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• • 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
  • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
• • 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
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
  • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
  • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

• the present invention relates to a method of constructing synthetic leader peptide sequences for secreting heterologous polypeptides in yeast, and yeast expression vectors for use in the method.
• Yeast organisms produce a number of proteins which are synthesized intracellularly, but which have 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-protein 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).
• European published patent application No. 88 632 describes a process by which proteins heterologous to yeast are expressed, processed and secreted by transforming a yeast organism with an expression vehicle 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 signal peptide of the desired protein itself, a heterologous signal peptide or a hybrid of native and heterologous signal peptide.
• a problem encountered with the use of signal peptides heterologous to yeast might be that the heterologous signal peptide does not ensure efficient translocation and/or cleavage after the signal peptide.
• the S. cerevisiae MF ⁇ 1 ( ⁇ -factor) is synthesized as a prepro form of 165 amino acids comprising signal-or prepeptide of 19 amino acids followed by a "leader” or propeptide of 64 amino aicds, 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 ⁇ 1 has been widely employed to obtain synthesis and secretion of heterologous proteins in S. cerivisiae.
• EP 16201, 123 294, 123 544, and 163 529 describe processes by which the ⁇ -factor signal-leader from Saccharomyces cerevisiae (MF ⁇ l or MF ⁇ 2) is utilized in the secretion process of expressed heterologous proteins in yeast.
• MF ⁇ l or MF ⁇ 2 Saccharomyces cerevisiae
• EP 206783 discloses a system for the secretion of polypeptides from S.
• 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 LysArgGluAlaGluAla.
• 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 polypeptide.
• a number of secreted proteins are routed so as to be exposed to a proteolytic processing system which can cleave the peptide 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 gene product is needed for the secretion of active S. cerevisiae mating factor ⁇ 1 (MF ⁇ 1 or ⁇ -factor) but is not involved in the secretion of active S. cerevisiae mating factor a.
• the present invention relates to a method of constructing a synthetic leader peptide sequence for secreting heterologous polypeptides in yeast, the method comprising
• X n is a DNA sequence encoding n amino acids, wherein n is O or an integer of from 1 to about 10 amino acids,
• RS is a restriction endonuclease recognition site for insertion of random DNA fragments, which site is provided at the junction of X n and X m ,
• X m is a DNA sequence encoding m amino acids, wherein m is O or an integer from 1 to about 10,
• NZT is a DNA sequence encoding Asn-Xaa-Thr, wherein p is O or 1,
• X is a DNA sequence encoding q amino acids, wherein q is O or an integer from 1 to about 10,
• PS is a DNA sequence encoding a peptide defining a yeast processing site
• step (b) transforming a yeast host cell with the expression vector of step (a);
• step (c) culturing the transformed host cell of step (b) under appropriate conditions
• step (d) screening the culture of step (c) for secretion of the heterologous polypeptide.
• leader peptide is understood to indicate a peptide whose function is to allow the heterologous polypeptide to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory veside for secretion into the medium, (i.e. exportation of the expressed polypeptide across the cell wall or at least through the cellular membrane into the periplasmic space of the cell).
• synthetic used in connection with leader peptides is intended to indicate that the leader peptide constructed by the present method is one not found in nature.
• signal peptide is understood to mean a presequence which is predominantly hydrophobic in nature and present as an N-terminal sequence of the precursor form of an extracellular protein expressed in yeast.
• the function of the signal peptide is to allow the heterologous 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 heterologous or homologous to the yeast organism producing the protein but, as explained above, a more efficient cleavage of the signal peptide may be obtained when it is homologous to the yeast organism in question.
• heterologous polypeptide is intended to indicate a polypeptide which is not produced by the host yeast organism in nature.
• the heterologous polypeptide is preferably one the secretion of which by transformed yeast cells may easily be detected, e.g. by established standard methods such as by immunological screening by means of antibodies reactive with the polypeptide in question (cf. for instance Sambrook, Fritsch and Maniatis, Molecular Cloning; A Laboratory Manual, Cold Spring Harbor, New York, 1989) or by screening for a specific biological activity of the heterologous polypeptide.
• a positive result of the screening indicates that a leader peptide useful for the secretion of heterologous polypeptides in yeast has been constructed.
• a random DNA fragment is intended to indicate any sequence of DNA at least 3 nucleotides in length, for instance obtained by digesting genomic DNA (of any organism) with restriction endonuclease(s) or by preparing synthetic DNA, e.g. by the phosphoamidite method described by S.L. Beaucage and M.H. Caruthers, Tetrahedron Letters 22, 1981, pp. 1859- 1869.
• the peptide Asn-Xaa-Thr encoded by "(NZT) p " is an asparaginelinked glycosylation site.
• "Xaa” denotes any one of the known amino acids except Pro.
• the present invention relates to a yeast expression cloning vector comprising the following sequence
• This vector may be used in the construction of leader peptide sequences according to the method described above.
• the present invention relates to a yeast expression vector comprising the following sequence
• ranDNA is a random DNA fragment inserted in a restriction endonuclease recognition site provided at the junction of X n and X m .
• the leader peptide sequence (once identified by the method of the invention) will be composed of the sequence X n -ranDNA-X m -(NZT) p -X q .
• Such a vector may be used in the production of a heterologous polypeptide of interest.
• the present invention relates to a process for producing a heterologous polypeptide in yeast, the process comprising culturing a yeast cell, which is capable of expressing a heterologous polypeptide and which is transformed with a yeast expression vector as described above including a leader peptide sequence constructed by the method of the invention, in a suitable medium to obtain expression and secretion of the heterologous polypeptide, after which the heterologous polypeptide is recovered from the medium.
• the length of the random DNA fragment inserted in the expression vector is not particularly critical. However, in order to be of a manageable length, the fragment preferably has a length of from 16 to about 600 base pairs. More preferably, the fragment has a length of from about 15 to about 300 base pairs. It is at present considered that a suitable length of the fragment is from about 30 to about 150 base pairs.
• the random DNA fragment preferably encodes a high proportion of polar amino acids. These are selected from the group consisting of Glu, Asp, Lys, Arg, His, Thr, Ser, Asn and Gin.
• a high proportion of is understood to indicate that the DNA fragment encodes a larger number of polar amino acids than do other DNA sequences of a corresponding length.
• the fragment encodes at least one proline.
• n and/or m and/or q are preferably ⁇ 1. In particular, all of n, m and q are ⁇ 1.
• the signal peptide sequence may encode any signal peptide which ensures an effective direction of the expressed heterologous polypeptide into the secretory pathway of the cell.
• the signal peptide may be a naturally occurring signal peptide or functional parts 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, the yeast BAR1 signal peptide or the Humicola lanuginosa lipase signal peptide, or a derivative thereof.
• the mouse salivary amylase signal sequence is described by 0. Hagenbüchle et al., Nature 289, 1981, pp. 643-646.
• the carboxypeptidase signal sequence is described by L.A. Valls et al., Cell 48, 1987, pp. 887-897.
• the BAR1 signal peptide is disclosed in WO 87/02670.
• the H. lanuginosa lipase signal peptide is disclosed in EP 305 216.
• the yeast processing site encoded by the DNA sequence PS may suitably be any paired combination of Lys and Arg, such as Lys-Arg, Arg-Lys, Lys-Lys or Arg-Arg, which permits processing of the heterologous polypeptide by the KEX2 protease of Saccharomyces cerevisiae or the equivalent protease in other yeast species (D.A. Julius et al., Cell 37, 1984, 1075 ff.). If KEX2 processing is not convenient, e.g. if it would lead to cleavage of the polypeptide product, a processing site for another protease may be selected instead comprising an amino acid combination which is not found in the polypeptide product, e.g. the processing site for FX a , Ile-Glu-Gly-Arg (cf. Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989).
• the heterologous protein produced by the method of the invention may be any protein which may advantageously be produced in yeast.
• examples of such proteins are aprotinin, tissue factor pathway inhibitor or other protease inhibitors, insulin or insulin precursors, human or bovine growth hormone, interleukin, glucagon, tissue plasminogen activator, transforming growth factor ⁇ or ⁇ , platelet-derived growth factor, enzymes, or a functional analogue thereof.
• 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.
• the random DNA fragment and the sequence 5'-SP-X n -3'-RS-5'-X m -(NZT) p -X q -PS-*gene*-3' may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by S.L. Beaucage 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. According to the phosphoamidite method, oligonucleotides are synthesized, e.g.
• sequence 5'-SP-X n -3'-RS-5'-X m -(NZT) p -X q -PS- *gene*-3' need not be prepared in a single operation, but may be assembled from two or more oligonucleotides prepared synthetically in this fashion.
• the random DNA fragment or one or more parts of the sequence 5l-SP-X n -3'-RS-5'-X m -(NZT) p -X q -PS-*gene*-3' 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 said parts (typically SP or *gene*) by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989).
• a genomic or cDNA sequence encoding a signal peptide may be joined to a genomic or cDNA sequence encoding the heterologous protein, after which the DNA sequence may be modified by the insertion of synthetic oligonucleotides encoding the sequence X n -3'-RS-5'-X m -(NZT) p -X q -PS in accordance with well-known procedures.
• the random DNA fragment and/ or the sequence 5'-SP-X n - 3'-RS-5'-X m -(NZT) p -X q -PS-*gene*-3' may be of mixed synthetic and genomic, 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 sequence, in accordance with standard techniques.
• the DNA sequence encoding the signal peptide or the heterologous polypeptide may be of genomic or cDNA origin, while the sequence X n -3'-RS-5'-X m -(NZT) p -X q -PS may be prepared synthetically.
• Preferred DNA constructs encoding insulin precursors are as shown in Sequence Listings ID Nos. 1-13, or suitable modifications thereof.
• suitable modifications of the DNA sequence are nucleotide substitutions which do not give rise to another amino acid sequence of the protein, but which may correspond to the codon usage of the yeast organism into which the DNA construct is inserted or nucleotide substitutions which do give rise to a different amino acid sequence and therefore, possibly, a different protein structure.
• Other examples of possible modifications are insertion of three or multiples of three nucleotides into the sequence, addition of three or multiples of three nucleotides at either end of the sequence and deletion of three or multiples of three nucleotides at either end of or within the sequence.
• the recombinant expression vector carrying the sequence 5'-SP-X n -3'-RS-5'-X m -(NZT) p -X q -PS-*gene*-3' or 5'-SP-X n -ranDNA-X m - (NZT) p -X q -PS-*gene*-3' may be any vector which is capable of replicating in yeast organisms.
• either DNA sequence 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 encoding a protein homologous to yeast. Examples of suitable promoters are the Saccharomyces cerevisiae MF ⁇ 1, TPI, ADH or PGK promoters.
• 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 vector may also comprise a selectable marker, e.g. the Schizosaccharomyces pombe TPI gene as described by P.R. Russell, Gene 40, 1985, pp. 125-130.
• the vector may be constructed either by first preparing a DNA construct containing the entire sequence 5'-SP-X n -3'-RS-5'-X m -(NZT) p -X q -PS-*gene*-3' 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 peptide, the sequence X n -3'-RS-5'-X m -(NZT) p -X q or the heterologous polypeptide) followed by ligation.
• DNA fragments containing genetic information for the individual elements such as the signal peptide, the sequence X n -3'-RS-5'-X m -(NZT) p -X q or the heterologous polypeptide
• the yeast organism used in the method of the invention may be any suitable yeast organism which, on cultivation, produces large amounts of the heterologous polypeptide in question.
• suitable yeast organisms may be strains of the yeast species Saccharomyces cerevisiae, Saccharomyces reteyveri, Schizosaccharomyces pombe or Saccharomyces 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
• Fig. 1 schematically shows the construction of pMT742 ⁇
• Fig. 2 schematically shows the construction of pLaC202
• Fig. 3 shows the DNA sequence and derived amino acid sequence at the cloning site in pLaC202 for random DNA fragments (it should be noted that the sequence is cleaved in the unique ClaI site and that ligation without insertion of random DNA will lead to a change in the reading frame);
• Fig. 4 schematically shows the construction of pLSC6315D#
• Plasmids and DNA materials are of the C-POT type. Such plasmids are described in EP patent application No. 171 142 and are characterized in containing the Schizosaccharomyces pombe triose phosphate isomerase gene (POT) for the purpose of plasmid selection and stabilization.
• POT Schizosaccharomyces pombe triose phosphate isomerase gene
• a plasmid containing the POT-gene is available from a deposited E. coli strain (ATCC 39685).
• the plasmids furthermore contain the S. cerevisiae triose phosphate isomerase promoter and terminator (P TPI and T TPI ). They are identical to pMT742 (M. Egel-Mitani et al., Gene 73, 1988, pp. 113-120) (see fig. 1) except for the region defined by the Sph-XbaI restriction sites encompassing the P TPI and the coding region for signal
• the P TPI has been modified with respect to the sequence found in pMT742, only in order to facilitate construction work.
• An internal SphI restriction site has been eliminated by SphI cleavage, removel of single stranded tails and religation.
• DNA sequences, upstream to and without any impact on the promoter have been removed by Bal31 exonuclease treatment followed by addition of an SphI restriction site linker.
• This promoter construction present on a 373 bp SphI- EcoRI fragment is designated P TPI ⁇ and when used in plasmids already described this promoter modification is indicated by the addition of a ⁇ to the plasmid name, e.g. pMT7425 (fig. 1).
• This vector containing a unique Clal site constitutes one embodiment of the random DNA cloning vector in which the product gene codes for the insulin precursor MI3 (B(1-29)-Ala-Ala-Lys-A(l-21)).
• MI3 insulin precursor
• the following examples concerns the leaders cloned via this construct.
• Total DNA was isolated from S. cerevisiae strain MT663, and digested by TaqI, HinPI or TaqI + HinP I. The digests were separated according to size on a 1% agarose gel, and fragments smaller than 600 bp were isolated from each of the three digestions.
• pLaC202 previously digested with ClaI, prevented from self ligation with Calf Intestine Alkaline Phosphatase (CIAP), dephosphorylation, was mixed with the fragment pools described above and ligated.
• Recombinant plasmids were prepared from each of the three types in pools encompassing all 5000 transformants. These plasmid pools were used to transform S. cerevisiae strain MT663 and the resulting TPI transformants were immunoscreened for MI3 secretion.
• Sequencing of the inserts of the eight isolated pLaC202 derivatives showed three different sequences, two of which, pLSC6315 and pLSC5210, most efficiently support MI3 secretion.
• the sequences of the cloned DNA and flanking regions are shown in Sequence Listings ID Nos. 2 and 4, respectively.
• pLSC6315 was chosen for further modification of the cloned synthetic leader sequence.
• pLSC6315 was digested with the ApaI endonuclease followed by treatment with the exonuclease Bal31. After phenol extraction the resulting DNA was digested with XbaI and DNA fragments smaller than the original 367 bp Apal-XbaI fragment, were isolated.
• pLaC202 was digested with ClaI, and the single stranded CG tails generated were removed, followed by XbaI digestion and isolation of the 11 Kb XbaI-]ClaI[ fragment ("] [" indicates that the single-stranded tails have been trimmed off). This fragment was mixed with the pLSC6415 fragments isolated above and ligated (fig. 6).
• Sequencing of the inserts showed 5 different sequences, two of which (pLAO2 and pLAO5) are more efficient where MI3 secretion is concerned.
• the sequences of the DNA inserts in pLAO2 and pLAO5 are shown, together with the flanking regions, in Sequence Listings ID Nos. 10 and 12, respectively.
• Example 5 Yeast strains harbouring plasmids as described above, were grown in YPD medium (Sherman, F. et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory 1981). For each strain 6 individual 5 ml cultures were shaken at 30oC for 60 hours, with a final OD 600 of approx. 15. After centrifugation the supernatant was removed for HPLC analysis by which method the concentration of secreted insulin precursor was measured by a method described by Leo Snel et al. (1987) Chromatographia 24, 329-332.
• MOLECULE TYPE protein
• TGT ACC TCC ATC TGC TCC TTG TAC CAA TTG GAA AAC TAC TGC AAC 348 cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn

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