amino acid residues, preferably only one aromatic amino acid residue. 48. An insulin precursor or insulin precursor analog of claim 41 , wherein the aromatic amino acid residues are selected from the group consisting of Trp and Tyr. 49. An insulin precursor or insulin precursor analog of claim 41, wherein one aromatic amino acid residues is less than 5 A away from at least one of the amino acid residues in position B11 , B12 or B26 in the B chain. 50. An insulin precursor or insulin precursor analog of claim 41 , wherein one aromatic amino acid residue is immediately N-terminal to Y. 51. An insulin precursor or insulin precursor analog according to claim 50, wherein the aromatic amino acid residue immediately N-terminal to Y is less than 5 A away from at least one of the amino acid residues in position B11 , B12 or B26 in the B chain. 52. An insulin precursor or insulin precursor analog of claim 41 , wherein X-i - Y is selected from the group of: (a) Met-Trp-Lys, (b) Ala-Trp-Lys, (c) Val-Trp-Lys, (d) lle-Trp-Lys, (e) Leu- Trp-Lys, (f) Glu-Glu-Phe-Lys (SEQ ID NO:15), (g) Glu-Phe-Lys, (h) Glu-Trp-Lys, (i) Ser-Trp- Lys, (j) Thr-Trp-Lys, (k) Arg-Trp-Lys, (I) Glu-Met-Trp-Lys (SEQ ID NO: 1), (m) Gln-Met-Trp- Lys (SEQ ID NO:2), and (n) Asp-Trp-Lys. 53. An insulin precursor or insulin precursor analog of claim 41 , wherein X 2 is Asp, X 3 is Lys and Xi is 1-3 amino acid residues of which one is Trp or Phe. 54. An insulin precursor or insulin precursor analog according to claim 41 , wherein the insulin precursor or insulin precursor analog exhibits an increased Cmid stability in solution relative to an insulin precursor or insulin precursor analog which does not comprise an aromatic ami- no acid residue in X^ 55. An insulin precursor or insulin precursor analog of claim 54, wherein Cmid is higher than about 5.5 M GuHCI. 56. An insulin precursor or insulin precursor analog of claim 54, wherein Cmid is higher than about 6.0 M GuHCI. 57. An insulin precursor or insulin precursor analog of claim 54, wherein Cmid is at least about 6.5 M GuHCI. 58. An insulin precursor or an insulin precursor analog comprising the formula: B(1-27) - X 2 - X 3 - Xi - Y - A(1-21) wherein Xi is a peptide sequence of 1 - 15 amino acid residues of which one is an aromatic amino acid residue immediately N-terminal to Y, X 2 is one of Pro, Asp, Lys, or lie at position 28 of the B chain, X 3 is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain, and Y is Lys or Arg. 59. An insulin precursor or insulin precursor analog of claim 58, wherein Xi is 1-9, preferably 1-5 amino acid residues. 60. An insulin precursor or insulin precursor analog of claim 58, wherein Xi is 1-3, preferably 1-2 amino acid residues. 61. An insulin precursor or insulin precursor analog of claim 58, wherein X 1 is 1-2 amino acid residues of which one is Trp or Phe. 62. An insulin precursor or insulin precursor analog of claim 58, wherein X - Y is selected from the group of: (a) Met-Trp-Lys, (b) Ala-Trp-Lys, (c) Val-Trp-Lys, (d) lle-Trp-Lys, (e) Leu- Trp-Lys, (f) Glu-Glu-Phe-Lys (SEQ ID NO:15), (g) Glu-Phe-Lys, (h) Glu-Trp-Lys, (i) Ser-Trp- Lys, G) Thr-Trp-Lys, (k) Arg-Trp-Lys, (I) Glu-Met-Trp-Lys (SEQ ID NO: 1), (m) Gln-Met-Trp- Lys (SEQ ID NO:2), and (n) Asp-Trp-Lys. 63. An insulin precursor or insulin precursor analog of claim 58, wherein X 2 is Asp, X 3 is Lys and X-i is of 1-3 amino acid residues of which one is Trp or Phe. 64. An insulin precursor or insulin precursor analog according to claim 58, wherein the insulin precursor or insulin precursor analog exhibits an increased Cmid stability in solution relative to an insulin precursor or insulin precursor analog which does not comprise an aromatic ami- no acid residue in Xi. 65. An insulin precursor or insulin precursor analog of claim 64, wherein Cmid is higher than about 5.5 M GuHCI. 66. An insulin precursor or insulin precursor analog of claim 64, wherein Cmid is higher than about 6.0 M GuHCI. 67. An insulin precursor or insulin precursor analog of claim 64, wherein Cmid is at least about 6.5 M GuHCI. 68. An insulin precursor or an insulin precursor analog comprising the formula : B(1-27) - X 2 - X 3 - X - Y - A(1-21) wherein Xi is a peptide sequence of 1 - 15 amino acid residues of which one is an aromatic amino acid residue which is less than 5 A away from at least one of the amino acid residues in position B11, B12 or B26 in the B chain, X 2 is one of Pro, Asp, Lys, or lie at position 28 of the B chain, X 3 is one of Pro, Lys, Ala, Arg or Pro-Thr at position 29 of the B chain, and Y is Lys or Arg. 69. An insulin precursor or insulin precursor analog of claim 68, wherein Xi is 1-9, preferably 1-5 amino acid residues. 70. An insulin precursor or insulin precursor analog of claim 68, wherein X-i is 1-4, preferably 1-3 amino acid residues. 71. An insulin precursor or insulin precursor analog of claim 68, wherein X-i is 1-2 amino acid residues. 72. An insulin precursor or insulin precursor analog of claim 68, wherein Xi comprises up to 5 aromatic amino acid residues. 73. An insulin precursor or insulin precursor analog of claim 68, wherein X-i comprises up to 3 aromatic amino acid residues, preferably only one aromatic amino acid residue. 74. An insulin precursor or insulin precursor analog of claim 68, wherein the aromatic amino acid residues are selected from the group consisting of Trp and Tyr. 75. An insulin precursor or insulin precursor analog of claim 75, wherein one aromatic amino acid residue is immediately N-terminal to Y. 76. An insulin precursor or insulin precursor analog according to claim 68, wherein the aromatic amino acid residue immediately N-terminal to Y is less than 5 A away from at least one of the amino acid residues in position B11, B12 or B26 in the B chain. IT. An insulin precursor or insulin precursor analog of claim 68, wherein Xi - Y is selected from the group of: (a) Met-Trp-Lys, (b) Ala-Trp-Lys, (c) Val-Trp-Lys, (d) lle-Trp-Lys, (e) Leu- Trp-Lys, (f) Glu-Glu-Phe-Lys (SEQ ID NO:15), (g) Glu-Phe-Lys, (h) Glu-Trp-Lys, (i) Ser-Trp- Lys, (j) Thr-Trp-Lys, (k) Arg-Trp-Lys, (I) Glu-Met-Trp-Lys (SEQ ID NO: 1), (m) Gln-Met-Trp- Lys (SEQ ID NO:2), and (n) Asp-Trp-Lys. 78. An insulin precursor or insulin precursor analog of claim 68, wherein X 2 is Asp, X 3 is Lys and Xi is 1-3 amino acid residues of which one is Trp or Phe. 79. An insulin precursor or insulin precursor analog according to claim 68, wherein the insulin precursor or insulin precursor analog exhibits an increased Cmid stability in solution relative to an insulin precursor or insulin precursor analog which does not comprise an aromatic ami- no acid residue in X-i. 80. An insulin precursor or insulin precursor analog of claim 79, wherein Cmid is higher than about 5.5 M GuHCI. 81. An insulin precursor or insulin precursor analog of claim 79, wherein Cmid is higher than about 6.0 M GuHCI. 82. An insulin precursor or insulin precursor analog of claim 79, wherein Cmid is at least about 6.5 M GuHCI. 83. A polynucleotide sequence encoding an insulin precursor or insulin precursor analog according to claim 1. 84. A polynucleotide sequence encoding an insulin precursor or insulin precursor analog according to claim 14. 85. A polynucleotide sequence encoding an insulin precursor or insulin precursor analog according to claim 27. 86. A polynucleotide sequence encoding an insulin precursor or insulin precursor analog ac- cording to claim 41. 87. A polynucleotide sequence encoding an insulin precursor or insulin precursor analog according to claim 58. 88. A polynucleotide sequence encoding an insulin precursor or insulin precursor analog according to claim 68. 89. An expression vector comprising a polynucleotide sequence according to any of claims 83 - 88. 90. A host cell transformed with a vector of claim 89. 91. A process for making an insulin precursor or an insulin precursor analog said method comprising (i) culturing a host cell comprising a polynucleotide sequence encoding an insulin precursor or an insulin precursor analog according to any of claims 1-82 under suitable cul- ture conditions for expression of said precursor or precursor analog; and (ii) isolating the expressed precursor or precursor analog. 92. A process according to claim 91, wherein the host cell is a yeast host cell . 93. A process for making insulin or an insulin analog, said method comprising (i) culturing a host cell comprising a polynucleotide sequence encoding an insulin precursor or an insulin precursor analog according to any of claims 1-82 under suitable culture conditions for expression of said precursor or precursor analog; (ii) isolating the precursor or precursor analog from the culture medium and (iii) converting the precursor or precursor analog into insulin or an insulin analog by in vitro chemical or enzymatic conversion. 94. A process according to claim 93, wherein the host cell is a yeast host cell .

Inventors: KAARSHOLM NIELS CHRISTIAN, KJELDSEN THOMAS BOERGLUM, LUDVIGSEN SVEND, KAARSHOLM, NIELS, CHRISTIAN, KJELDSEN, THOMAS, BOERGLUM, LUDVIGSEN, SVEND
Applicants: NOVO NORDISK AS, NOVO NORDISK A/S
Application year: 2000
Main classification: C07K14
IPC codes: C07K14, C12N1, C12N5, C12N15, C12P21