WO1995035384A1 - Proteines comportant une extension a l'extremite n-terminale, exprimees par une levure - Google Patents

Proteines comportant une extension a l'extremite n-terminale, exprimees par une levure Download PDF

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WO1995035384A1
WO1995035384A1 PCT/DK1995/000250 DK9500250W WO9535384A1 WO 1995035384 A1 WO1995035384 A1 WO 1995035384A1 DK 9500250 W DK9500250 W DK 9500250W WO 9535384 A1 WO9535384 A1 WO 9535384A1
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glu
ala
lys
asp
yeast
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PCT/DK1995/000250
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English (en)
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Knud Vad
Jakob Brandt
Thomas Børglum KJELDSEN
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Novo Nordisk A/S
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Priority to AU27335/95A priority Critical patent/AU2733595A/en
Priority to EP95922440A priority patent/EP0765395A1/fr
Priority to BR9508053A priority patent/BR9508053A/pt
Priority to JP50150596A priority patent/JP3730255B2/ja
Priority to MX9606536A priority patent/MX9606536A/es
Priority to PL95317761A priority patent/PL317761A1/xx
Publication of WO1995035384A1 publication Critical patent/WO1995035384A1/fr
Priority to FI965030A priority patent/FI965030A/fi
Priority to NO965411A priority patent/NO965411L/no

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention relates to polypeptides expressed and processed in yeast, a DNA construct comprising a DNA sequence encoding such polypeptides, vectors carrying such DNA frag ⁇ ments and yeast cells transformed with the vectors, as well as a process of producing heterologous proteins in yeast.
  • Yeast organisms produce a number of proteins synthesized in- tracellularly, but having a function outside the cell. Such extracellular 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 presequence ensuring effective direction of the expressed product across the membrane of the endoplasmic reticulum (ER) .
  • the presequence 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.
  • 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, S.R. and Rothman, J.E. Ann.Rev.Biochem. 56 (1987), 829-852) .
  • Euro ⁇ pean Publication No. 0088632A describes a process by which proteins heterologous to yeast are expressed, processed and secreted by transforming a yeast organism with an expression vector harbouring DNA encoding the desired protein and a signal peptide, preparing a culture of the transformed organism, growing the culture and recovering the protein from the culture medium.
  • the signal peptide may be the desired proteins own signal peptide, a heterologous signal peptide or a hybrid of native and heterologous signal peptide.
  • a problem encountered with the use of signal peptides hetero- logous to yeast might be that the heterologous signal peptide does not ensure efficient translocation and/or cleavage after the signal peptide.
  • the Saccharomyces cerevisiae MF ⁇ l ( ⁇ -factor) is synthesized as a prepro form of 165 amino acids comprising a 19 amino acids long signal- or prepeptide followed by a 64 amino acids long "leader” or propeptide, encompassing three N-linked glycosylation sites followed by (LysArg(Asp/Glu, Ala) 2.3 ⁇ - factor) 4 (Kurjan, J. and Herskowitz, I. Cell 30 (1982), 933- 943) .
  • the signal-leader part of the preproMF ⁇ l has been widely employed to obtain synthesis and secretion of heterologous proteins in S ⁇ cerivisiae.
  • EP 0123289A utilization of the S_ s. cerevisiae ⁇ -factor pre ⁇ cursor is described whereas EP 100561 describes the utilization of the Saccharomyces cerevisiae PH05 signal and PCT Publication No. WO 95/02059 describes the utilization of YAP3 signal peptide for secretion of foreign proteins.
  • EP 206,783 discloses a system for the secretion of polypep ⁇ tides from £>_;_ cerevisiae whereby the ⁇ -factor leader sequence has been truncated to eliminate the four ⁇ -factor peptides present on the native leader sequence so as to leave the leader peptide itself fused to a heterologous polypeptide via the ⁇ -factor processing site Lys-Arg-Glu-Ala-Glu-Ala. This construction is indicated to lead to an efficient process of smaller peptides (less than 50 amino acids) .
  • the native ⁇ - factor leader sequence has been truncated to leave one or two ⁇ -factor peptides between the leader peptide and the polypep ⁇ tide.
  • a number of secreted proteins are routed so as to be exposed to a proteolytic processing system which can cleave the pep ⁇ tide bond at the carboxy end of two consecutive basic amino acids.
  • This enzymatic activity is in S_ ⁇ _ cerevisiae encoded by the KEX 2 gene (Julius, D.A. et al. , Cell 37 (1984b), 1075).
  • Processing of the product by the KEX 2 protease is needed for the secretion of active S_;_ cerevisiae mating factor ⁇ l (MF ⁇ l or ⁇ -factor) but is not involved in the secretion of active S. cerevisiae mating factor a.
  • WO 90/10075 describes a yeast expression system with improved processing of a heterologous polypeptide obtained by providing certain modifications near the processing site at the C-terminal end of the leader peptide and/or the N- terminal end of a heterologous polypeptide fused to the leader peptide.
  • a heterologous polypeptide obtained by providing certain modifications near the processing site at the C-terminal end of the leader peptide and/or the N- terminal end of a heterologous polypeptide fused to the leader peptide.
  • the present invention describes modifications of the N- terminal end of the heterologous polypeptide designed as extensions which can be cleaved off either by naturally occurring yeast proteases before purification from the culture media or by in vitro proteolysis during or subsequently to purification of the product from the culture media.
  • the present invention relates to a DNA construct encoding a polypeptide having the following structure signal peptide-leader peptide-X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -heterologous protein wherein X 1 is Lys or Arg; X 2 is Lys or Arg, X 1 and X 2 together defining a yeast processing site; X 3 is Glu or Asp;
  • X 4 is a sequence of amino acids with the following structure (A " B) n wherein
  • A is Glu or Asp
  • X 4 is a sequence of amino acids with the following structure
  • C is Glu or Asp, and m is 0 or an integer from 1 to 5;
  • X 5 is a peptide bond or is one or more amino acids which may be the same or different;
  • X 6 is a peptide bond or an amino acid residue selected from the group consisting of Pro, Asp, Thr, Glu, Ala and Gly; and
  • X 7 is Lys or Arg.
  • the term "signal peptide” is under ⁇ stood to mean a presequence which is predominantly hydropho- bic in nature and present as an N-terminal sequence on the precursor form of an extracellular protein expressed in yeast.
  • the function of the signal peptide is to allow the he ⁇ terologous protein to be secreted to enter the endoplasmic reticulum.
  • the signal peptide is normally cleaved off in the course of this process.
  • the signal peptide may be hetero ⁇ logous or homologous to the yeast organism producing the pro ⁇ tein but a more efficient cleavage of the signal peptide may be obtained when it is homologous to the yeast organism in question.
  • leader peptide is understood to indicate a predominantly hydrophilic peptide whose function is to allow the heterologous protein to be secreted to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory vesicle for secretion into the medium, (i.e. exportation of the expressed protein or polypeptide across the cell wall or at least through the cellular membrane into the periplasmic space of the cell) .
  • heterologous protein is intended to indicate a protein or polypeptide which is not produced by the host yeast organism in nature.
  • X 3 -X 4 -X 5 -X 6 -X 7 together form an extension at the N-terminal of the heterologous polypeptide.
  • This extension not only increases the fermentation yield but is due to the presence of X 3 , protected against dipeptidyl aminopeptidase (DPAP A) processing, resulting in a homogenous N-terminal of the polypeptide.
  • DPAP A dipeptidyl aminopeptidase
  • the extension may be constructed in such a way that it will be cleaved off by naturally occurring yeast proteases other than DPAP such as, for instance, yeast aspartic protease 3 (YAP3) before purification of the heterologous protein product from the culture media.
  • the extension may be constructed in such a way that it is resistant to proteolytic cleavage during fermentation so that the N-terminally extended heterologous protein product may be purified from the culture media for subsequent in vitro maturation, e.g. by trypsin, Achromobacter lyticus protease I or enterokinase.
  • the invention relates to a process for producing a heterologous protein in yeast, comprising cultivating the transformed yeast strain in a suitable medium to obtain expression and secretion of the heterologous protein, after which the protein is isolated from the medium.
  • n is preferably 2-4 and more preferably 3.
  • X 3 may be Glu
  • A may be Glu
  • B may be Ala
  • X 5 may be a peptide bond or Glu
  • Glu Pro Lys Ala or X 6 may be Pro or a peptide bond.
  • Examples of possible N-terminal extensions X 3 -X 4 -X 5 -X 6 -X 7 are:
  • the signal peptide sequence of the polypeptide of the inven- tion may be any signal peptide which ensures an effective di- rection of the expressed polypeptide into the secretory path ⁇ way of the cell.
  • the signal peptide may be a naturally oc ⁇ curring signal peptide or a functional part thereof, or it may be a synthetic peptide.
  • Suitable signal peptides have been found to be the ⁇ -factor signal peptide, the signal peptide of mouse salivary amylase, a modified carboxypeptidase signal peptide or the yeast BAR1 signal peptide and the yeast aspartic protease 3 (YAP3) signal peptide.
  • the mouse salivary amylase signal sequence is desc- ribed by 0.
  • the leader peptide sequence of the polypeptide of the inven ⁇ tion may be any leader peptide which is functional in direct ⁇ ing the expressed polypeptide through the endoplasmic reticulum and further along the secretory pathway.
  • Possible leader sequences which are suited for this purpose are natural leader peptides derived from yeast or other organisms, such as the ⁇ -factor leader or a functional analogue thereof.
  • the leader peptide may also be a synthetic leader peptide, e.g. one of the synthetic leader peptides disclosed in PCT Publication No. WO 89/02463 or WO 92/11378.
  • the N-terminally extended heterologous protein produced by the method of the invention may be any protein which may advantageously be produced in yeast.
  • pro ⁇ teins are aprotinin, tissue factor pathway inhibitor or other protease inhibitors, and insulin or insulin precursors, insulin analogues, insulin-like growth factor I or II, human or bovine growth hormone, interleukin, tissue plasminogen activator, glucagon, glucagon-like peptide-1 (GLP-1) , Factor VII, Factor VIII, Factor XIII, platelet-derived growth fac- tor, enzymes, or a functional analogue of anyone of these proteins.
  • the term "functional analogue” is meant to indicate a polypeptide with a similar function as the native protein (this is intended to be understood as relating to the nature rather than the level of biological activity of the native protein) .
  • the polypeptide may be structurally similar to the native protein and may be derived from the native protein by addition of one or more amino acids to either or both the C- and N-terminal end of the native protein, substitution of one or more amino acids at one or a number of different sites in the native amino acid sequence, deletion of one or more amino acids at either or both ends of the native protein or at one or several sites in the amino acid sequence, or insertion of one or more amino acids at one or more sites in the native amino acid sequence.
  • modifications are well known for several of the proteins mentioned above.
  • suitable insulin precursors and insulin analogues are B(l-29) -Ala-Ala-Lys-A(l-21) (as described in, e.g., EP 163 529), B(l-27)-Asp-Lys-Ala-Ala-Lys-A(l-21) (as described in, e.g., PCT Publication No. 95/00550), B(l-29)-Ala-Ala-Arg- A(l-21) (as described in , e.g., PCT Publication No. 95/07931) , and B(1-29) -Ser-Asp-Asp-Ala-Arg-A(1-21) .
  • the DNA construct of the invention encoding the polypeptide of the invention may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by S.L. Bea ⁇ cage and M.H. Caruthers, Tetrahedron Letters 22, 1981, pp. 1859-1869, or the method described by Matthes et al., EMBO Journal 3. 1984, pp. 801-805.
  • oligonucleotides are synthesized, e.g. 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), e.g.
  • the DNA construct of the invention may also be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the polypeptide of the invention by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1989) .
  • a genomic or cDNA sequence encoding a signal and leader peptide may be joined to a genomic or cDNA sequence encoding the heterologous protein, after which the DNA sequence may be modified at a site corresponding to the amino acid sequence X 1 -X -X 3 -X 4 -X 5 -X 6 -X 7 of the polypeptide, e.g. by inserting synthetic oligonucleotides encoding the desired amino acid sequence for homologous recombination in accordance with well-known procedures.
  • the DNA construct may be of mixed synthetic and ge ⁇ nomic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by annealing fragments of synthetic, genomic or cDNA origin (as appropriate) , the fragments corresponding to various parts of the entire DNA construct, in accordance with standard techniques.
  • the DNA sequence encoding the heterologous protein may be of ge ⁇ nomic or cDNA origin, while the sequence encoding the signal and leader peptide as well as the sequence encoding the N- terminal extension X-X 2 -X 3 -X 4 -X 5 -X 6 -X 7 may be prepared synthetically.
  • the invention relates to a recombinant expression vector which is capable of replicating in yeast and which carries a DNA construct encoding the above-defined polypeptide.
  • the recombinant expression vector may be any vector which is capable of replicating in yeast organisms.
  • the DNA sequence encoding the polypeptide of the invention should be operably connected to a suitable promoter sequence.
  • the promoter may be any DNA sequence which shows transcriptional activity in yeast and may be derived from genes encoding proteins either homologous or heterologous to yeast.
  • the promoter is preferably derived from a gene en ⁇ coding a protein homologous to yeast. Examples of suitable promoters are the Saccharomyces cerevisiae M ⁇ l, TPI, ADH or PGK promoters.
  • the DNA sequence encoding the polypeptide of the invention may also be operably connected to a suitable terminator, e.g. the TPI terminator (cf. T. Alber and G. Kawasaki, J. Mol. Appl. Genet. 1. 1982, pp. 419-434).
  • a suitable terminator e.g. the TPI terminator (cf. T. Alber and G. Kawasaki, J. Mol. Appl. Genet. 1. 1982, pp. 419-434).
  • the recombinant expression vector of the invention further comprises a DNA sequence enabling the vector to replicate in yeast.
  • yeast sequences are the yeast plasmid 2 ⁇ replication genes REP 1-3 and origin of replication.
  • the vec ⁇ tor may also comprise a selectable marker, e.g. the Schizo- saccharomyces pombe TPI gene as described by P.R. Russell, Gene 40, 1985, pp. 125-130.
  • the procedures used to ligate the DNA sequences coding for the polypeptide of the invention, the promoter and the ter ⁇ minator, respectively, and to insert them into suitable yeast vectors containing the information necessary for yeast re ⁇ plication are well known to persons skilled in the art (cf. , for instance, Sambrook et al., op.cit. ) . It will be under ⁇ stood that the vector may be constructed either by first pre ⁇ paring a DNA construct containing the entire DNA sequence coding for the polypeptide 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, leader or heterologous protein) followed by ligation.
  • the yeast organism used in the process of the invention may be any suitable yeast organism which, on cultivation, pro ⁇ considers large amounts of the heterologous protein or polypep ⁇ tide in question.
  • suitable yeast organisms may be strains of the yeast species Saccharomyces cerevisiae, Sac ⁇ charomyces reteyveri, Schizosaccharo yces pombe or Saccharo ⁇ myces uvarum.
  • 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 heterologous protein may be recovered from the medium by conventional procedures including separating the yeast cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, followed by purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, affinity chromatography, or the like.
  • a salt e.g. ammonium sulphate
  • the protein After secretion to the culture medium, the protein may be subjected to various procedures to remove the sequence X 3 -X 4 - X 5 -X 6 -X 7 .
  • the extensions are found to be stably attached to the heterologous protein during fermentation, protecting the N-terminal of the heterological protein against the proteolytic activity of yeast proteases such as DPAP.
  • the presence of an N-terminal extension on the heterologous protein may also serve as a protection of the N-terminal amino group of the heterologous protein during chemical processing of the protein, i.e. it may serve as a substitute for a BOC or similar protecting group.
  • the amino acid sequence X 3 -X 4 -X 5 -X 6 -X 7 may be removed from the recovered heterologous protein by means of a proteolytic enzyme which is specific for a basic amino acid (e.g.
  • proteolytic enzymes are trypsin, Achromobacter lyticus protease I, Enterokinase, Fusarium oxysporum trypsin-like protease, and yeast aspartic protease 3 (YAP3) of Saccharomyces cerevisiae.
  • YAP3 has been characterized applying various prohormones and expressions of heterologous protein in Saccharomyces cerevisiae (Niamh, X.C. et al., FEBS Letters, 332 , p. 273- 276, 1993; Bourbonnais, Y. et al., EMBO Journal, .12, p. 285- 294, 1993; Egel-Mitani, M. et al. , YEAST, 6 , p. 127-137, 1990) .
  • Saccharomyces cerevisiae It is active when the pH is from pH 3 to about pH 6.
  • the appearance of YAP3 is most pronounced using minimal types of media for production. In such media the cells are starved of glucose and/or nitrogen, but other conditions which limit the growth conditions may be used.
  • the YAP3 protease requires a defined motif flanking the cleavage site. Such a motif is present in the N-terminal extension when the amino acid immediately preceding X 7 is Ala or Glu (but not when X 6 is Pro, Thr, Ser, Gly or Asp) .
  • the extensions may to be cleaved from the heterologous protein during fermentation, most likely subsequent to the secretion thereof from the yeast cells - a process which depends on YAP3 acting on its substrate (the peptide bond after lysine or arginine) in the yeast cells or in the growth media.
  • YAP3 inside, attached to, or escaped from the yeast cells may selectively cleave off the extensions such that the non-extended product may be purified from the growth media.
  • the amino acid sequence X 3 -X 7 wherein X 6 Ala or Glu or is a peptide bond (and the amino acid preceding X 7 is Ala or Glu) may be removed from the heterologous protein in the medium by a process involving subjecting the yeast cells to stress to make them release YAP3 into the medium, whereby the amino acid sequence X 3 -X 7 is cleaved off.
  • the stress to which the yeast cells are subjected may comprise reducing the pH of the culture medium to below 6.0, preferably below 5, or starving the yeast cells of glucose and/or nitrogen.
  • the yeast cell may further be transformed with one or more genes encoding a protease which is specific for basic amino acid residues so that, on cultivation of the cell the gene or genes are expressed, the consequent production of protease ensuring a mo're complete cleavage of X 3 - X 7 from the heterologous polypeptide.
  • one or more additional genes encoding YAP3 may be used to transform the yeast cell so that, on cultivation of the cell the gene or genes are overexpressed, the consequent overproduction of YAP3 ensuring a more complete cleavage of X 3 -X 7 from the heterologous polypeptide.
  • yeast aspartic protease 3 embraces the native YAP3 enzyme as well as enzymes derived from the native enzyme, wherein the C-terminal has been modified in order to ensure efficient release of the YAP3 protein from the cell or wherein any modification has taken place maintaining the proteolytic activity.
  • protease may be A.lyticus protease I, Enterokinase or trypsin.
  • FIG. 1 shows a general scheme for the construction of plasmids containing genes expressing N-terminally extended polypeptides.
  • Fig. 1 Denotes the TPI gene promoter sequence from S. cerevisiae.
  • 2 Denotes the region encoding a signal/leader peptide
  • 3* Denotes the region encoding a N-terminal extended heterologous polypeptide.
  • POT Denotes TPI gene from S. pombe..
  • 2 ⁇ Ori Denotes a sequence from S. cerevisiae 2 ⁇ plasmid including its origin of DNA replication in S. cerevisiae.
  • Ap R Sequence from pBR322 /pUC13 including the ampicilli resistance gene and an origin of DNA replication in
  • Fig. 2 shows the DNA sequence in pJB59 encoding the insulin precursor B chain (l-27) -Asp-Lys-Ala-Ala-Lys- A chain (l-21) N-terminally fused to the 85 residues which make up the ⁇ -factor signal/leader peptide in which Leu in position 82 and Asp in position 83 have been substituted by Met and Ala, respectively, and Fig. 3 the DNA sequence of pAK623 encoding GLP-l 7 _ 36 a N- terminally fused to the synthetic signal/leader sequence " YAP3/Sl pAVA "
  • Fig. 4 the DNA sequence of pKV142 encoding B chajn (l-29) -Ala- Ala-Arg-A chajn (l-21) N-terminally fused to the 85 residues which make up the ⁇ -factor signal/leader peptide in which Leu in position 82 and Asp in position 83 have been substituted by Met and Ala, respectively.
  • Fig. 5 the DNA sequence of pAK679 encoding B chain (l-29) -Ala- Ala-Lys-A cha ⁇ - n (l-21) N-terminally fused to the synthetic signal/leader sequence " YAP3/LA19"
  • Fig. 6 shows HPLC chromatogra s of culture supernatants containing the insulin precursor B chajn (l-27)Asp-Lys- Ala-Ala-Lys-A chain (l-21) with or without N-terminal extensions; and with or without in vivo or in vitro processing of the N-terminal extensions.
  • Fig. 7 shows the products according to figure 4 separated by size on a 10% Tricine-SDS-PAGE gel.
  • Fig. 8 shows the effect of the presence of YAP3 coexpression on the yield derived from the HPLC data in pJB176 compared to pJB64.
  • Plasmids and DNA All expressions plasmids are of the C-POT type (see figure 1), similar to those described in WO EP 171 142, which are characterized by containing the Schizosaccharomyces pombe triose phosphate iso erase gene (POT) for the purpose of plasmid selection and stabilization in S. cerevisiae.
  • the plasmids furthermore contain the S. cerevisiae triose phosphate isomerase promoter (region 1 in figure 1) and terminator (region 4 in figure 1) .
  • sequences are identical to the corresponding sequences in plasmid pKFN1003 (described in WO 90/100075) as are all sequences except the sequence of the EcoRI-Xbal fragment encoding the signal/leader/product (region 2 and 3 in figure 1) .
  • the EcoRI-Xbal fragment of pKFN1003 is simply replaced by an EcoRI-Xbal fragment encoding the signal/leader/product of interest.
  • EcoRI-Xbal fragments may be synthesized using synthetic oligonucleotides and PCR according to standard techniques (cf. Sambrook et al., 1989 supra) .
  • Figure 1 shows the general scheme used for the construction of plasmids containing genes expressing N-terminally extended polypeptides, the scheme including the following steps.
  • a sample of the C-POT plasmid vector is digested with restriction nucleases EcoRV and Xbal and the largest DNA fragment is isolated using standard molecular techniques (Sambrook J, Fritsch EL and Maniatis T, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989) .
  • C-POT plasmid (which may be the same or different from the plasmid mentioned above) is digested with restriction nucleases EcoRV and Ncol and the fragment comprising region 1 and 2 is isolated.
  • PCR Polymerase Chain Reaction
  • AATTTATTTTACATAACACTAG-3 ' is designed in such a way that it amplifies region 3 and the flanking recognition site for restriction nuclease Xbal (5 ' - TCTAGA-3 ' ) in PCRs with Pl using standard techniques described in Sambrook et al., supra.
  • the PCR product is digested with restriction nucleases Ncol and Xbal and the digested fragment is isolated.
  • the fragments isolated are ligated together by T4 DNA ligase under standard conditions (described in
  • the ligation mixture is used to transform competent E.coli cells ap r" and selected for ampicillin resistance according to Sambrook et al., supra.
  • Plasmids are isolated from the resulting E.coli clones using standard molecular techniques (described in Sambrook et al., supra.
  • DNA Sequencing is performed using enzymatic chain termination (Sequenase, United States Biochemicals) according to the manufacturer's instructions in order to verify (or determine) the DNA sequences encoding the N-terminal extended polypeptide and to ensure that it is in frame with the DNA sequence encoding the signal/leader peptide of region 2 .
  • enzymatic chain termination Sequenase, United States Biochemicals
  • the plasmid is used to transform the yeast strain MT663 and selected for growth on glucose, as described in detail below:
  • Yeast transformation S. cerevisiae strain MT663 (E2-7B XE11- 36 a/ ⁇ , ⁇ tpi/ ⁇ tpi, pep 4-3/pep 4-3) (the yeast strain MT663 was deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen in connection with filing WO 92/11378 and was given the deposit number DSM 6278) was grown on YPGaL (1% Bacto yeast extract, 2% Bacto peptone, 2% galactose, 1% lactate) to an O.D. at 600 nm of 0.6.
  • YPGaL 1% Bacto yeast extract, 2% Bacto peptone, 2% galactose, 1% lactate
  • 1 ml of CAS-suspended cells was mixed with approx. 0.1 ⁇ g of plasmid DNA and left at room temperature for 15 minutes.
  • Plasmid pJB59 is a derivative of pKFN1003 in which the EcoRI-
  • Xbal fragment encodes the insulin precursor B chal . n (l-27) -Asp- Lys-Ala-Ala-Lys-A cha ⁇ - n (l-21) N-terminally fused to a signal/leader sequence corresponding to the 85 residues of the ⁇ -factor prepro signal peptide in which Leu in position 82 and Asp in position 83 have been substituted by Met and Ala, respectively ( Figure 2) .
  • the EcoRI-Xbal fragment is synthesized in an applied biosystems DNA synthesizer (Perkin Elmer DNA Thermal Cycler) in accordance with the manufacturer's instructions.
  • Plasmid constructs designed to express N-terminally extended insulin precursor B C a i n ( 1-27 ) -Asp-Lys-Ala-Ala-Lys-A chajn (l-21) were obtained by means of a Pl-primer with the following sequence
  • the P2-primer 5' -AATTTATTTTACATAACACTAG-3' and the plasmid pJB59 according to the general scheme described above.
  • PCR and cloning resulted in a construct wherein the DNA sequence encoding the insulin precursor B chain (l-27) -Asp-Lys-Ala-Ala- Lys-A cha ⁇ - n (l-21) is preceded by a DNA sequence encoding the N- terminal extension Glu-Glu-Ala-Glu-Ala-Glu-Ala-Xaa-Lys, where Xaa is either Pro (pJB108; Pro encoded by CCA) or Thr (pJB109; Thr encoded by ACA).
  • Xaa is either Pro (pJB108; Pro encoded by CCA) or Thr (pJB109; Thr encoded by ACA).
  • Plasmid constructs designed to express additional N- terminally extended versions of the insulin precursor B cha - n (l- 27) -Asp-Lys-Ala-Ala-Lys-A chain (l-21) were made by means of a Pl-primer with the following sequence
  • plasmids encode the insulin precursor B chain (l-27) -Asp-Lys-Ala-Ala-Lys-A chain (l-21) preceded by the N-terminal extension Glu-Glu-Ala-Glu-Ala-Glu- Ala-Xaa-Lys, where Xaa is either Glu (pJB44 ; Glu encoded by GAA) , Asp (pJB126; Asp encoded by GAC) or Gly (pJB107; Gly encoded by GGC) .
  • Plasmid constructs designed to express N-terminally extended insulin precursor B chajn (l-27) -Asp-Lys-Ala-Ala-Lys-A chai - n (l-21) were obtained by means of a Pl-primer with the following sequence
  • plasmids encode N- terminal extensions of B chain (l-27) -Asp-Lys-Ala-Ala-Lys-A chain (l- 21) preceded by a DNA sequence encoding the N-terminal extension Glu-Glu-Ala-Glu-Ala-Glu-Xaa-Lys, where Xaa is either Glu (pJBHO; Glu encoded by GAA) or Ala (pJB64 ; Ala encoded by GCA) .
  • Xaa is either Glu (pJBHO; Glu encoded by GAA) or Ala (pJB64 ; Ala encoded by GCA) .
  • Plasmid pAK623 is a derivative of pKNF 1003 in which the EcoRI- Xbal fragment encodes GLP-l 7.36Ala N-terminally fused to a synthetic signal leader sequence YAP3/Sl pAVA (Fig. 3) .
  • the EcoRI-Xbal fragment was synthesized in an Applied Biosystems DNA synthesizer according to the manufacturer's instructions.
  • Plasmid constructs designed to express GLP-1 7.36ALA with the N- terminal extension in form of Glu-Glu-Ala-Glu-Ala-Glu-Ala-Glu- Arg was obtained by means of a Pl-primer with the following sequence
  • yeast results illustrate the ability of yeast to cleave off N- terminal extensions selectively in vivo when the extension is either Glu-Glu-Ala-Glu-Ala-Glu-Ala-Lys (pJB64) or Glu-Glu-Ala- Glu-Ala-Glu-Ala-Glu-Lys (pJB44) and the inability of yeast to cleave off an N-terminal extension in the form of Glu-Glu-Ala- Glu-Ala-Glu-Ala-Pro-Lys (pJBlO ⁇ ) .
  • the proteolytic activity responsible for cleaving off the extensions may be associated with enzymes in the secretory pathway such as membrane-bound YAP3 in the trans-Golgi system of the yeast cells.
  • the culture supernatants described above were used as substrates for proteolytic cleavage with either partially purified YAP3 enzyme isolated from yeast strain ME7 ⁇ 3 overex- pressing YAP3 (Egel-Mitani et al. 1990) or Achromobacter lyti- cus protease I.
  • YAP3 assay was performed as follows: 4 ⁇ l of YAP3 enzyme 800 ⁇ l of cell free growth media Samples were incubated for 15 h at 37"C in 0,1 M Na citrate buffer, pH 4.0
  • Achromobacter lvticus protease I assay performed as follows: lO ⁇ g A. lvticus protease I lml of cell free growth media Samples were incubated for 1 h at 37°C in 0,1 M Tris buffer, pH 8.75
  • Figure 4b and 4c show the results evaluated by HPLC chro ⁇ matography obtained from the YAP3 and A. lyticus protease I digestions, respectively. Parallel samples were run on 10% Tricine-SDS-PAGE (Fig. 5 lane 5-12) .
  • the YAP3 gene cloned by Egel-Mitani et al. (Yeast 6 (1990) pp. 127-137) was inserted into the C-POT plasmid pJB64 encoding Glu-Glu-Ala-Glu-Ala-Glu-Ala-Lys-B chain (l-27) -Pro-Lys-Ala-Ala-Lys- A chajn (l-21) (see example 3) in the following way:
  • the 2.5 kb Sall/SacI fragment containing the YAP3 gene was isolated from plasmid pME76 ⁇ (Egel-Mitani et al. Yeast 6 . (1990) pp. 127-137) and inserted into the Sail and SacI site of plasmid pIC19R (March et al. Gene 32 (19 ⁇ 4) pp. 4 ⁇ l-4 ⁇ 5) . From the resulting plasmid designated pME834 a 2.5 kb Sall/Xhol fragment containing the YAP3 gene was isolated and inserted into the unique Sail site placed between the POT and the Ap R sequences of pJB64. One resulting plasmid was designated pJB176.
  • Yeast strain MT663 was transformed with pJB176 and analyzed as described in example 5.
  • Plasmid pKV142 is a derivative of pKFN1003 in which the EcoRI- Xbal fragment encodes the insulin precursor B chain (l-29)-Ala-Ala- Arg-A cha ⁇ . n (l-21) N-terminally fused to a signal/leader sequence corresponding to the 85 residues of the ⁇ -factor prepro signal peptide in which Leu in position ⁇ 2 and Asp in position ⁇ 3 have been substituted by Met and Ala, respectively ( Figure 4) .
  • Plasmid constructs designed to express B chain (l-29)-Ala-Ala-Arg- A cha ⁇ - n (l-21) with a N-terminal extension in form Asp-Asp-Ala-Asp- Ala-Asp-Ala-Asp-Pro-Arg was obtained by means of a Pl-primer with the following sequence
  • the P2-primer 5' -AATTTATTTTACATAACACTAG-3' and the plasmid pKV142 according to the general scheme described above.
  • Plasmid constructs designed to express B cha1n (l-29)-Ala-Ala-Arg- A cha1n (l-21) with a N-terminal extension in form Glu-Glu-Ala-Glu- Ala-Glu-Ala-Glu-Pro-Lys-Ala-Thr-Arg was obtained by means of a Pl-primer with the following sequence
  • Yeast strain MT663 transformed with the C-POT plasmids pKV142 pKV143 and pKV102 and analyzed as described in example 5.
  • Plasmid N-terminal extension Yield pKV142 100% pKV143:Asp-Asp-Ala-Asp-Ala-Asp-Ala-Asp-Pro-Arg 263% pKV102:Glu-Glu-Ala-Glu-Ala-Glu-Ala- Glu-Pro-Lys-Ala-Thr-Arg 400%
  • Plasmid pAK679 is a derivative of pKFN1003 in which the EcoRI- Xbal fragment encodes the insulin precursor B chain (l-29) -Ala-Ala- Lys-A cha - n (l-21) N-terminally fused to a synthetic signal/leader sequence YAP3/LA19 ( Figure 5) .
  • Plasmid constructs designed to express N-terminally extended insulin precursor B chain (l-29) -Ala-Ala-Lys-A chajn (l-21) were obtained by a procedure involving two successive PCR reaction. The first PCR reaction was performed by means of the primer with the following sequence
  • the second PCR reaction was performed by means of the Pl-primer
  • the PCR product of the second PCR reaction was cut with Ncol and Xbal and ligated into pAK679 according to the general scheme described above.
  • pIM70 Glu-Glu-Glu-Pro-Lys
  • pIM69 Glu-Glu-Glu-Glu-Pro-Lys
  • Yeast strain MT663 was transformed with the C-POT plasmids pAK579, pIM69 and pIM70 and analyzed as described in example 5.
  • yeast with pIM69 and pIM70 was found to produce large quantity of Glu-Glu-Glu-Glu-Pro-Lys-B chain (l-29) -Ala-Ala-Lys- A chain (l-21) and Glu-Glu-Glu-Pro-Lys-B chain (l-29)-Ala-Ala-Lys- A chain (l-21) respectively.
  • ORGANISM Saccharomyces cerevisiae
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal 4 ⁇
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • V FRAGMENT TYPE: internal
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTT-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal

Abstract

La présente invention concerne des polypeptides exprimés et transformés par une levure, et des ADN de recombinaison comprenant une séquence d'ADN codant pour des polypeptides ayant la structure suivante: peptide signal - peptide leader -X1-X2-X3-X4-X5-X6-X7- protéine hétérologue. Dans cette formule X1 est Lys ou Arg; X2 est Lys ou Arg, X1 et X2 constituant ensemble un site de transformation par la levure; X3 est Glu ou Asp; X4 est une séquence d'acides aminés ayant la structure (A-B)n, où A est Glu ou Asp, B est Ala, Val, Leu ou Pro et n est égal à 0 ou à un nombre entier entre 1 et 5 et lorsque n > 2, A et B sont identiques ou différents des autres A(s) et B(s); X4 est une séquence d'acides aminés ayant la structure (C)m, où C est Glu ou Asp et m est égal à 0 ou à un nombre entier entre 1 et 5; X5 est une liaison peptidique ou un ou plusieurs acides aminés qui peuvent être identiques ou différents; X6 est une liaison peptidique ou un acide aminé choisi dans le groupe constitué par Pro, Asp, Thr, Ser, Glu, Ala, et Gly; et X7 est Lys ou Arg.
PCT/DK1995/000250 1994-06-17 1995-06-16 Proteines comportant une extension a l'extremite n-terminale, exprimees par une levure WO1995035384A1 (fr)

Priority Applications (8)

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AU27335/95A AU2733595A (en) 1994-06-17 1995-06-16 N-terminally extended proteins expressed in yeast
EP95922440A EP0765395A1 (fr) 1994-06-17 1995-06-16 Proteines comportant une extension a l'extremite n-terminale, exprimees par une levure
BR9508053A BR9508053A (pt) 1994-06-17 1995-06-16 Construção de DNA vetor de expressão recombinante cepa de levedura e processo para a produção de uma proteina heteróloga
JP50150596A JP3730255B2 (ja) 1994-06-17 1995-06-16 イーストにおいて発現されたｎ末端に伸長した蛋白質
MX9606536A MX9606536A (es) 1994-06-17 1995-06-16 Proteinas n-terminalmente extendidas expresadas en levaduras.
PL95317761A PL317761A1 (en) 1994-06-17 1995-06-16 In yeast exprimed proteins elongated at the end n
FI965030A FI965030A (fi) 1994-06-17 1996-12-16 Hiivassa ekspressoituneet N-terminaalisesti pidennetyt proteiinit
NO965411A NO965411L (no) 1994-06-17 1996-12-16 N-terminalt forlengede proteiner uttrykt i gjær

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DK0712/94 1994-06-17
DK71294 1994-06-17

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CA (1) CA2192943A1 (fr)
CZ (1) CZ363796A3 (fr)
FI (1) FI965030A (fr)
HU (1) HUT75840A (fr)
IL (1) IL114160A (fr)
MX (1) MX9606536A (fr)
NO (1) NO965411L (fr)
PL (1) PL317761A1 (fr)
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WO1997022706A1 (fr) * 1995-12-20 1997-06-26 Novo Nordisk A/S Vecteur pour l'expression de proteines a prolongement n-terminal dans les cellules de levure
WO1998026080A1 (fr) * 1996-12-13 1998-06-18 Chiron Corporation Procedes permettant d'exprimer des proteines heterologues dans la levure
WO1998028429A1 (fr) * 1996-12-20 1998-07-02 Novo Nordisk A/S Proteines a extension d'extremite n-terminal exprimees dans la levure
WO1999037793A1 (fr) * 1998-01-23 1999-07-29 Novo Nordisk A/S Procede servant a preparer des polypeptides souhaites dans de la levure
US6500645B1 (en) 1994-06-17 2002-12-31 Novo Nordisk A/S N-terminally extended proteins expressed in yeast
WO2003010185A2 (fr) * 2001-07-24 2003-02-06 Novo Nordisk A/S Procede pour produire des polypeptides acyles
WO2004085472A1 (fr) * 2003-03-27 2004-10-07 Novo Nordisk A/S Procede de fabrication de precurseurs de l'insuline humaine et insuline humaine
WO2008037735A1 (fr) 2006-09-27 2008-04-03 Novo Nordisk A/S Procédé de fabrication de polypeptides d'insuline mature
US7572884B2 (en) 2001-07-24 2009-08-11 Novo Nordisk A/S Method for making acylated polypeptides
WO2011064282A1 (fr) 2009-11-25 2011-06-03 Novo Nordisk A/S Procédé de production de polypeptides
US8153395B2 (en) 2006-11-22 2012-04-10 Novo Nordisk A/S Method of making activated carboxypeptidases
US8710001B2 (en) 2006-07-31 2014-04-29 Novo Nordisk A/S PEGylated, extended insulins
US8835132B2 (en) 2001-07-24 2014-09-16 Novo Nordisk A/S Method for making acylated polypeptides
US9056921B2 (en) 2005-08-16 2015-06-16 Novo Nordisk A/S Method for making mature insulin polypeptides
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US8883722B2 (en) 2010-06-23 2014-11-11 Novo Nordisk A/S Human insulin containing additional disulfide bonds
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JP2018531899A (ja) 2015-08-25 2018-11-01 ノヴォ ノルディスク アー／エス 新規インスリン誘導体及びその医学的使用
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BR112018072034A2 (pt) * 2016-05-24 2019-02-12 Novo Nordisk A/S compostos mic-1 e usos dos mesmos
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Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6500645B1 (en) 1994-06-17 2002-12-31 Novo Nordisk A/S N-terminally extended proteins expressed in yeast
WO1997022706A1 (fr) * 1995-12-20 1997-06-26 Novo Nordisk A/S Vecteur pour l'expression de proteines a prolongement n-terminal dans les cellules de levure
US6706496B2 (en) 1996-12-13 2004-03-16 Chiron Corporation Method for expression of heterologous proteins in yeast
WO1998026080A1 (fr) * 1996-12-13 1998-06-18 Chiron Corporation Procedes permettant d'exprimer des proteines heterologues dans la levure
US6897043B2 (en) 1996-12-13 2005-05-24 Chiron Corporation Method for expression of heterologous proteins in yeast
US6017731A (en) * 1996-12-13 2000-01-25 Chiron Corporation Method for expression of heterologous proteins in yeast
US6083723A (en) * 1996-12-13 2000-07-04 Chiron Corporation Method for expression of heterologous proteins in yeast
US6312923B1 (en) 1996-12-13 2001-11-06 Chiron Corporation Method for expression of heterologous proteins in yeast
US7166446B2 (en) 1996-12-13 2007-01-23 Chiron Corporation Method for expression of heterologous proteins in yeast
EP1365028A1 (fr) * 1996-12-13 2003-11-26 Chiron Corporation Procédés permettant d'exprimer des protéines PDGF ou IGF dans la levure
WO1998028429A1 (fr) * 1996-12-20 1998-07-02 Novo Nordisk A/S Proteines a extension d'extremite n-terminal exprimees dans la levure
WO1999037793A1 (fr) * 1998-01-23 1999-07-29 Novo Nordisk A/S Procede servant a preparer des polypeptides souhaites dans de la levure
US6183989B1 (en) 1998-01-23 2001-02-06 Novo Nordisk A/S Process for making desired polypeptides in yeast
WO2003010185A3 (fr) * 2001-07-24 2004-03-25 Novo Nordisk As Procede pour produire des polypeptides acyles
WO2003010185A2 (fr) * 2001-07-24 2003-02-06 Novo Nordisk A/S Procede pour produire des polypeptides acyles
US7572884B2 (en) 2001-07-24 2009-08-11 Novo Nordisk A/S Method for making acylated polypeptides
US8835132B2 (en) 2001-07-24 2014-09-16 Novo Nordisk A/S Method for making acylated polypeptides
WO2004085472A1 (fr) * 2003-03-27 2004-10-07 Novo Nordisk A/S Procede de fabrication de precurseurs de l'insuline humaine et insuline humaine
US9056921B2 (en) 2005-08-16 2015-06-16 Novo Nordisk A/S Method for making mature insulin polypeptides
US8710001B2 (en) 2006-07-31 2014-04-29 Novo Nordisk A/S PEGylated, extended insulins
WO2008037735A1 (fr) 2006-09-27 2008-04-03 Novo Nordisk A/S Procédé de fabrication de polypeptides d'insuline mature
US8518668B2 (en) 2006-09-27 2013-08-27 Novo Nordisk A/S Method for making maturated insulin polypeptides in a fungal cell
US8153395B2 (en) 2006-11-22 2012-04-10 Novo Nordisk A/S Method of making activated carboxypeptidases
WO2011064282A1 (fr) 2009-11-25 2011-06-03 Novo Nordisk A/S Procédé de production de polypeptides
CN112105635A (zh) * 2018-06-18 2020-12-18 联合化学实验室有限公司 用于重组蛋白的更高表达的前导序列

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MX9606536A (es) 1997-03-29
HU9603476D0 (en) 1997-02-28
FI965030A0 (fi) 1996-12-16
SG47883A1 (en) 1998-04-17
JP3730255B2 (ja) 2005-12-21
IL114160A (en) 2006-12-31
FI965030A (fi) 1997-02-14
HUT75840A (en) 1997-05-28
JPH10501695A (ja) 1998-02-17
CA2192943A1 (fr) 1995-12-28
CZ363796A3 (en) 1997-05-14
PL317761A1 (en) 1997-04-28
IL114160A0 (en) 1995-10-31
BR9508053A (pt) 1997-08-12
AU2733595A (en) 1996-01-15
CN1152942A (zh) 1997-06-25
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ZA954983B (en) 1996-02-14

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