CA2192942C - Synthetic leader peptide sequences - Google Patents
Synthetic leader peptide sequences Download PDFInfo
- Publication number
- CA2192942C CA2192942C CA002192942A CA2192942A CA2192942C CA 2192942 C CA2192942 C CA 2192942C CA 002192942 A CA002192942 A CA 002192942A CA 2192942 A CA2192942 A CA 2192942A CA 2192942 C CA2192942 C CA 2192942C
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- Prior art keywords
- seq
- ser
- sequence
- thr
- leu
- Prior art date
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- RXUOAOOZIWABBW-XGEHTFHBSA-N Ser-Thr-Arg Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N RXUOAOOZIWABBW-XGEHTFHBSA-N 0.000 description 1
- PCMZJFMUYWIERL-ZKWXMUAHSA-N Ser-Val-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O PCMZJFMUYWIERL-ZKWXMUAHSA-N 0.000 description 1
- 102000005924 Triose-Phosphate Isomerase Human genes 0.000 description 1
- XBWKCYFGRXKWGO-SRVKXCTJSA-N Tyr-Cys-Asn Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(O)=O XBWKCYFGRXKWGO-SRVKXCTJSA-N 0.000 description 1
- TWAVEIJGFCBWCG-JYJNAYRXSA-N Tyr-Gln-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC1=CC=C(C=C1)O)N TWAVEIJGFCBWCG-JYJNAYRXSA-N 0.000 description 1
- 102100020705 WD repeat-containing protein 11 Human genes 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
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- SKEFKEOTNIPLCQ-LWIQTABASA-N mating hormone Chemical compound C([C@@H](C(=O)NC(CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCS(C)=O)C(=O)NC(CC=1C=CC(O)=CC=1)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CN=CN1 SKEFKEOTNIPLCQ-LWIQTABASA-N 0.000 description 1
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Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to synthetic leader peptide sequences for secreting polypeptides in yeast.
Description
W O 95/34666 ~ 219 2 9 4 2 BYNTHETIC LEADER PEPTIDE SEQUENCES
FIELD OF INVENTION
The present invention relates to synthetic leader peptide sequences for secreting polypeptides in yeast.
BACKGROUND OF THE INVENTION
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 10-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. 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, S.R. and Rothman, J.E. Ann.Rev.Biochem. ~ø
(1987) 829-852).
Several approaches have been suggested for the expression and secretion in yeast of proteins heterologous to yeast: 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 wo 9sr~4~ss ~ ~ ~ 9 2 9 4 2 p"~' z peptide or a hybrid of native and heterologous signal peptide., Ir probles encountered with ttae use of signal peptides hetero logous to yeast eight be that the heterologous signal peptide does not ansnsa e!licieat translocation and/or cleavage after the sigsa3 peptide.
The ~~,g cerevisiae HFal (a-factor) is synthesized as a prapro form of 165 amino acids comprising signal- or prepeptide of 19 amino acids followed by a "leader" or propeptide of 64 amino acids, encompassing three N-linked glycosylation sites followed by (Lys7lrg((Asp/Glu)l~la)Z-3a-tactor)4 (ICurjan, J. and Aerskowitz, I. ~, ~Q (1981) 933-943). The signal-leader part of the preproMFal has been widely employed to obtain synthesis and secretion of heterologous proteins in ~,,. ~yy~. .
Use o! signal/lsader paptidea hoaolog~s to yeast is known lrom i.a. D6 patent specification No. a,5as,081, European published patent applfo~!tions Nos. 116 201. 123 294, u3 544, 163 529 and iz3 zes and European Patent No. 0100 561.
In EP 123 289 utilization of the ,~,, cerevisiae a-factor prr 2o cursor is described whereas WO &4/01153 indicates utilization of the ~ ~~revisiae invgrtase signal peptide and DR 3614/83 utilisation o! the ~ ~ F805 signal peptide for secretion of foreign proteins.
U& patent specification No. 4,546,OBZ, EP 16 101, 123 29a, 123 544 and 163 529. describe processes by which the e-!actor signal-leader from j~.cerevisiae.,(!LP'al or xFn1) ie utilised in the secretion process of expressed heterologous proteins in yaact. By fusing a DNA sequence encoding the $,,cerevisiae NFal signal/leader sequence at the 5~ end of the gene for the 3o desired protein secretion and processing of the desired protein was demonstrated.
R'O 95/34666 ~ 219 2 9 4 2 PCTIDK95100249 EP 206 783 discloses a system for the secretion of polypeptides.
from ,~ cerevisiae using an a-factor leader sequence which has been truncated to eliminate the four a-factor units present on the native leader sequence so as to leave the leader peptide itself fused to a heterologous polypeptide via the a-factor processing site LysArgGluAlaGluAla. This construction is indicated to lead to an efficient processing of smaller peptides (less than 50 amino acids). For the secretion and processing of larger polypeptides, the native a-factor leader sequence has been truncated to leave one or two of the a-factor units 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 ~ cerevisiae encoded by the KEX
FIELD OF INVENTION
The present invention relates to synthetic leader peptide sequences for secreting polypeptides in yeast.
BACKGROUND OF THE INVENTION
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 10-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. 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, S.R. and Rothman, J.E. Ann.Rev.Biochem. ~ø
(1987) 829-852).
Several approaches have been suggested for the expression and secretion in yeast of proteins heterologous to yeast: 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 wo 9sr~4~ss ~ ~ ~ 9 2 9 4 2 p"~' z peptide or a hybrid of native and heterologous signal peptide., Ir probles encountered with ttae use of signal peptides hetero logous to yeast eight be that the heterologous signal peptide does not ansnsa e!licieat translocation and/or cleavage after the sigsa3 peptide.
The ~~,g cerevisiae HFal (a-factor) is synthesized as a prapro form of 165 amino acids comprising signal- or prepeptide of 19 amino acids followed by a "leader" or propeptide of 64 amino acids, encompassing three N-linked glycosylation sites followed by (Lys7lrg((Asp/Glu)l~la)Z-3a-tactor)4 (ICurjan, J. and Aerskowitz, I. ~, ~Q (1981) 933-943). The signal-leader part of the preproMFal has been widely employed to obtain synthesis and secretion of heterologous proteins in ~,,. ~yy~. .
Use o! signal/lsader paptidea hoaolog~s to yeast is known lrom i.a. D6 patent specification No. a,5as,081, European published patent applfo~!tions Nos. 116 201. 123 294, u3 544, 163 529 and iz3 zes and European Patent No. 0100 561.
In EP 123 289 utilization of the ,~,, cerevisiae a-factor prr 2o cursor is described whereas WO &4/01153 indicates utilization of the ~ ~~revisiae invgrtase signal peptide and DR 3614/83 utilisation o! the ~ ~ F805 signal peptide for secretion of foreign proteins.
U& patent specification No. 4,546,OBZ, EP 16 101, 123 29a, 123 544 and 163 529. describe processes by which the e-!actor signal-leader from j~.cerevisiae.,(!LP'al or xFn1) ie utilised in the secretion process of expressed heterologous proteins in yaact. By fusing a DNA sequence encoding the $,,cerevisiae NFal signal/leader sequence at the 5~ end of the gene for the 3o desired protein secretion and processing of the desired protein was demonstrated.
R'O 95/34666 ~ 219 2 9 4 2 PCTIDK95100249 EP 206 783 discloses a system for the secretion of polypeptides.
from ,~ cerevisiae using an a-factor leader sequence which has been truncated to eliminate the four a-factor units present on the native leader sequence so as to leave the leader peptide itself fused to a heterologous polypeptide via the a-factor processing site LysArgGluAlaGluAla. This construction is indicated to lead to an efficient processing of smaller peptides (less than 50 amino acids). For the secretion and processing of larger polypeptides, the native a-factor leader sequence has been truncated to leave one or two of the a-factor units between the leader peptide and the polypeptide.
A number of secreted proteins are routed so as to be exposed to a proteolytic processing system which can cleave the peptide bond at the carboxy end of two consecutive basic amino acids.
This enzymatic activity is in ~ cerevisiae encoded by the KEX
2 gene (Julius, D.A. et al., Cell ~7 (1984bj 1075). Processing of the product by the KEX 2 protease is needed for the secretion of active S,~ ~erevisia~ mating factor ai (MFa1 or a-facto;) whereas KEX 2 is not involved in the secretion of ac-tive Sue. cerevisiae mating factor a.
Secretion and correct processing of a polypeptide intended to be secreted is obtained in some cases when culturing a yeast organism which is transformed with a vector constructed as indicated in the references given above. In many cases, how-ever, the level of secretion is very low or there is no se-cretion, or the proteolytic processing may be incorrect or incomplete. It is therefore the object of the present invention to provide leader peptides which ensure a more efficient expression and/or processing of polypeptides.
SUMMARY OF T$E INVENTION
Surprisingly, a new type of leader peptide has been found which allows secretion in high yield of a polypeptide in yeast.
WO 95134666 219 2 9 G~ 2 PCT/DK95100249 Accordingly, the present invention relates to a DNA expression cassette comprising the following sequence:
5'-P-SP-LS-PS-*gene*-(T)i-3' wherein P is a promoter sequence, SP is a DNA sequence encoding a signal peptide, LS is a DNA sequence encoding a leader peptide with the general formula I:
GlnProIle(Asp/Glu)(Asp/Glu)X1(Glu/Asp)X2AsnZ(Thr/Ser)X3 (I) l0 wherein X1 is a peptide bond or a codable amino acid;
X2 is a peptide bond, a codable amino acid or a sequence of up to 4 codable amino acids which may be the same or different;
Z is a codable amino acid except Pro; and X3 is a sequence of from 4 to 30 codable amino acids which may be the same or different:
PS is a DNA sequence encoding a processing site:
*gene* is a DNA sequence encoding a polypeptide;
T is a terminator sequence; and i is 0 or 1.
In the present context, the expression "leader peptide" is understood to indicate a peptide whose function is to allow the expressed polypeptide to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory ve-sicle 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).
The term "synthetic" used in connection with leader peptides is intended to indicate that the leader peptide is one not found in nature.
WO 95/34666 219 2 9 4 2 PCT~~5100249 The term "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 5 is to allow the expressed 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.
The expression "polypeptide" is intended to indicate a heterologous polypeptide, i.e. a polypeptide which is not pro-duced by the host yeast organism in nature as well as a homologous polypeptide, i.e. a polypeptide which is produced by the host yeast organism in nature and any preform thereof. In a preferred embodiment, the expression cassette of the present invention encodes a heterologous polypeptide.
The expression "a codable amino acid" is intended to indicate an amino acid which can be coded for by a triplet ("codon") of nucleotides.
When, in the amino acid sequences given in the present specification, the three letter codes of two amino acids, separated by a slash, are given in brackets, e.g. (Asp/Glu), this is intended to indicate that the sequence has either the one or the other of these amino acids in the pertinent position.
In a further aspect, the present invention relates to a process for producing a polypeptide in yeast, the process comprising culturing a yeast cell, which is capable of expressing a polypeptide and which is transformed with a yeast expression vector as described above including a leader peptide sequence of the invention, in a suitable medium to obtain expression and secretion of the polypeptide, after which the polypeptide is recovered from the medium.
wo>s2192942 BEIEF DEiCRIpTI0~1 OF TEE DR11~I~
The present invention is turthar illustrated with ratarenas to the appended drawings wherein Fig. 1 schematically chows the placmid p11Kd9Zt Fig. 2 shows part of the DNA sequence encoding the cign6l peptfde/leader/MI7 insulin precursor:
Fig. 3 shows the construction of the plasmid pAR546r Fig. 4 shows the aaifno acfd sequence at the leader SEQ ID lro.
43 and the corresponding DNA SEQ ID No.: 42 io Fig. 5 shows the amino acid SEQ ID No. :45 and the corresponding DNA SEQ ID NO.: 44.
Fig. 6 shows the amino acid SEQ ID NO.: 47 and the corresponding DNA SEQ ID NO.: 46.
Fig. 7 shows the amino acid sequence of the leader SEQ ID No.
8 and the DNA sequence sncoding itt Pig. 8 chows the aaino acid sequence of the leader SEQ ID No.
17 and the DNA sequence encoding itt Fig. 9 shows the amino acid sequence o! the leader SEQ ID No.
is and the DNA aeguance encoding it;
Fig. 1o shows the amino acid sequence of the leader SEQ ID No.
19 and the DNA sequence encoding it:
Fig. 11 shows the amino acid sequence of the leader SEQ ID No.
20 and the DNA sequence encoding itt WO 95/34666 219 2 9 4 2 P~~~S~OO1A9 Fig. 12 shows the amino acid sequence of the leader SEQ ID No.
21 and the DNA sequence encoding it;
Fig. 13 shows the DNA fragment of pAK527 used as the direct template in the construction of SEQ ID Nos. 4 and 6;
Fig. 14 shows the DNA fragment of pAK531 used as the direct template in the construction of SEQ .ID No. 8;
Fig. 15 shows the DNA fragment of pAK555 used as the direct template in the construction of SEQ ID Nos. 16 and 17;
Fig. 16 shows the DNA fragment of pAK559 used as the direct l0 template in the construction of SEQ ID Nos. 19 and 20; and Fig. 17 shows the DNA fragment of pAK562 used as the direct template in the construction of SEQ ID No. 21;
Fig. 18 shows the amino acid sequence of the leader SEQ ID No.
27 and the DNA sequence SEQ ID No. 66 encoding it;
Fig. 19 shows the amino acid sequence SEQ ID No. 70 of an N-terminally extended MI3 insulin precursor and the DNA sequence SEQ ID No. 71 encoding it;
Fig. 20 shows the amino acid sequence of the leader SEQ ID No.
67 and the DNA sequence SEQ ID No. 69 encoding it;
Fig. 21 shows the DNA fragment SEQ ID No. 72 of pAK614 used as the direct template in the construction of SEQ ID No. 27; and Fig. 22 shows the DNA fragment SEQ ID No. 73 of pAK625 used as the direct template in the construction of SEQ ID No. 67.
R'O 95134666 ~ ~ 9 2 9 4 2 PCT/DK95100249 DETAINED DI8CLOSORE Of THE INVENTION
When X1 in general formula I designates an amino acid, it is preferably Ser, Thr or Ala. When X2 in general formula I
designates one amino acid, it is preferably Ser, Thr or Ala.
When X2 in general formula I designates a sequence of two amino acids, it is preferably SerIle. When X2 in general formula I
designates a sequence of three amino acids, it is preferably SerAlaIle. When X2 in general formula I designates a sequence of four amino acids it is preferably SerPheAlaThr. In a preferred embodiment, X3 is an amino acid sequence of the general formula a X4_X5_X6 (II) wherein X4 is a sequence of from 1 to 21 codable amino acids which may be the same or different, XS is Pro or one of the amino acid sequences ValASnLeu or LeuAlaASnValAlaMetAla, and X6 is a sequence of from 1 to 8 codable amino acids which may be the same or different.
In general formula II, X4 is preferably an amino acid sequence which includes one or more of the motifs LeuValAsnLeu, SerValAsnLeu, MetAlaAsp, ThrGluSer, ArgPheAlaThr or ValAlaMetAla; or X4 is an amino acid sequence which includes the sequence AsnSerThr or AsnThrThr: or X4 is an amino acid sequence which includes the sequence (Ser/Leu)ValAsnLeu, (Ser/Leu)ValAsnLeuMetAlaAsp, (Ser/Leu)ValAsnLeuMetAlaAspAsp, (Ser/Leu)ValAsnLeuMetAlaAspASpThrGluSer, (Ser/Leu)ValAsnLeuMetAlaAspAspThrGluSerIle or (Ser/Leu)ValAsnLeuMetAlaAspAspThrGluSerArgPheAlaThr:
or X4 is an amino acid sequence which includes the sequence Asn(Thr/Ser)ThrLeu, Asn(Thr/Ser)ThrLeuAsnLeu or Asn(Thr/Ser)ThrLeuValAsnLeu~ or any combination thereof.
In general formula II, X5 is preferably Pro or an amino acid sequence which includes the sequence ValAsnLeu, LeuAlaAsnValAlaMetAla, LeuAspValValAsnLeuProGly or LeuAspValValAsnLeuIleSerMet.
When X6~ in general formula II, designates one amino acid, it is preferably Ala, Gly, Leu, Thr, Val- or Ser. When X6~ in general formula II, designates a sequence of two amino acids, it is preferably GlyAla or SerAla. When X6~ in general formula II, designates a sequence of three amino acids, it is preferably AlaValAla. When X6, in general formula II, designates a sequence of eight amino acids, it is preferably GlyAlaAspSerLysThrValGlu.
Examples of preferred leader peptides coded for by the DNA
sequence LS are:
SEQ ID No. 1 GlnProIleAspGluAspAsnAspThrSerValAsnLeuProAla:
SEQ ID No. 2 GlnProIleAspAspGluAsnThrThrSerValAsnLeuProAla;
SEQ ID No. 3 GlnProIleAspAspGluSerAsnThrThrSerValAsnLeuProAla;
SEQ ID No. 4 GlnProIleAspAspGluAsnThrThrSerValAsnLeuProVal:
SEQ ID No. 5 GlnProIleAspAspThrGluAsnThrThrSerValAsnLeuProAla;
SEQ ID No. 6 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuPro-Ala;
SEQ ID No. 7 GlnProIleAspAspGluAsnThrThrSerValAsnLeuMetAla;
SEQ ID No. 8 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuPro-GlyAla;
WO 95134666 ~ ~ ~ 219 2 9 4 2 PCT~~5100249 SEQ ID No. 9 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-Ala:
SEQ ID No. 10 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnValPro-Thr;
5 SEQ ID No. 11 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnValPro-Thr:
SEQ ID No. 12 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuPro-Thr;
SEQ ID No. 13 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnValPro-10 GlyAla;
SEQ ID No. 14 GlnProIleAspAspThrGluSerASnThrThrSerValAsnLeuMet-AlaProAlaValAla;
SEQ ID No. 15 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuNet-AspLeuAlaValGlyLeuProGlyAla;
SEQ ID No. 16 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuNet-AlaAspAspThrGluSerIleAsnThrThrLeuValAsnLeuProGly-Ala;
SEQ ID No. 17 GlnProIleAspAspThrGluSerIleASnThrThrLeuValAsnLeu-ProGlyAla;
SEQ ID No. 18 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnLeuPro-GlyAla;
SEQ ID No. 19 GlnProIleAspASpThrGluSerAsnThrThrSerValAsnLeuNet-AlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuValAsn-LeuProLeu;
SEQ ID No. 20 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-AlaAspAspThrGluSerIleAsnThrThrLeuValAsnLeuAlaAsn-ValAlaMetAla;
SEQ ID No. 21 GlnProIleAspAspThrGluSerAlaIleASnThrThrLeuValAsn-LeuProGlyAla;
SEQ ID No. 22 GlnProIleAspAspThrGluSerPheAlaThrAsnThrThrLeuVa1-AsnLeuProGlyAla;
SEQ ID No. 23 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsnLeu-MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuVa1-AsnLeuProLeu;
SEQ ID No. 24 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsnLeu-MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuAsp-ValValAsnLeuProGlyAla;
SEQ ID No. 25 GlnProIleAspAspThrGluSerAlaAlaIleAsnThrThrLeuVa1-AsnLeuProGlyAla;
SEQ ID No. 26 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-AlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuValAsn-LeuAlaAsnValAlaMetAla;
SEQ ID No. 27 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-AlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuAspVa1-ValAsnLeuIleSerMetAla;
SEQ ID No. 28 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-AlaAsnThrThrGluSerArgPheAlaThrAsnThrThrLeuAspVa1-ValAsnLeuIleSerMetAla; and SEQ ID No. 67 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-AlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuAlaLeu-AspValValAsnLeuIleSerMetAlaLysArg.
Particularly preferred leader peptides coded for by the DNA
sequence LS are:
- 2.~ 92942 SEQ ID No. 15 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-AspLeuAlaValGlyLeuProGlyAla:
SEQ ID No. 16 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-AlaAspAspThrGluSerIleAsnThrThrLeuValAsnLeuProGly- , Ala:
SEQ ID No. 17 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsnLeu-ProGlyAla:
SEQ ID No. 18 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnLeuPro-GlyAla:
SEQ ID No. 19 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-AlaAspAspThrGluSerArgPheAlaThrASnThrThrLeuValAsn-LeuProLeu:
SEQ ID No. 20 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet AlaAspAspThrGluSerIleAsnThrThrLeuValASnLeuAlaAsn ValAlaMetAla:
SEQ ID No. 21 GlnProIleAspAspThrGluSerAlaIleAsnThrThrLeuValAsn-LeuProGlyAla:
SEQ ID No. 22 GlnProIleAspAspThrGluSerPheAlaThrAsnThrThrLeuVa1-AsnLeuProGlyAla:
SEQ ID No. 23 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsnLeu-MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuVal-AsnLeuProLeu:
SEQ ID No. 24 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsnLeu-MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuAsp-ValValAsnLeuProGlyAla;
SEQ ID No. 25 GlnProIleAspAspThrGluSerAlaAlaIleAsnThrThrLeuVa1-AsnLeuProGlyAla:
~ wo» ~ 2192942 SEQ ID No. 26 GlnProIleAspASpThrGluSerAenThrThrSeYValAanLauMat-AlaAspAspThxGluserArgPheAlaThrhsnThtThrLeuValAsn-LeuAlaAsnValAlaMetAla: and S~Q ID No. 28 GinProIleAspAspTtuCluSfrAsnThM'hrSeYValAsnLauMet AlaAsr~IhrTttrGluSerArgPheAlaThrASnThrT~srLeuAspVal ValAsnLeuIleSerlietAla.
SEQ ID No. 67 GlnProIleAspAspThrGluSerASnThYfhrSer'ValAsnheuNet-AlnAspAspThrGluSerArgPheAlaThrASnThYIhrLeuAlaLsu-AspValVelAsnLeuIle&erlietAla l0 The signal sequence (SP) may encode any signal peptide which ensures an effective direction of the expressed 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 a-factor signal peptide, the signal peptide of mouse salivary amylase, a modilie:d carboxypeptidase signal peptide, the yeast ~~$y signal peptide or the lanuainosa lipase signal peptide or a derivative thereof. The mouse salivary aaylase signal sequence is described by Hagenbnchle, O, et al., ~yrg ~g,Q (1981) 6C3 s4s. The carboxypeptidase signal sequence is described by Valls, L.A. et al., C~I1 ~ (1987) 887-897. The g8g1 signal peptide is disclosed in WO 87/OZ670. The yeast aspartic protease 3 signal peptide is described in European Patent No.
z5 0 792 367.
The yeast processing site encoded by the DNA sequence PS may suitably be any paired combination of Lys and Arg, such as LysArg, ArgLys, ArgArg or LysLys which peraits processing of the polypeptide by, the 1X2 protease of Saccharomyces cerevisiae or the equivalent protease in other yeast species (Julius, D.A. et al., Cell ~? (1984) 1075). If 1X2 processing is not convenient, e.g. if itr would lead to cleavage of the polypeptide product, e.g. .due to the presence of two WO 95134666 219 2 9 4 2 P~~°~sl~0~a9 consecutive basic amino acid internally in the desired product, a processing site for another protease may be selected comprising an amino acid combination which is not found in the polypeptide product, e.g. the processing site for FXa, IleGluGlyArg (cf. Sambrook, J., Fritsch, E.F. and Maniatis, T., , MW P~niar Cloning: A Laboratorv Manual, Cold Spring Harbor Laboratory Press, New York, 1989).
The protein produced by the method of the invention may be any protein which may advantageously be produced in yeast. Examples of such proteins are heterologous proteins such as aprotinin, tissue factor pathway inhibitor or other protease inhibitors, insulin or insulin precursors, human or bovine growth hormone, interleukin, glucagon, GLP-1, IGF-I, IGF-II, tissue plasminogen activator, transforming growth factor a or p, platelet-derived growth factor, enzymes or a functional analogue thereof. In the present context, the term "functional analogue'° is meant to indicate a protein 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 protein 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. Such modifications are well known for several of the proteins mentioned above. Also, precursors or intermediates for other proteins may be produced by the method of the invention. An example of such a precursor is the MI3 insulin precursor which comprises the amino acid sequence B(1-29)AlaAlaLysA(1-21) wherein A(1-21) is the A chain of human insulin and B(1-29) is the B chain of human insulin in which Thr(B30) is missing.
W O 95134666 219 2 9 4 2 pCT~~5100249 Preferred DNA constructs encoding leader sequences are as shown in Figs. 4 - 12 or suitable modifications thereof. Examples of suitable modifications of the DNA sequence are nucleotide substitutions which do not give rise to another amino acid 5 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 10 modifications are insertion of one or more codons into the sequence, addition of one or more codons at either end of the sequence and deletion of one or more codons at either end of or within the sequence.
The recombinant expression vector carrying the expression 15 casette 5'-P-SP-LS-PS-*gene*-(T)i-3' wherein P, SP, LS, *gene*, T and i are as defined above may be any vector which is capable of replicating in yeast organisms.
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 $accharomvces cerevisiae MFal, TPI, ADH or PGK promoters.
The sequences shown above should preferably also be operably connected to a suitable terminator, e.g. the TPI terminator (cf. Alber, T: and Kawasaki, G., d. Mol. Apol. Genet. ~ (1982) 419-434).
The recombinant expression vector of the invention further comprises a DNA sequence enabling the vector to replicate in yeast. Examples of such sequences are the yeast plasmid 2u replication genes REP 1-3 and origin of replication. The vector may also comprise a selectable marker, e.g. the Schizo-WO 95134666 f 219 2 9 4 2 P~~~~80~9 saccharomyces pombe TPI gene as described by Russell, P.R., Gene ~Q (1985) 125-130.
The methods used to ligate the sequence 5'-P-SP-LS-PS-*gene*-(T)i-3' and to insert it into suitable yeast vectors containing the information necessary for yeast replication, are well known to persons skilled in the art (cf., for instance, Sambrook, J., Fritsch, E.F. and Maniatis, T., ~.cit.). It will be understood that the vector may be constructed either by first preparing a DNA construct containing the entire sequence 5'-P-SP-LS-PS-*gene*-(T)i-3' and subsequently inserting this fragment into a suitable expression vector, or by sequentially inserting DNA
fragments into a suitable vector containing genetic information for the individual elements (such as the promoter sequence, the signal peptide, the leader sequence GlnProIle(Asp/Glu)(Asp/Glu)X1(Glu/Asp)X2AsnZ(Thr/Ser)X3, the processing site, the polypeptide, and, if present, the terminator sequence) followed by ligation.
The yeast organism used in the method of the invention may be any suitable yeast organism which, on cultivation, produces large amounts of the desired polypeptide. Examples of suitable yeast organisms may be strains of the yeast species Saccharoniyces cerevisiae, Saccharomvces kluyveri, Schiaosaccharomvces pombe or Saccharomvces 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 polypeptide, 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 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, WO 95134666 ~ 2 PCTlDK95100?A9 affinity chromatography or the like.
The invention is further described in the following examples which are not to be construed as limiting the scope of the invention as claimed.
EXAMPLES
plasm~ds and DNA material All expression plasmids are of the C-POT type. Such plasmids are described in EP patent application No. 171 142 and are characterized in containing the Schizosaccharomvces pomhe _.
triose phosphate isomerase gene (POT) for the purpose of plasmid selection and stabilization. A plasmid containing the POT-gene is available from a deposited E. coli strain (ATCC
39685). The plasmids furthermore contain the ~. cerevisiae triose phosphate isomerase promoter and terminator (PTP1 and TTP~). They are identical to pMT742 (Egel-Mitani, M. et al., Gene (1988) 113-120) (see Fig. 1) except for the region defined by the EcoR I-Xba I restriction sites encompassing the coding region for signal/leader/product.
The plasmids pAK527, pAK531, pAK555, pAK559, pAK562, pAK614 and pAK625 were used as DNA templates in the PCR reactions applied in the construction of the leaders described in the examples.
The synthetic DNA fragments serving as the direct template are shown in Figs. 13 - 17. With the exception of the shown DNA
regions the plasmids are identical to pAK492 shown in Fig. 1.
Synthetic DNA fragments were synthesized on an automatic DNA
synthesizer (Applied Biosystems model 380A) using phosphoramidite chemistry and commercially available reagents (Beaucage, S.L. and Caruthers, M.H., Tetrahedron Letters ~ __ (1981) 1859-1869).
wo 9sr3~s. . ~ ~ ' v~ 19 2 9 4 2 rcrmxssioe~
All other methods and matsrials used are coaston state o! ttfe art knowledge (sea, s.g. &ambrook, J., Fritsch, E.F. and I~Ianiatis, T., ~c~ecu~ar Cloning: A Laborato~~nual, Cold Spring Harbor Lelboratory Press, Pew York, 1989).
EZaliPLE i Synthesis of the leader SEQ ID'No. 4 for expression of the xI3 insulin precursor in ~ c red (strain yAR546).
The leader SEQ ID No. 4 has the following amino acid sequence:
GlnProIleAspAspGluAsnThYrhrSsrValAsnLeuProVa1 The following oligonuclaotides wars synthesised:
94 5'-TJW1TCTATJ1ACTAC11AAAAACACAT11-3' SSQ ID xo. t!
# 333 5'-GACTCTCTTAACTGGCAAGTTGACA-3' 8Efl ID lio. ~0 / 312 5'-AAGTACAAAGCTTCAACCAAGTGAGAACCACACAAGTaiT .
GGTT7U1CGAATCTC1'f-3' SEQ ID xo. ~i t 1845 5'-CATACACAATATAAACGACGG-3' SSQ ID No. 3Z
The following poTynerase -cbain reactions (PCR) xere.perlorasd using the Gene Amp~PCR reagent kit (Psrkin Elmer~' 761 plain Avewalk, CT 06859, SSA) according to the manulacturers instructions. During the reaction, the PCR mixturaa xara overlayed with 100 ~tl of mineral oil (Sigma Chemical CO, St.
Louie NO, U6A):
lRivnerasa cram raacz.avn nv. i 5 ~1 0! oligonucleotide f 94 (50 pmol) 5 ul of oligonucleotids 4 333 (50 pmol) 10 ~l o! 10X PCR buffer 16 ~l of dNTP mix 0.5 xl of Taq ensy~
0.5 ~t1 of pAK527 plasmid (Fig. 13) as template (0.2 ug of DtIA) w't- = -T~'1 2192942 .
63 ul of water A total of 12 cycles were performed, one cycle was 94'C for 1 sin: 37'C for 2 min: 72'C for 3 min. The PCR mixture was then loaded onto a 2~ agarose gel and electrophoresis was performed using standard techniques (Sambrook, J., Fritsch, E.F. and Maniatis, T., o_p.cit.). The resulting DNA fragment was cut out of the agairose gel and isolated using the Gene Clean kit (Bio 101 inc., PO BOX 2284, La Jolla, CA 92038, USA) according to the manufacturers instructions.
Polvmerase chain reaction No. 2 5 ~1 of oligonucleotide ~ 312 (50 pmol) 5 pl of oligonucleotide ; 94 (50 pmol) io ul of lox ~ buffer i6 ~l of dNTP mix 0.5 ~1 of Taq enzyme l0 ~C1 of purified DNA lragment from PCR No. 1 53.5 ~tl of water A total o! 12 cycles Were performed, one cycle was 94'C for 1 min; 37'C for 2 min: 72'C for 3 min.
The DNA fragment from polymerase chain reaction No. 2 was isolated.and purified using the Gens Clear~kit (Bio 101 inc., ~_.
PO 80X 2284, La Jolla, CA 92038, USA) according to the manufacturers instructions. ' The purified pCR DNA fragment was dissolved in l0 pl o! water and restriction endonuclease buffer and cut with the restriction endonuclsases Asp 718 and Hind iII in a total volume of 15 pl according to standard techniques (Sanbrook, J., Fritsch, E.F. and Maniatis, T., on.cit.). The 167 by Asp 718/Hind III DNA fragment was subjected to electrophoresis on agarose gel and purified using The Gene Clean Kit as described.
The ~ oaravi_si_"e_ expression plasmid pAK492 (shown in Fig. 1) is a derivative of the previously described plasmid pMT742 in ..._ ..._........... ......______ _.___. _ ___._ .
WO 95/34666 ~ i ~ 2 9 4 2 PCTIDK95100249 which the fragment encoding the signal/leader/insulin precursor has been replaced by the EcoR I-Xba I fragment shown in Fig. 2.
This fragment has been synthesized on an Applied Biosystems DNA
synthesizer in accordance with the manufacturer's instructions.
5 The plasmid pAK492 was cut with the restriction endonucleases Asp 718 and Xba I and the vector fragment of 10986 by was isolated. The plasmid pAK492 was cut with the restriction endonucleases Hind III and Xba I and the DNA fragment of 140 by encoding part of the MI3 insulin precursor was isolated. The 10 three DNA fragments were ligated together using T4 DNA ligase under standard conditions (Sambrook, J., Fritsch, E.F. and Maniatis, T., oo.cit.). The ligation mixture was then transformed into a competent E. coli strain (R-, M+) and transformants were identified by ampicillin resistance.
15 Plasmids were isolated from the resulting E. coli colonies using standard DNA miniprep technique (Sambrook, J., Fritsch, E.F. and Maniatis, T., op.cit.), checked with appropriate restrictions endonucleases i.e. EcoR I, Xba I, Nco I and Hind III. The selected plasmid, pAK546, was shown by DNA sequencing 20 analysis (Sequenase, U.S. Biochemical Corp.) using the primer # 94 to contain a DNA sequence encoding the leader SEQ ID No.
4. For the DNA sequence encoding the leader SEQ ID No. 4, see Fig. 4). The plasmid pAK546 was transformed into ,~ cerevisiae strain MT663 as described in European published patent application No. 214 826 and the resulting strain was named yAK546. The DNA sequence of the protein coding region of the expression plasmid is given in Fig. 5.
Synthesis of the leader SEQ ID No. 6 for expression of the MI3 insulin precursor in ~ cerevisiae (strain,yAK531).
The leader SEQ ID No. 6 has the following amino acid sequence:
GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuProAla WO 95/34666 2 ~ 9 2 9 q. 2 PCTIDK95100249 The following oligonucleotide was synthesised:
# 331 5'-GAATCTCTTAGCTGGCAAGTTGACAGAAGTAGTGTTAG
TTTCAGAGTCGTCAATT-3' SEQ ID No. 33 The polymerase chain reaction was performed as described in Example 1 with the expection that oligonucleotide # 331 was used insted of oligonucleotide # 333.
The Asp 718/Hind III DNA fragment of 168 by was subjected to electrophoresis on agarose gel and purified as described in Example 1. The Asp 718/Hind III DNA fragment was subcloned into the Sz cerevisiae expression plasmid as described in Example 1.
The selected plasmid, pAK531, was shown by DNA sequencing analysis, as described in Example 1, to contain a DNA sequence encoding the leader SEQ ID No. 6. For the DNA sequence encoding the leader SEQ ID No. 6, see Fig. 6. The plasmid pAK531 was transformed into ~ ~erevisiae strain MT663 as described in European patent application 86306721.1 and the resulting strain was named yAK531. The DNA sequences encoding the signal peptide and the insulin precursor MI3 were the same as those shown in Fig. 5.
Synthesis of the leader SEQ ID No. 8 for expression of the MI3 insulin precursor in ~ v~~~a (strain yAK547).
The leader SEQ ID No. 8 has the following amino acid sequence:
GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuProGlyAla The following oligonucleotide was synthesised:
# 345 5'-AACGAATCTCTTAGCACCTGGCAAGTTGACAGAAGT-3' SEQ ID No. 34 WO 95134666 ~-19 2 9 4 2 PCT1DK95100249 The polymerise chain reaction was performed as described in Example 1 with the expection that oligonucleotide # 345 was used insted of oligonucleotide # 333 and plasmid pAK531 (Fig.
14) was used as template.
The Asp 718/Hind III DNA fragment of 171 by was subjected to electrophoresis on agarose gel and purified as described in Example 1. The Asp 718/Hind III DNA fragment was subcloned into the ~ cerevisiae,expression plasmid as described in Example 1.
The selected plasmid, pAK547, was shown by DNA sequencing analysis, as described in Example 1, to contain a DNA sequence encoding the leader SEQ ID No. 8. For the DNA sequence encoding the leader SEQ ID No. 8, see Fig. 7. The plasmid pAR547 was transformed into ~ gerevisiae strain MT663 as described in European patent application No. 86306721.1 and the resulting strain was named yAK547. The DNA sequences encoding the signal peptide and the insulin precursor MI3 were the same as those shown in Fig. 5.
Synthesis of the leader SEQ ID No. 17 for expression of the MI3 insulin precursor in ~ cerevisiae (strain yAK561).
The leader SEQ ID No. 17 has the following amino acid sequence:
GlnProIleAspAspThrGluSerIleAsnThrThrLeuValASnLeuProGlyAla The following oligonucleotide was synthesised:
# 376 5'-AACGAATCTCTTAGCACCTGGCAAGTTGACCAAAGTAG
TGTTGATAGATTCAGTGTCGTC-3' SEQ ID No. 35 The polymerise chain reaction was performed as described in Example 1 with the expection that oligonucleotide # 376 was used insted of oligonucleotide # 333 and plasmid pAK555 (Fig.
15) was used as template.
R'O 95/34666 219 2 9 4 2 PCTIDK95100249 The Asp 718/Hind III DNA fragment of 180 by was subjected to electrophoresis on agarose gel and purified as described in Example 1. The Asp 718/Hind III DNA fragment was subcloned into the S~ cerevisiae expression plasmid as described in Example 1.
The selected plasmid, pAK561, was 'shown by DNA sequencing analysis, as described in Example 1, to contain a DNA sequence encoding the leader SEQ ID No. 17. For the DNA sequence encoding the leader SEQ ID No. 17, see Fig. 8. The plasmid pAK561 was transformed into Sue. cerevisiae strain MT663 as described in European patent application No. 86306721.1 and the resulting strain was named yAK561. The DNA sequences encoding the signal peptide and the insulin precursor MI3 were the same as those shown in Fig. 5.
Synthesis of the leader SEQ ID No. 16 for expression of the MI3 insulin precursor in ;Z cerevisiae (strain yAK559j.
The leader SEQ ID No. 16 has the following amino acid sequence:
GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMetAlaAspAspThr-GluSerIleAsnThrThrLeuValAsnLeuProGlyAla The following oligonucleotide was synthesised:
# 375 5'-AACGAATCTCTTAGCACCTGGCAAGTTAACCAAAGTAGT
GTTGATAGATTCAGTGTCGTCAGCCATCAAGTTGAC-3' 8EQ ID No. 36 The polymerase chain reaction was performed as described in Example 1 with the expection that oligonucleotide # 375 was used insted of oligonucleotide # 333 and plasmid pAK555 (Fig.
15j was used as template.
The Asp 718/Hind III DNA fragment of 222 by was subjected to electrophoresis on agarose gel and purified as described in Example 1. The Asp 718/Hind III DNA fragment was subcloned into the ~ cerevisiae expression plasmid as described in Example 1.
The selected plasmid, pAK559, was shown by DNA sequencing analysis, as described in Example 1, to contain a DNA sequence encoding the leader SEQ ID No. 16. For the DNA sequence encoding the leader SEQ ID No. 16, see Fig. 9. The plasmid pAK559 was transformed into ~ cerevisiae strain MT663 as described in European patent application No. 86306721.1 and the resulting strain was named yAK559. The DNA sequences encoding the signal peptide and the insulin precursor MI3 were the same as those shown in Fig. 5.
Synthesis of the leader SEQ ID No. 19 for expression of the MI3 insulin precursor in ~ cerevisiae (strain yAK580).
The leader SEQ ID No. 19 has the following amino acid sequence:
GlnProIleAspAspThrGluSerAsnThrThrSerValASnLeuMetAlaAspAspThr-GluSerArgPheAlaThrAsnThrThrLeuValAsnLeuProLeu The folloiaing oligonucleotide was synthesised:
# 384 5'-AACGAATCTCTTCAATGGCAAGTTAACCAAAGTAGTGT
TAGTAGCGAATCTAGATTCAGTGTCGTCAGCCAT-3' 8EQ ID No. 37 The polymerase chain reaction was performed as described in Example 1 with the expection that oligonucleotide # 384 was used insted of oligonucleotide # 333 and plasmid pAK559 (Fig.
16) was used as template.
The Asp 718/Hind III DNA fragment of 228 by was subjected to electrophoresis on agarose gel and purified as described in Example 1. The Asp 718/Hind III DNA fragment was subcloned into the ~ cerevisiae expression plasmid as described in Example 1.
The selected plasmid, pAK580, was shown by DNA sequencing analysis, as described in Example 1, to contain a DNA sequence 219 2 9 4 2 pCT~~S/00249 encoding the leader SEQ ID No. 19. For the DNA sequence.
encoding the leader SEQ ID No. 19, see Fig. 10. The plasmid pAK580 was transformed into ,~ cerevisiae strain MT663 as described in European patent application No. 86306721.1 and the 5 resulting strain was named yAK580. The DNA sequences encoding the signal peptide and the insulin precursor MI3 were the same as those shown in Fig. 5.
Synthesis of the leader SEQ ID No. 20 for expression of the MI3 10 insulin precursor in ~ s sa (strain yAK583).
The leader SEQ ID No. 20 has the following amino acid sequence:
GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMetAlaAspAspThr-GluSerIleAsnThrThrLeuValAsnLeuAlaAsnValAlaMetAla 15 The following oligonucleotide was synthesised:
# 390 5'-AACGAATCTCTTAGCCATGGCAACGTTAGCCAAGTTAA
~CCAAAGT-3' SEQ ID No. 38 The polymerase chain reaction was performed as described in Example 1 with the expection that oligonucleotide # 390 was 20 used insted of oligonucleotide # 333 and plasmid pAK559 (Fig.
16) was used as template.
The Asp 718/Hind III DNA fragment of 231 by was subjected to electrophoresis on agarose gel and purified as described in Example 1. The Asp 718/Hind III DNA fragment was subcloned into 25 the ~ cerevisiae expression plasmid as described in Example 1.
The selected plasmid, pAK583, was shown by DNA sequencing analysis, as described in Example 1, to contain a DNA sequence encoding the leader SEQ ID No. 20. For the DNA sequence encoding the leader SEQ ID No. 20, see Fig. 11. The plasmid pAK583 was transformed into Sue. ~erevisiae strain MT663 as a described in European patent application No. 86306721.1 and the resulting strain was named yAK583. The DNA sequences encoding the signal peptide and the insulin precursor MI3 were the same as those shown in Fig. 5.
E%AMPLE 8 Synthesis of the leader SEQ ID No. 21 for expression of the MI3 insulin precursor in ~ cerevisiae (strain yAK586).
The leader SEQ ID No. 21 has the following amino acid sequence:
GlnProIleAspAspThrGluSerAlaIleAsnThrThrLeuValAsnLeuProGlyAla The following oligonucleotide was synthesised:
# 401 5'-AACGAATCTCTTAGCACCTGGCAAGTTGACCAAAGTAG
TGTTGATAGCAGATTCAGTGTCG-3° SEQ ID No. 39 The palymerase chain reaction was performed as described in Example 1 with the exception that oligonucleotide # 401 was used insted of oligonucleotide # 333 and plasmid pAK562 (Fig.
17) was used as template.
The Asp 718/Hind III DNA fragment of 183 by was subjected to electrophoresis on agarose gel and purified as described in Example 1. The Asp 718/Hind III DNA fragment,was subcloned into the ~ cerevisiae expression plasmid as described in Example 1.
The selected plasmid, pAK586,. was shown by DNA sequencing analysis, as described in Example 1, to contain a DNA sequence encoding the leader SEQ ID No. 21, see Fig. 12. The plasmid pAK586 was transformed into ~ cerevisiae strain MT663 as described in European patent application No. 86306721.1 and the resulting strain was named yAK586. The DNA sequences encoding , the signal peptide and the insulin precursor MI3 were the same as those shown in Fig. 5.
R'O 95/34666 PCTIDK95100249 Expression of the MI3 insulin precursor using selected leader sequences according to the present invention.
Yeast strains harbouring plasmids as described above, were grown in YPD medium (Sherman, F. et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, 1981). For each strain 6 individual 5 ml cultures were shaken at 30°C for 72 hours, with a final OD6oo 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 Snel, L. et al. Chromatoaraphia ~ (1987) 329-332.
In Table 1 the expression levels of the insulin precursor, MI3, obtained by use of selected leader sequences according to the present invention, are given as a percentage of the level obtained with transformants of pMT742, utilizing the MFa(1) leader of ~ cerevisiae.
Table 1 ~ Leader Expression level, %
MT748 a-leader 100 SEQ ID No. 15 87 SEQ ID No. 16 215 SEQ ID No. 17 157 SEQ ID No. 19 166 SEQ ID No. 20 86 SEQ ID No. 21 145 SEQ ID No. 22 137 SEQ ID No. 23 121 WO 95134666 219 2 9 4 2 PCT~~5100249 Synthesis of the leader SEQ ID No. 27 for expression of the extended MI3 insulin precursor in S~ cerevisiae (strain yAK677).
The leader SEQ ID No. 27 has the following amino acid sequence:
GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMetAlaAspAspThr-GluSerArgPheAlaThrAsnThrThrLeuASpValValASnLeuIleSerMetAla The following oligonucleotides were synthesised:
# 440 5'-GGTTAACGAACTTTGGAGCTTCAGCTTCAGCTTCTTCTCTCTTAGCCAT
GGAGATCAAGTTAACAACATCCAAAGTAGTGTT-3' BEQ ID No. 64 and # 441 5'-CAAGTACAAAGCTTCAACCAAGTGGGAACCGCACAAGTGTTGGTTAACG
AACTT-3' SEQ ID No. 65 Polymerise chain reactions were performed as described in Example in 1 with the exception that oligonucleotide # 440 was used instead of oligonucleotide # 333 and plasmid pAK614 was used as template. For the second polymerise chain reaction, oligonucleotide # 441 was used instead of oligonucleotide #
312.
The purified PCR DNA fragment was isolated and digested with the restriction endonucleases Asp 718 and Hind III as described in Example 1. The Asp 718/Hind III DNA fragment of 268 by was subjected to electrophoresis on agarose gel and purified as descYibed in Example 1. The Asp 718/Hind III DNA
fragment was subcloned into the ~ cerevisiae expression plasmid as described in Example 1, with the exception that the 140 by Hind III/Xba I DNA fragment was derived from pAK602 and encodes Asp$28 human insulin. The selected plasmid, pAK616, was shown by DNA sequencing analysis, as described in Example 1, to contain the DNA sequence encoding the leader SEQ ID No. 27. For the DNA sequence, SEQ ID No.
66, encoding the leader SEQ ID No. 27, see Fig. 18. The Asp 718/Hind III DNA fragment of 268 by from pAK616 was isolated and ligated with the 10986 by Asp 718/Xba I DNA fragment from pAK601 and the 140 by DNA fragment Hind III/Xba I from pAK464 (encoding an extended version of AspB28 human insulin) and named pAK 625. The 180 by Asp 718/Nco I DNA fragment from pAK625 was isolated and ligated with the 221 by Nco I/Xba I
DNA fragment from pJB146 (encoding and extended version of the insulin precursor) and the 10824 by Asp 718/Xba I DNA
fragment from pAK601 and the resulting plasmid was named pAK677. The plasmid pAK677 was transformed into S~ cerevisiae strain MT663 as described in European patent application 86306721.1 and the resulting strain was named yAK677. With the exception of the DNA sequence encoding the leader, the DNA sequence encoding the signal peptide is as described in Fig. 5. The DNA sequence coding for the extended MI3 insulin precursor is as described in Fig. 19.
EgAMPLE ~1 Synthesis of the leader SEQ ID No. 67 for expression of the extended MI3 insulin precursor in Sue. cerevisiae (yAK680) The leader SEQ ID No. 67 has the following amino acid sequence:
GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMetAlaAspAspThr GluSerArgPheAlaThrAsnThrThrLeuAlaLeuAspValValAsnLeuIleSerMet Ala The following oligonucleotide was synthesised:
# 577 5'-TCTCTTAGCCATGGAGATCAAGTTAACAACATCCAAAG
CCAAAGTAGTGTT-3' SEQ ID No. 68 wo>s~ . . 2192942 so The PCR vas performed es daecribed in Example fn 1 with the exception that oligonucleotlde ~ 577 vas need instead of oligonucleotide ~ 933 and plasmid puC625 vas used as te~lata and the second PcR vas not perforaad. The FC7t lragment vas 3 digested with the restriction endonuclaases Asp 718 and Nco I
as described in Exeaplo 1:
The Asp 71~%ll0o I DNA fragsent o! 19o by vas subjected to electrophoresis on agarose gei and puriliad as described in Example 1 expect that the 10824 by Asp 718/Xba I vector DNA
iragsem 'vas isolated iron and tros pA1~601. Tba 190 by Asp 718/Nco I DNA fragment vas subclonsd into the ~ ~YifiiA!
expression plasmid as described in Exaaple 1, expect that the 221 by DNA fragosnt Nco I/Xba I (encoding an extended version of the !!I3 insulin precursor) vas isolated lram pAR677 and used instead of the Nind III/Xba I DNA lrsgsant. The selected plasmid vas shown by DNA sequencing analysis as described in Example I to contain the DNA sequence encoding the leader SEQ
ID No. 67 a~ named pA1C680. For the DNA saquanoe, BEQ ID No.
69, encoding the feeder SEQ ID No. 67, sea Fig. 20. Tha plasmid pA1C680 vas transforsed into $z cereviaiae strain 1~T663 as' described in European patent Tlo. 0214 $26.
and the resulting strain vas named yAR68o. lPith the exception of the DNA sequence encoding the leader, the DNA sequence encoding the signal peptide is as described in Fig. 5 and the extended insulin precursor liI3 DNA saqusnae is as described 1n rig. 19. _ LIIUIPLE l2 Exprasslon of N-terminally extended 1~I3 insulin precursors using the leader sequences SEQ ID No. 27 and SEQ ID No. 67 3o according to flee preset invention.
Yeast strains harbouring plasmids as described above, Ware grown in YPD medium (Shernan, F. at al., Methodg,in Yeast W0 95/34666 PCTlDK95100249 Genetics, Cold Spring Harbor Laboratory Press, 1981). For each strain 6 individual 5 ml cultures were shaken at 30'C
for 72 hours, with a final oDboo of approximately 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 Snel, L. et al. Chromatog!raphia ,fig (1987) 329-332.
In Table 2 the expression levels of some N-terminally extended MI3 insulin precursors, obtained by use of the leader sequences SEQ ID No. 27 and SEQ ID No. 67 according to the present invention, are given as a percentage of the level obtained with transformants of pMT742, utilizing the MFa(ij leader of S_s Cerevisiae.
Table 2 Strain Signal peptideLeader Extension Relative to MT748 a a yAK675 YAP3 SEQ ID EEAEAEAP 251%
No.27 K
yAK677 YAP3 SEQ ID EEAEAEAE 224%
No.27 PK
yAK681 YAP3 SEQ ID EEAEAEAP 248%
No.67 K
2o yAK680 YAP3 SEQ ID EEAEAEAE 362%
No.67 PK
WO 95134666 219 2 9 4 2 PCT~~SI00249 SEQUENCE LISTING
(1) GENERAL INFORMATION:
{i) APPLICANT:
(A) NAME: Novo Nordisk A/S
(B) STREET: Novo Alle (C) CITY: DK-2880 Bagsvaerd (E) COUNTRY: Denmark (G) TELEPHONE: +45 44448888 (H) TELEFAX: +45 44490555 (I) TELEX: 37173 (ii) TITLE OF INVENTION: SYNTHETIC LEADERS PEPTIDE SEQUENCES
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{A) APPLICATdON NUMBER: DK 0705/94 and US 08/282,852 (B) FILING DATE: 16-JUN-1994 and 29-JUL-1994 (viii} ATTORNEY/AGENT INFORMATION:
A) NAME: Jorgensen, Dan et a1.
~C) REFERENCE/DOCKET NUMBER: 4085-WO, DJ
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: +45 44448888 (B) TELEFAX: +45 44493256 (2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear WO 95/34666 219 2 9 4 2 PC'I'~I~95100249 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
itn Pro I1e Asp 51u Asp Asn Asp Thr Ser Va1 Asn Leu Pro A1a (2) INFORMATION FOR SEQ ID N0:2:
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R'O 95134666 219 2 9 4 2 PCT~~S/00149 (A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5: -Gtn Pro I1e Asp Asp Thr G1u Asn Thr Thr Ser Ya1 Asn Leu Pro A1a (2) INFORMATION FOR SEQ ID N0:6:
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G1n Pro I1e Asp Asp Thr G1u Ser Asn Thr Thr Ser Va1 Asn Leu Pro Ala (2) INFORMATION FOR SEQ ID N0:7:
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G1n Pro I1e Asp Asp Glu Asn Thr Thr Ser Yal Asn Leu Met Ala (2) INFORMATION FOR SEQ ID N0:8:
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WO 95/34666 219 2 9 4 2 PCTJDK95l00249 Gln Pro Ile Asp Asp Thr GIu Ser Asn Thr Thr Ser Va1 Asn Leu Pro G1y A1a (2) INFORMATION FOR SEQ ID N0:9:
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G1n Pro I1e Asp Asp Thr G1u Ser Asn Thr Thr Ser Ya1 Asn Leu Met i 5 10 15 A1a (2) INFORMATION FOR SEQ ID N0:10:
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G1n Pro I1e Asp Asp Thr G1u Ser Asn Thr Thr Leu Ya1 Asn Ya1 Pro 61y A1a (2) INFORMATION FOR SEQ ID N0:14:
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WO 95/34666 219 2 9 4 2 PCT11~K95100249 G1n Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Ser Va1 Asn Leu Met A1a Pro A1a Va1 A1a (2) INFORMATION FOR SEQ ID N0:15:
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Gln Pro I1e Asp Asp Thr G1u Ser Asn Thr Thr Ser Val Asn Leu Met Asp Leu A1a Va1 Gly Leu Pro G1y A1a (2) INFORMATION FOR SEQ ID N0:16:
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G1n Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Ser Va1 Asn Leu Met Ala Asp Asp Thr Glu Ser I1e Asn Thr Thr Leu Yal Asn Leu Pro Gty A1a (2) INFORMATION FOR SEQ ID N0:17:
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R'O 95/34666 . 219 2 9 4 2 p~~gg~p0249 G1n Pro I1e Asp Asp Thr G1u Ser I1e Asn Thr Thr Leu Ya1 Asn Leu Pro Gly A1a (2) INFORMATION FOR SEQ ID N0:18:
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G1n Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Leu Yal Asn Leu Pro G1y A1a (2) INFORMATION FOR SEQ ID N0:19:
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G1n Pro IIe Asp Asp Thr G1u Ser Asn Thr Thr Ser Ya1 Asn Leu Met A1a Asp Asp Thr G1u Ser Arg Phe Ala Thr Asn Thr Thr Leu Ya1 Asn Leu Pro Leu (2) INFORMATION FOR SEQ ID N0:20:
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i1n Pro I1e Asp 5sp Thr G1u Ser Asn Thr Thr Ser Va1 Asn Leu Met A1a Asp Asp Thr G1u Ser I1e Asn Thr Thr Leu Ya1 Asn Leu A1a Asn Ya1 A1a Met Ala (2) INFORMATION FOR SEQ ID N0:21:
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(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
i1n Pro I1e Asp 5sp Thr Glu Ser A1a Ile Asn Thr Thr Leu Yal Asn Leu Pro G1y A1a (2) INFORMATION FOR SEQ ID N0:22:
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(A) LENGTH: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
i1n Pro I1e Asp 5sp Thr GIu Ser Phe A1a Thr Asn Thr Thr Leu Yal Asn Leu Pro Gly A1a (2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide WO 95!34666 219 2 9 4 2 PCT~~5100249 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
G1n Pro I1e Asp Asp Thr G1u Ser Ite Asn Thr Thr Leu Ya1 Asn Leu Met A1a Asp Asp Thr Glu Ser Arg Phe A1a Thr Asn Thr Thr Leu Ya1 Asn Leu Pro Leu (2) INFORMATION FOR SEQ ID N0:24:
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G1n Pro I1e Asp Asp Thr G1u Ser I1e Asn Thr Thr Leu Vat Asn Leu Met A1a Asp Asp Thr G1u Ser Arg Phe A1a Thr Asn Thr Thr Leu Asp Va1 Va1 Asn Leu Pro G1y A1a (2) INFORMATION FOR SEQ ID N0:25:
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G1n Pro I1e Asp Asp Thr Glu Ser Ata A1a Ite Asn Thr Thr Leu Va1 Asn Leu Pro G1y Ala (2) INFORMATION FOR SEQ ID N0:26:
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G1n Pro Ile Asp Asp Thr G1u Ser Asn Thr Thr Ser Yat Asn Leu Met Ala Asp Asp Thr Glu Ser Arg Phe A1a Thr Asn Thr Thr Leu Va1 Asn Leu A1a Asn Yat Ala Met Ala (2) INFORMATION FOR SEQ ID N0:27:
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Gln Pro I1e Asp Asp Thr Glu Ser Asn Thr Thr Ser Va1 Asn Leu Met Ata Asp Asp Thr G1u Ser Arg Phe A1a Thr Asn Thr Thr Leu Asp Va1 Val Asn Leu I1e Ser Met A1a (2) INFORMATION FOR SEQ ID N0:28:
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G1n Pro IIe Asp Asp Thr G1u Ser Asn Thr Thr Ser Ya1 Asn Leu Met Ata Asn Thr Thr G1u Ser Arg Phe A1a Thr Asn Thr Thr Leu Asp Va1 WO 95134666 2 ~ ~ 9 2 9 4 2 PCTI1DK95I00249 Ya1 Asn Leu I1e Ser Met Ata (2) INFORMATION FOR SEQ ID N0:29:
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(2) INFORMATION FOR SEQ ID N0:36:
S
W O 95Y34666 219 2 9 4 2 PCT~~~00249 (i) SEQUENCE CHARACTERISTICS:
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6TCGTCAGCC AT ~ 72 (2) INFORMATION FOR SEQ ID N0:38:
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G sl (2) INFORMATION FOR SEQ ID N0:40:
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(A) LENGTH: 372 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 82..351 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:
GAATTCATTC AAGAATAGTT TATCAATTTC ATACACAATA
CAAACAAGAA
GATTACAAAC
A
Met LysLeuLys ThrYalArgSer A1aYa1 TCA
LeuSer Leu PheA1aSer GtnYa1Leu G1yGtnProIle AspG1u Ser GAC
AspAsn Thr SerSerMet AtaLysArg PheYa1AsnGtn HisLeu Asp TCC
CysGly His LeuVa1G1u A1aLeuTyr .LeuVa1CysG1y G1uArg Ser TTC
G1yPhe Tyr ThrProLys AtaAlaLys G1yI1eYa1G1u G1nCys Phe TGTACC ATC TGCTCCTTG TACCAATTG GAAAACTACTGC AACTAGACGCAGC
TCC
CysThr I1e CysSerLeu TyrG1nLeu G1uAsnTyrCys Asn Ser TAGA
(2)INFORMATION FORSEQID :
N0:41 (i) CHARACTERIST ICS:
SEQUENCE
R'O 95134666 2 , ~ 9 2 9 4 2 PCT~~~I00249 (A) LENGTH: 89 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:
Met Lys Leu Lys Thr Ya1 Arg Ser Ala Ya1 Leu Ser Ser Leu Phe A1a Ser G1n Ya1 Leu G1y G1n Pro I1e Asp Glu Asp Asn Asp Thr Ser Ser Met Ala Lys Arg Phe Va1 Asn G1n His Leu Cys G1y Ser His Leu Ya1 G1u A1a Leu Tyr Leu Va1 Cys G1y G1u Arg G1y Phe Phe Tyr Thr Pro Lys A1a A1a Lys Gty I1e Val G1u G1n Cys Cys Thr Ser I1e Cys Ser Leu Tyr G1n Leu G1u Asn Tyr Cys Asn (2) INFORMATION FOR SEQ ID N0:42:
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~(A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..45 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:
Gln Pro I1e Asp Asp Gtu Asn Thr Thr Ser Ya1 Asn Leu Pro Va1 (2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear WO 95/34666 2-192 9 4 2 PCT~1~95/0.0249 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
G1n Pro I1e Asp Asp G1u Asn Thr Thr Ser Va1 Asn Leu Pro Va1 (2) INFORMATION FOR SEQ ID N0:44:
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(A) LENGTH: 297 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..276 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:
ACT
MeiLysLeuLys Th Va1ArgSer AlaVa1Leu SerSerLeuPhe Ata SerG1nVa1Leu GlyGlnProI1e AspAspGlu AsnThrThrSer Va1 AsnLeuProVa1 LysArgPheYa1 AsnG1nHis LeuCysG1ySer His LeuVa1G1uA1a LeuTyrLeuYat CysG1yG1u ArgGlyPhePhe Tyr ThrProLysA1a A1aLysG1yIle YalGtuGtn GysCysThrSer I1e CCGCAGGGTC
CysSerLeuTyr G1nLeuG1uAsn TyrCysAsn TAGA
(2)INFORMAT ION FORSEQID
N0:45:
( i) EQUENCE CHARACTE RISTICS:
S
(A) LENGTH:91 amino ids ac (B) TYPE: aci d amino (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:
Met Lys Leu Lys Thr Ya1 Arg Ser Ata Ya1 Leu Ser Ser Leu Phe A1a Ser G1n Va1 Leu Gty Gtn Pro I1e Asp Asp G1u Asn Thr Thr Ser Ya1 Asn Leu Pro Va1 Lys Arg Phe Ya1 Asn Gln His Leu Cys G1y Ser His Leu Va1 Gtu Ala Leu Tyr Leu Va1 Cys G1y G1u Arg G1y Phe Phe Tyr Thr Pro Lys A1a A1a Lys G1y I1e Va1 G1u Gtn Cys Cys Thr Ser I1e Cys Ser Leu Tyr G1n Leu G1u Asn Tyr Cys Asn (2) INFORMATION FOR SEQ ID N0:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..51 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:46:
G1n Pro I1e Asp Asp Thr Glu Ser Asn Thr Thr Ser Va1 Asn Leu Pro A1a (2) INFORMATION FOR SEQ ID N0:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: Linear R'O 95134666 PC"lYDK95100249 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:
Gln Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Ser Ya1 Asn Leu Pro A1a (2) INFORMATION FOR SEQ ID N0:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..54 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:
Gln Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Ser Yal Asn Leu Pro GGT GCT ~ 54 G1y A1a (2) INFORMATION FOR SEQ ID N0:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
~ (A) NAME/KEY: CDS
(B) LOCATION: 1..57 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:
61n Pro I1e Asp Asp Thr Glu Ser I1e Asn Thr Thr Leu Va1 Asn Leu 2x92942 ccA GGT Gcr Pro G1y A1a (2) INFORMATION FOR SEQ ID N0:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 99 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..99 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:
G1n Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Ser Vat Asn Leu Met Ata Asp Asp Thr G1u Ser I1e Asn Thr Thr Leu Va1 Asn Leu Pro G1y A1a (2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 105 base pairs B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATIDN: 1..105 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:
G1n Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Ser Va1 Asn Leu Met WO 95134666 219 2 9 4 2 pCTIDK95100249 A1a Asp Asp Thr G1u Ser Arg Phe Ata Thr Asn Thr Thr Leu Ya1 Asn Leu Pro Leu (2) INFORMATION FOR SEQ ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 108 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..108 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:52:
G1n Pro I1e Asp Asp Thr G1u Ser Asn Thr Thr Ser Va1 Asn Leu Met A1a Asp Asp Thr G1u Ser I1e Asn Thr Thr Leu Ya1 Asn Leu A1a Asn ' 20 25 30 Ya1 Ata Met A1a (2) INFORMATION FOR SEQ ID N0:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..60 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:53:
WO 95134666 ~ s w219 2 9 4 2 PCT~~5100249 G1n Pro I1e Asp Asp Thr G1u Ser A1a Ite Asn Thr Thr Leu Yal Asn Leu Pro G1y Ata (2) INFORMATION FOR 5EQ ID N0:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 276 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 113..274 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:54:
AACTACAAAA TCAAACAAGA
AACACATACA
CTATCAATTT TA
CATACACAAT ATG
Met AGA GCG TCG
LysLeuLys Ya1 Ser Ya1Leu Ser LeuPheA1a Ser Thr Arg A1a Ser GGC CCA GAC AAC
GlnYa1Leu Gln I1e AspG1u Thr ThrSerYal Asn G1y Pro Asp Asn AAG TTC AAC TTG
LeuProA1a Arg Va1 G1nHis Cys GlySerHis Leu Lys Phe Asn Leu TTG TT
YalGluA1a Tyr Leu (2) INFORMATION FOR SEQ ID N0:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 amino acids (B) TYPE: amino acid (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:55:
Met Lys Leu Lys Thr Va1 Arg Ser Ala Yal Leu Ser Ser Leu Phe A1a Ser G1n Ya1 Leu Gly G1n Pro I1e Asp Asp Gtu Asn Thr Thr Ser Ya1 Asn Leu Pro A1a Lys Arg Phe Yal Asn G1n His Leu Cys Gty Ser His Leu Ya1 G1u Ala Leu Tyr (2) INFORMATION FOR SEQ ID N0:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 282 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 113..280 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:56:
TTAAATCTAT AACTACAAAA CAAGAATAGT TCAAACAAGA
AACACATACA
GGAATTCATT
AGATTACAAA CTATCAATTT GGTACCAAAA
CATACACAAT TA
ATAAACGACG ATG
Met GTC
LysLeu LysThrYa1 ArgSer A1a LeuSerSer LeuPheA1aSer Va1 GAC
G1nYal LeuG1yGln ProI1e Asp ThrGtuSer AsnThrThrSer Asp GTT
YalAsn LeuProA1a LysArg Phe AsnG1nHis LeuCysG1ySer Ya1 CACTTG GTTGAAGCT TTGTAC TT 2g2 HisLeu Va1G1uA1a LeuTyr (2)INFORMATION FOR SEQID N0:57:
W095134666 - - w-21 9 2 9 4 2 P~'~''~~s~~~'''9 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 56 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:57:
Met Lys Leu Lys Thr Ya1 Arg Ser A1a Ya1 Leu Ser Ser Leu Phe A1a Ser Gtn Ya1 Leu G1y G1n Pro I1e Asp Asp Thr G1u Ser Asn Thr Thr Ser Va1 Asn Leu Pro A1a Lys Arg Phe Ya1 Asn Gtn His Leu Cys G1y Ser His Leu Va1 G1u A1a Leu Tyr (2) INFORMATION FOR SEQ ID N0:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 282 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single {D) TOPOLOGY: linear {ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 113..280 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:
Met Lys Leu Lys Thr Ya1 Arg Ser A1a Ya1 Leu Ser Ser Leu Phe Ala Ser Gln Yai Leu Gly Gln Pro Ile Asp Asp Thr Gtu Ser Asn Thr Thr Ser Ya1 Asn Leu Met A1a Lys Arg Phe Ya1 Asn G1n His Leu Cys G1y Ser His Leu Ya1 Glu A1a Leu Tyr (2) INFORMATION FOR SEQ ID N0:59:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 56 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:59:
Mei Lys Leu Lys Th5 Yat Arg Ser A1a Ya1 Leu Ser Ser Leu Phe A1a Ser Gln Va1 Leu Giy G1n Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Ser Val Asn Leu Met A1a Lys A4g0 Phe Ya1 Asn G1n His Leu Cys G1y Ser His Leu Ya1 G1u A1a Leu Tyr (2) INFORMATION FOR SEQ ID N0:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 330 base pairs {B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 113..328 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:60:
Met Lys Leu Lys Thr Ya1 Arg Ser Ata Va1 Leu Ser Ser Leu Phe A1a Ser WO 95134666 219 2 9 4 2 PCT~~5100249 i GlnYa1LeuG1yG1n ProI1eAsp AspThrG1uSer AsnThrThrSer Va1AsnLeuMetA1a AspAspThr G1u5erI1eAsn ThrThrLeuYa1 AsnLeuProGtyA1a LysArgPhe YatAsnGtnHis LeuCysG1ySer HisLeuVa1GtuA1a LeuTyr (2) INFORMATION FOR SEQ ID N0:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 72 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:61:
Met Lys Leu Lys Thr Ya1 Arg Ser Ata Ya1 Leu Ser Ser Leu Phe A1a Ser G1n Yal~Leu G1y Gtn Pro Ite Asp Asp Thr G1u Ser Asn Thr Thr Ser Ya1 Asn Leu Met A1a Asp Asp Thr Gtu Ser Ite Asn Thr Thr Leu Va1 Asn Leu Pro G1y A1a Lys Arg Phe Ya1 Asn G1n His Leu Cys G1y Ser His Leu Va1 G1u A1a Leu Tyr (2) INFORMATION FOR SEQ ID N0:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 288 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
WO 95/34666 ~ 19 2 9 4 2 p~~g9g100249 (B) LOCATION: 113..286 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:62:
Met Lys Leu Lys Thr Va1 Arg Ser A1a Ya1 Leu Ser Ser Leu Phe A1a Ser 61n Va1 Leu Gly G1n Pro I1e Asp Asp Thr G1u Ser Ile Asn Thr Thr Leu Ya1 Asn Leu Pro G1y A1a Lys Arg Phe Va1 Asn G1n His Leu Cys GGT TCC CAC TTG GTT GAA GCT TTG TAC TT 2gg G1y Ser His Leu Ya1 61u A1a Leu Tyr (2) INFORMATION FOR SEQ ID N0:63:
(i) SEQUENCE CHARACTERISTICS:
~ (A) LENGTH: 58 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:63:
Met Lys Leu Lys Thr Val Arg Ser A1a Va1 Leu Ser Ser Leu Phe Ala Ser Gln Yal Leu G1y Gln Pro Ile Asp Asp Thr G1u Ser I1e Asn Thr Thr Leu Va1 Asn Leu Pro G1y A1a Lys Arg Phe Ya1 Asn Gtn His Leu Cys G1y Ser His Leu Ya1 G1u A1a Leu Tyr (2) INFORMATION FOR SEQ ID N0:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 82 base pairs W095134666 ' ' " PCTIDK95100?A9 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D} TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:64:
(2) INFORMATION FOR SEQ ID N0:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
{xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:
(2) INFORMATION FOR SEQ ID NO:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 117 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:66:
(2) INFORMATION FOR SEQ ID NO:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acids {B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:67:
WO 95/34666 219 2 9 ~ 2 pCTIDK95100249 iln Pro I1e Asp 5sp Thr G1u Ser Asn Thr Thr Ser Va1 Asn Leu Met A1a Asp Asp Thr G1u Ser Arg Phe A1a Thr Asn Thr Thr Leu Ata Leu Asp Ya1 Yal Asn Leu Ile Ser Met Ala 35 . 40 (2) INFORMATION FOR SEQ ID N0:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:68:
(2) INFORMATION FOR SEQ ID N0:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 123 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:69:
GCT . 123 (2) INFORMATION FOR SEQ ID N0:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 65 amino acids (B) TYPE: amino acid {D) TOPOLOGY: Linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:70:
WO 95!34666 219 2 9 4 2 PCT~~5~00249 Lys Arg Gtu G1u Ala G1u A1a Gtu A1a G1u Pro Lys Phe Va1 Asn 61n His Leu Cys Gly Ser His Leu Vat Glu A1a Leu Tyr Leu Va1 Cys Gty G1u Arg G1y Phe Phe Tyr Thr Pro Lys A1a A1a Lys G1y I1e Va1 61u Gln Cys Cys Thr Ser I1e Cys Ser Leu Tyr Gln Leu G1u Asn Tyr Cys Asn (2) INFORMATION FOR SEQ IO N0:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 219 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID N0:71:
(2) INFORMATION FOR SEQ ID NO:72:
(1) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 348 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:72:
(2) INFORMATION FOR SEQ ID N0:73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 379 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:73:
GGTTGAAGCTTTGTACTTG 37g
Secretion and correct processing of a polypeptide intended to be secreted is obtained in some cases when culturing a yeast organism which is transformed with a vector constructed as indicated in the references given above. In many cases, how-ever, the level of secretion is very low or there is no se-cretion, or the proteolytic processing may be incorrect or incomplete. It is therefore the object of the present invention to provide leader peptides which ensure a more efficient expression and/or processing of polypeptides.
SUMMARY OF T$E INVENTION
Surprisingly, a new type of leader peptide has been found which allows secretion in high yield of a polypeptide in yeast.
WO 95134666 219 2 9 G~ 2 PCT/DK95100249 Accordingly, the present invention relates to a DNA expression cassette comprising the following sequence:
5'-P-SP-LS-PS-*gene*-(T)i-3' wherein P is a promoter sequence, SP is a DNA sequence encoding a signal peptide, LS is a DNA sequence encoding a leader peptide with the general formula I:
GlnProIle(Asp/Glu)(Asp/Glu)X1(Glu/Asp)X2AsnZ(Thr/Ser)X3 (I) l0 wherein X1 is a peptide bond or a codable amino acid;
X2 is a peptide bond, a codable amino acid or a sequence of up to 4 codable amino acids which may be the same or different;
Z is a codable amino acid except Pro; and X3 is a sequence of from 4 to 30 codable amino acids which may be the same or different:
PS is a DNA sequence encoding a processing site:
*gene* is a DNA sequence encoding a polypeptide;
T is a terminator sequence; and i is 0 or 1.
In the present context, the expression "leader peptide" is understood to indicate a peptide whose function is to allow the expressed polypeptide to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory ve-sicle 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).
The term "synthetic" used in connection with leader peptides is intended to indicate that the leader peptide is one not found in nature.
WO 95/34666 219 2 9 4 2 PCT~~5100249 The term "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 5 is to allow the expressed 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.
The expression "polypeptide" is intended to indicate a heterologous polypeptide, i.e. a polypeptide which is not pro-duced by the host yeast organism in nature as well as a homologous polypeptide, i.e. a polypeptide which is produced by the host yeast organism in nature and any preform thereof. In a preferred embodiment, the expression cassette of the present invention encodes a heterologous polypeptide.
The expression "a codable amino acid" is intended to indicate an amino acid which can be coded for by a triplet ("codon") of nucleotides.
When, in the amino acid sequences given in the present specification, the three letter codes of two amino acids, separated by a slash, are given in brackets, e.g. (Asp/Glu), this is intended to indicate that the sequence has either the one or the other of these amino acids in the pertinent position.
In a further aspect, the present invention relates to a process for producing a polypeptide in yeast, the process comprising culturing a yeast cell, which is capable of expressing a polypeptide and which is transformed with a yeast expression vector as described above including a leader peptide sequence of the invention, in a suitable medium to obtain expression and secretion of the polypeptide, after which the polypeptide is recovered from the medium.
wo>s2192942 BEIEF DEiCRIpTI0~1 OF TEE DR11~I~
The present invention is turthar illustrated with ratarenas to the appended drawings wherein Fig. 1 schematically chows the placmid p11Kd9Zt Fig. 2 shows part of the DNA sequence encoding the cign6l peptfde/leader/MI7 insulin precursor:
Fig. 3 shows the construction of the plasmid pAR546r Fig. 4 shows the aaifno acfd sequence at the leader SEQ ID lro.
43 and the corresponding DNA SEQ ID No.: 42 io Fig. 5 shows the amino acid SEQ ID No. :45 and the corresponding DNA SEQ ID NO.: 44.
Fig. 6 shows the amino acid SEQ ID NO.: 47 and the corresponding DNA SEQ ID NO.: 46.
Fig. 7 shows the amino acid sequence of the leader SEQ ID No.
8 and the DNA sequence sncoding itt Pig. 8 chows the aaino acid sequence of the leader SEQ ID No.
17 and the DNA sequence encoding itt Fig. 9 shows the amino acid sequence o! the leader SEQ ID No.
is and the DNA aeguance encoding it;
Fig. 1o shows the amino acid sequence of the leader SEQ ID No.
19 and the DNA sequence encoding it:
Fig. 11 shows the amino acid sequence of the leader SEQ ID No.
20 and the DNA sequence encoding itt WO 95/34666 219 2 9 4 2 P~~~S~OO1A9 Fig. 12 shows the amino acid sequence of the leader SEQ ID No.
21 and the DNA sequence encoding it;
Fig. 13 shows the DNA fragment of pAK527 used as the direct template in the construction of SEQ ID Nos. 4 and 6;
Fig. 14 shows the DNA fragment of pAK531 used as the direct template in the construction of SEQ .ID No. 8;
Fig. 15 shows the DNA fragment of pAK555 used as the direct template in the construction of SEQ ID Nos. 16 and 17;
Fig. 16 shows the DNA fragment of pAK559 used as the direct l0 template in the construction of SEQ ID Nos. 19 and 20; and Fig. 17 shows the DNA fragment of pAK562 used as the direct template in the construction of SEQ ID No. 21;
Fig. 18 shows the amino acid sequence of the leader SEQ ID No.
27 and the DNA sequence SEQ ID No. 66 encoding it;
Fig. 19 shows the amino acid sequence SEQ ID No. 70 of an N-terminally extended MI3 insulin precursor and the DNA sequence SEQ ID No. 71 encoding it;
Fig. 20 shows the amino acid sequence of the leader SEQ ID No.
67 and the DNA sequence SEQ ID No. 69 encoding it;
Fig. 21 shows the DNA fragment SEQ ID No. 72 of pAK614 used as the direct template in the construction of SEQ ID No. 27; and Fig. 22 shows the DNA fragment SEQ ID No. 73 of pAK625 used as the direct template in the construction of SEQ ID No. 67.
R'O 95134666 ~ ~ 9 2 9 4 2 PCT/DK95100249 DETAINED DI8CLOSORE Of THE INVENTION
When X1 in general formula I designates an amino acid, it is preferably Ser, Thr or Ala. When X2 in general formula I
designates one amino acid, it is preferably Ser, Thr or Ala.
When X2 in general formula I designates a sequence of two amino acids, it is preferably SerIle. When X2 in general formula I
designates a sequence of three amino acids, it is preferably SerAlaIle. When X2 in general formula I designates a sequence of four amino acids it is preferably SerPheAlaThr. In a preferred embodiment, X3 is an amino acid sequence of the general formula a X4_X5_X6 (II) wherein X4 is a sequence of from 1 to 21 codable amino acids which may be the same or different, XS is Pro or one of the amino acid sequences ValASnLeu or LeuAlaASnValAlaMetAla, and X6 is a sequence of from 1 to 8 codable amino acids which may be the same or different.
In general formula II, X4 is preferably an amino acid sequence which includes one or more of the motifs LeuValAsnLeu, SerValAsnLeu, MetAlaAsp, ThrGluSer, ArgPheAlaThr or ValAlaMetAla; or X4 is an amino acid sequence which includes the sequence AsnSerThr or AsnThrThr: or X4 is an amino acid sequence which includes the sequence (Ser/Leu)ValAsnLeu, (Ser/Leu)ValAsnLeuMetAlaAsp, (Ser/Leu)ValAsnLeuMetAlaAspAsp, (Ser/Leu)ValAsnLeuMetAlaAspASpThrGluSer, (Ser/Leu)ValAsnLeuMetAlaAspAspThrGluSerIle or (Ser/Leu)ValAsnLeuMetAlaAspAspThrGluSerArgPheAlaThr:
or X4 is an amino acid sequence which includes the sequence Asn(Thr/Ser)ThrLeu, Asn(Thr/Ser)ThrLeuAsnLeu or Asn(Thr/Ser)ThrLeuValAsnLeu~ or any combination thereof.
In general formula II, X5 is preferably Pro or an amino acid sequence which includes the sequence ValAsnLeu, LeuAlaAsnValAlaMetAla, LeuAspValValAsnLeuProGly or LeuAspValValAsnLeuIleSerMet.
When X6~ in general formula II, designates one amino acid, it is preferably Ala, Gly, Leu, Thr, Val- or Ser. When X6~ in general formula II, designates a sequence of two amino acids, it is preferably GlyAla or SerAla. When X6~ in general formula II, designates a sequence of three amino acids, it is preferably AlaValAla. When X6, in general formula II, designates a sequence of eight amino acids, it is preferably GlyAlaAspSerLysThrValGlu.
Examples of preferred leader peptides coded for by the DNA
sequence LS are:
SEQ ID No. 1 GlnProIleAspGluAspAsnAspThrSerValAsnLeuProAla:
SEQ ID No. 2 GlnProIleAspAspGluAsnThrThrSerValAsnLeuProAla;
SEQ ID No. 3 GlnProIleAspAspGluSerAsnThrThrSerValAsnLeuProAla;
SEQ ID No. 4 GlnProIleAspAspGluAsnThrThrSerValAsnLeuProVal:
SEQ ID No. 5 GlnProIleAspAspThrGluAsnThrThrSerValAsnLeuProAla;
SEQ ID No. 6 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuPro-Ala;
SEQ ID No. 7 GlnProIleAspAspGluAsnThrThrSerValAsnLeuMetAla;
SEQ ID No. 8 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuPro-GlyAla;
WO 95134666 ~ ~ ~ 219 2 9 4 2 PCT~~5100249 SEQ ID No. 9 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-Ala:
SEQ ID No. 10 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnValPro-Thr;
5 SEQ ID No. 11 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnValPro-Thr:
SEQ ID No. 12 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuPro-Thr;
SEQ ID No. 13 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnValPro-10 GlyAla;
SEQ ID No. 14 GlnProIleAspAspThrGluSerASnThrThrSerValAsnLeuMet-AlaProAlaValAla;
SEQ ID No. 15 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuNet-AspLeuAlaValGlyLeuProGlyAla;
SEQ ID No. 16 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuNet-AlaAspAspThrGluSerIleAsnThrThrLeuValAsnLeuProGly-Ala;
SEQ ID No. 17 GlnProIleAspAspThrGluSerIleASnThrThrLeuValAsnLeu-ProGlyAla;
SEQ ID No. 18 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnLeuPro-GlyAla;
SEQ ID No. 19 GlnProIleAspASpThrGluSerAsnThrThrSerValAsnLeuNet-AlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuValAsn-LeuProLeu;
SEQ ID No. 20 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-AlaAspAspThrGluSerIleAsnThrThrLeuValAsnLeuAlaAsn-ValAlaMetAla;
SEQ ID No. 21 GlnProIleAspAspThrGluSerAlaIleASnThrThrLeuValAsn-LeuProGlyAla;
SEQ ID No. 22 GlnProIleAspAspThrGluSerPheAlaThrAsnThrThrLeuVa1-AsnLeuProGlyAla;
SEQ ID No. 23 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsnLeu-MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuVa1-AsnLeuProLeu;
SEQ ID No. 24 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsnLeu-MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuAsp-ValValAsnLeuProGlyAla;
SEQ ID No. 25 GlnProIleAspAspThrGluSerAlaAlaIleAsnThrThrLeuVa1-AsnLeuProGlyAla;
SEQ ID No. 26 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-AlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuValAsn-LeuAlaAsnValAlaMetAla;
SEQ ID No. 27 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-AlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuAspVa1-ValAsnLeuIleSerMetAla;
SEQ ID No. 28 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-AlaAsnThrThrGluSerArgPheAlaThrAsnThrThrLeuAspVa1-ValAsnLeuIleSerMetAla; and SEQ ID No. 67 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-AlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuAlaLeu-AspValValAsnLeuIleSerMetAlaLysArg.
Particularly preferred leader peptides coded for by the DNA
sequence LS are:
- 2.~ 92942 SEQ ID No. 15 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-AspLeuAlaValGlyLeuProGlyAla:
SEQ ID No. 16 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-AlaAspAspThrGluSerIleAsnThrThrLeuValAsnLeuProGly- , Ala:
SEQ ID No. 17 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsnLeu-ProGlyAla:
SEQ ID No. 18 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnLeuPro-GlyAla:
SEQ ID No. 19 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-AlaAspAspThrGluSerArgPheAlaThrASnThrThrLeuValAsn-LeuProLeu:
SEQ ID No. 20 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet AlaAspAspThrGluSerIleAsnThrThrLeuValASnLeuAlaAsn ValAlaMetAla:
SEQ ID No. 21 GlnProIleAspAspThrGluSerAlaIleAsnThrThrLeuValAsn-LeuProGlyAla:
SEQ ID No. 22 GlnProIleAspAspThrGluSerPheAlaThrAsnThrThrLeuVa1-AsnLeuProGlyAla:
SEQ ID No. 23 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsnLeu-MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuVal-AsnLeuProLeu:
SEQ ID No. 24 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsnLeu-MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuAsp-ValValAsnLeuProGlyAla;
SEQ ID No. 25 GlnProIleAspAspThrGluSerAlaAlaIleAsnThrThrLeuVa1-AsnLeuProGlyAla:
~ wo» ~ 2192942 SEQ ID No. 26 GlnProIleAspASpThrGluSerAenThrThrSeYValAanLauMat-AlaAspAspThxGluserArgPheAlaThrhsnThtThrLeuValAsn-LeuAlaAsnValAlaMetAla: and S~Q ID No. 28 GinProIleAspAspTtuCluSfrAsnThM'hrSeYValAsnLauMet AlaAsr~IhrTttrGluSerArgPheAlaThrASnThrT~srLeuAspVal ValAsnLeuIleSerlietAla.
SEQ ID No. 67 GlnProIleAspAspThrGluSerASnThYfhrSer'ValAsnheuNet-AlnAspAspThrGluSerArgPheAlaThrASnThYIhrLeuAlaLsu-AspValVelAsnLeuIle&erlietAla l0 The signal sequence (SP) may encode any signal peptide which ensures an effective direction of the expressed 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 a-factor signal peptide, the signal peptide of mouse salivary amylase, a modilie:d carboxypeptidase signal peptide, the yeast ~~$y signal peptide or the lanuainosa lipase signal peptide or a derivative thereof. The mouse salivary aaylase signal sequence is described by Hagenbnchle, O, et al., ~yrg ~g,Q (1981) 6C3 s4s. The carboxypeptidase signal sequence is described by Valls, L.A. et al., C~I1 ~ (1987) 887-897. The g8g1 signal peptide is disclosed in WO 87/OZ670. The yeast aspartic protease 3 signal peptide is described in European Patent No.
z5 0 792 367.
The yeast processing site encoded by the DNA sequence PS may suitably be any paired combination of Lys and Arg, such as LysArg, ArgLys, ArgArg or LysLys which peraits processing of the polypeptide by, the 1X2 protease of Saccharomyces cerevisiae or the equivalent protease in other yeast species (Julius, D.A. et al., Cell ~? (1984) 1075). If 1X2 processing is not convenient, e.g. if itr would lead to cleavage of the polypeptide product, e.g. .due to the presence of two WO 95134666 219 2 9 4 2 P~~°~sl~0~a9 consecutive basic amino acid internally in the desired product, a processing site for another protease may be selected comprising an amino acid combination which is not found in the polypeptide product, e.g. the processing site for FXa, IleGluGlyArg (cf. Sambrook, J., Fritsch, E.F. and Maniatis, T., , MW P~niar Cloning: A Laboratorv Manual, Cold Spring Harbor Laboratory Press, New York, 1989).
The protein produced by the method of the invention may be any protein which may advantageously be produced in yeast. Examples of such proteins are heterologous proteins such as aprotinin, tissue factor pathway inhibitor or other protease inhibitors, insulin or insulin precursors, human or bovine growth hormone, interleukin, glucagon, GLP-1, IGF-I, IGF-II, tissue plasminogen activator, transforming growth factor a or p, platelet-derived growth factor, enzymes or a functional analogue thereof. In the present context, the term "functional analogue'° is meant to indicate a protein 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 protein 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. Such modifications are well known for several of the proteins mentioned above. Also, precursors or intermediates for other proteins may be produced by the method of the invention. An example of such a precursor is the MI3 insulin precursor which comprises the amino acid sequence B(1-29)AlaAlaLysA(1-21) wherein A(1-21) is the A chain of human insulin and B(1-29) is the B chain of human insulin in which Thr(B30) is missing.
W O 95134666 219 2 9 4 2 pCT~~5100249 Preferred DNA constructs encoding leader sequences are as shown in Figs. 4 - 12 or suitable modifications thereof. Examples of suitable modifications of the DNA sequence are nucleotide substitutions which do not give rise to another amino acid 5 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 10 modifications are insertion of one or more codons into the sequence, addition of one or more codons at either end of the sequence and deletion of one or more codons at either end of or within the sequence.
The recombinant expression vector carrying the expression 15 casette 5'-P-SP-LS-PS-*gene*-(T)i-3' wherein P, SP, LS, *gene*, T and i are as defined above may be any vector which is capable of replicating in yeast organisms.
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 $accharomvces cerevisiae MFal, TPI, ADH or PGK promoters.
The sequences shown above should preferably also be operably connected to a suitable terminator, e.g. the TPI terminator (cf. Alber, T: and Kawasaki, G., d. Mol. Apol. Genet. ~ (1982) 419-434).
The recombinant expression vector of the invention further comprises a DNA sequence enabling the vector to replicate in yeast. Examples of such sequences are the yeast plasmid 2u replication genes REP 1-3 and origin of replication. The vector may also comprise a selectable marker, e.g. the Schizo-WO 95134666 f 219 2 9 4 2 P~~~~80~9 saccharomyces pombe TPI gene as described by Russell, P.R., Gene ~Q (1985) 125-130.
The methods used to ligate the sequence 5'-P-SP-LS-PS-*gene*-(T)i-3' and to insert it into suitable yeast vectors containing the information necessary for yeast replication, are well known to persons skilled in the art (cf., for instance, Sambrook, J., Fritsch, E.F. and Maniatis, T., ~.cit.). It will be understood that the vector may be constructed either by first preparing a DNA construct containing the entire sequence 5'-P-SP-LS-PS-*gene*-(T)i-3' and subsequently inserting this fragment into a suitable expression vector, or by sequentially inserting DNA
fragments into a suitable vector containing genetic information for the individual elements (such as the promoter sequence, the signal peptide, the leader sequence GlnProIle(Asp/Glu)(Asp/Glu)X1(Glu/Asp)X2AsnZ(Thr/Ser)X3, the processing site, the polypeptide, and, if present, the terminator sequence) followed by ligation.
The yeast organism used in the method of the invention may be any suitable yeast organism which, on cultivation, produces large amounts of the desired polypeptide. Examples of suitable yeast organisms may be strains of the yeast species Saccharoniyces cerevisiae, Saccharomvces kluyveri, Schiaosaccharomvces pombe or Saccharomvces 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 polypeptide, 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 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, WO 95134666 ~ 2 PCTlDK95100?A9 affinity chromatography or the like.
The invention is further described in the following examples which are not to be construed as limiting the scope of the invention as claimed.
EXAMPLES
plasm~ds and DNA material All expression plasmids are of the C-POT type. Such plasmids are described in EP patent application No. 171 142 and are characterized in containing the Schizosaccharomvces pomhe _.
triose phosphate isomerase gene (POT) for the purpose of plasmid selection and stabilization. A plasmid containing the POT-gene is available from a deposited E. coli strain (ATCC
39685). The plasmids furthermore contain the ~. cerevisiae triose phosphate isomerase promoter and terminator (PTP1 and TTP~). They are identical to pMT742 (Egel-Mitani, M. et al., Gene (1988) 113-120) (see Fig. 1) except for the region defined by the EcoR I-Xba I restriction sites encompassing the coding region for signal/leader/product.
The plasmids pAK527, pAK531, pAK555, pAK559, pAK562, pAK614 and pAK625 were used as DNA templates in the PCR reactions applied in the construction of the leaders described in the examples.
The synthetic DNA fragments serving as the direct template are shown in Figs. 13 - 17. With the exception of the shown DNA
regions the plasmids are identical to pAK492 shown in Fig. 1.
Synthetic DNA fragments were synthesized on an automatic DNA
synthesizer (Applied Biosystems model 380A) using phosphoramidite chemistry and commercially available reagents (Beaucage, S.L. and Caruthers, M.H., Tetrahedron Letters ~ __ (1981) 1859-1869).
wo 9sr3~s. . ~ ~ ' v~ 19 2 9 4 2 rcrmxssioe~
All other methods and matsrials used are coaston state o! ttfe art knowledge (sea, s.g. &ambrook, J., Fritsch, E.F. and I~Ianiatis, T., ~c~ecu~ar Cloning: A Laborato~~nual, Cold Spring Harbor Lelboratory Press, Pew York, 1989).
EZaliPLE i Synthesis of the leader SEQ ID'No. 4 for expression of the xI3 insulin precursor in ~ c red (strain yAR546).
The leader SEQ ID No. 4 has the following amino acid sequence:
GlnProIleAspAspGluAsnThYrhrSsrValAsnLeuProVa1 The following oligonuclaotides wars synthesised:
94 5'-TJW1TCTATJ1ACTAC11AAAAACACAT11-3' SSQ ID xo. t!
# 333 5'-GACTCTCTTAACTGGCAAGTTGACA-3' 8Efl ID lio. ~0 / 312 5'-AAGTACAAAGCTTCAACCAAGTGAGAACCACACAAGTaiT .
GGTT7U1CGAATCTC1'f-3' SEQ ID xo. ~i t 1845 5'-CATACACAATATAAACGACGG-3' SSQ ID No. 3Z
The following poTynerase -cbain reactions (PCR) xere.perlorasd using the Gene Amp~PCR reagent kit (Psrkin Elmer~' 761 plain Avewalk, CT 06859, SSA) according to the manulacturers instructions. During the reaction, the PCR mixturaa xara overlayed with 100 ~tl of mineral oil (Sigma Chemical CO, St.
Louie NO, U6A):
lRivnerasa cram raacz.avn nv. i 5 ~1 0! oligonucleotide f 94 (50 pmol) 5 ul of oligonucleotids 4 333 (50 pmol) 10 ~l o! 10X PCR buffer 16 ~l of dNTP mix 0.5 xl of Taq ensy~
0.5 ~t1 of pAK527 plasmid (Fig. 13) as template (0.2 ug of DtIA) w't- = -T~'1 2192942 .
63 ul of water A total of 12 cycles were performed, one cycle was 94'C for 1 sin: 37'C for 2 min: 72'C for 3 min. The PCR mixture was then loaded onto a 2~ agarose gel and electrophoresis was performed using standard techniques (Sambrook, J., Fritsch, E.F. and Maniatis, T., o_p.cit.). The resulting DNA fragment was cut out of the agairose gel and isolated using the Gene Clean kit (Bio 101 inc., PO BOX 2284, La Jolla, CA 92038, USA) according to the manufacturers instructions.
Polvmerase chain reaction No. 2 5 ~1 of oligonucleotide ~ 312 (50 pmol) 5 pl of oligonucleotide ; 94 (50 pmol) io ul of lox ~ buffer i6 ~l of dNTP mix 0.5 ~1 of Taq enzyme l0 ~C1 of purified DNA lragment from PCR No. 1 53.5 ~tl of water A total o! 12 cycles Were performed, one cycle was 94'C for 1 min; 37'C for 2 min: 72'C for 3 min.
The DNA fragment from polymerase chain reaction No. 2 was isolated.and purified using the Gens Clear~kit (Bio 101 inc., ~_.
PO 80X 2284, La Jolla, CA 92038, USA) according to the manufacturers instructions. ' The purified pCR DNA fragment was dissolved in l0 pl o! water and restriction endonuclease buffer and cut with the restriction endonuclsases Asp 718 and Hind iII in a total volume of 15 pl according to standard techniques (Sanbrook, J., Fritsch, E.F. and Maniatis, T., on.cit.). The 167 by Asp 718/Hind III DNA fragment was subjected to electrophoresis on agarose gel and purified using The Gene Clean Kit as described.
The ~ oaravi_si_"e_ expression plasmid pAK492 (shown in Fig. 1) is a derivative of the previously described plasmid pMT742 in ..._ ..._........... ......______ _.___. _ ___._ .
WO 95/34666 ~ i ~ 2 9 4 2 PCTIDK95100249 which the fragment encoding the signal/leader/insulin precursor has been replaced by the EcoR I-Xba I fragment shown in Fig. 2.
This fragment has been synthesized on an Applied Biosystems DNA
synthesizer in accordance with the manufacturer's instructions.
5 The plasmid pAK492 was cut with the restriction endonucleases Asp 718 and Xba I and the vector fragment of 10986 by was isolated. The plasmid pAK492 was cut with the restriction endonucleases Hind III and Xba I and the DNA fragment of 140 by encoding part of the MI3 insulin precursor was isolated. The 10 three DNA fragments were ligated together using T4 DNA ligase under standard conditions (Sambrook, J., Fritsch, E.F. and Maniatis, T., oo.cit.). The ligation mixture was then transformed into a competent E. coli strain (R-, M+) and transformants were identified by ampicillin resistance.
15 Plasmids were isolated from the resulting E. coli colonies using standard DNA miniprep technique (Sambrook, J., Fritsch, E.F. and Maniatis, T., op.cit.), checked with appropriate restrictions endonucleases i.e. EcoR I, Xba I, Nco I and Hind III. The selected plasmid, pAK546, was shown by DNA sequencing 20 analysis (Sequenase, U.S. Biochemical Corp.) using the primer # 94 to contain a DNA sequence encoding the leader SEQ ID No.
4. For the DNA sequence encoding the leader SEQ ID No. 4, see Fig. 4). The plasmid pAK546 was transformed into ,~ cerevisiae strain MT663 as described in European published patent application No. 214 826 and the resulting strain was named yAK546. The DNA sequence of the protein coding region of the expression plasmid is given in Fig. 5.
Synthesis of the leader SEQ ID No. 6 for expression of the MI3 insulin precursor in ~ cerevisiae (strain,yAK531).
The leader SEQ ID No. 6 has the following amino acid sequence:
GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuProAla WO 95/34666 2 ~ 9 2 9 q. 2 PCTIDK95100249 The following oligonucleotide was synthesised:
# 331 5'-GAATCTCTTAGCTGGCAAGTTGACAGAAGTAGTGTTAG
TTTCAGAGTCGTCAATT-3' SEQ ID No. 33 The polymerase chain reaction was performed as described in Example 1 with the expection that oligonucleotide # 331 was used insted of oligonucleotide # 333.
The Asp 718/Hind III DNA fragment of 168 by was subjected to electrophoresis on agarose gel and purified as described in Example 1. The Asp 718/Hind III DNA fragment was subcloned into the Sz cerevisiae expression plasmid as described in Example 1.
The selected plasmid, pAK531, was shown by DNA sequencing analysis, as described in Example 1, to contain a DNA sequence encoding the leader SEQ ID No. 6. For the DNA sequence encoding the leader SEQ ID No. 6, see Fig. 6. The plasmid pAK531 was transformed into ~ ~erevisiae strain MT663 as described in European patent application 86306721.1 and the resulting strain was named yAK531. The DNA sequences encoding the signal peptide and the insulin precursor MI3 were the same as those shown in Fig. 5.
Synthesis of the leader SEQ ID No. 8 for expression of the MI3 insulin precursor in ~ v~~~a (strain yAK547).
The leader SEQ ID No. 8 has the following amino acid sequence:
GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuProGlyAla The following oligonucleotide was synthesised:
# 345 5'-AACGAATCTCTTAGCACCTGGCAAGTTGACAGAAGT-3' SEQ ID No. 34 WO 95134666 ~-19 2 9 4 2 PCT1DK95100249 The polymerise chain reaction was performed as described in Example 1 with the expection that oligonucleotide # 345 was used insted of oligonucleotide # 333 and plasmid pAK531 (Fig.
14) was used as template.
The Asp 718/Hind III DNA fragment of 171 by was subjected to electrophoresis on agarose gel and purified as described in Example 1. The Asp 718/Hind III DNA fragment was subcloned into the ~ cerevisiae,expression plasmid as described in Example 1.
The selected plasmid, pAK547, was shown by DNA sequencing analysis, as described in Example 1, to contain a DNA sequence encoding the leader SEQ ID No. 8. For the DNA sequence encoding the leader SEQ ID No. 8, see Fig. 7. The plasmid pAR547 was transformed into ~ gerevisiae strain MT663 as described in European patent application No. 86306721.1 and the resulting strain was named yAK547. The DNA sequences encoding the signal peptide and the insulin precursor MI3 were the same as those shown in Fig. 5.
Synthesis of the leader SEQ ID No. 17 for expression of the MI3 insulin precursor in ~ cerevisiae (strain yAK561).
The leader SEQ ID No. 17 has the following amino acid sequence:
GlnProIleAspAspThrGluSerIleAsnThrThrLeuValASnLeuProGlyAla The following oligonucleotide was synthesised:
# 376 5'-AACGAATCTCTTAGCACCTGGCAAGTTGACCAAAGTAG
TGTTGATAGATTCAGTGTCGTC-3' SEQ ID No. 35 The polymerise chain reaction was performed as described in Example 1 with the expection that oligonucleotide # 376 was used insted of oligonucleotide # 333 and plasmid pAK555 (Fig.
15) was used as template.
R'O 95/34666 219 2 9 4 2 PCTIDK95100249 The Asp 718/Hind III DNA fragment of 180 by was subjected to electrophoresis on agarose gel and purified as described in Example 1. The Asp 718/Hind III DNA fragment was subcloned into the S~ cerevisiae expression plasmid as described in Example 1.
The selected plasmid, pAK561, was 'shown by DNA sequencing analysis, as described in Example 1, to contain a DNA sequence encoding the leader SEQ ID No. 17. For the DNA sequence encoding the leader SEQ ID No. 17, see Fig. 8. The plasmid pAK561 was transformed into Sue. cerevisiae strain MT663 as described in European patent application No. 86306721.1 and the resulting strain was named yAK561. The DNA sequences encoding the signal peptide and the insulin precursor MI3 were the same as those shown in Fig. 5.
Synthesis of the leader SEQ ID No. 16 for expression of the MI3 insulin precursor in ;Z cerevisiae (strain yAK559j.
The leader SEQ ID No. 16 has the following amino acid sequence:
GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMetAlaAspAspThr-GluSerIleAsnThrThrLeuValAsnLeuProGlyAla The following oligonucleotide was synthesised:
# 375 5'-AACGAATCTCTTAGCACCTGGCAAGTTAACCAAAGTAGT
GTTGATAGATTCAGTGTCGTCAGCCATCAAGTTGAC-3' 8EQ ID No. 36 The polymerase chain reaction was performed as described in Example 1 with the expection that oligonucleotide # 375 was used insted of oligonucleotide # 333 and plasmid pAK555 (Fig.
15j was used as template.
The Asp 718/Hind III DNA fragment of 222 by was subjected to electrophoresis on agarose gel and purified as described in Example 1. The Asp 718/Hind III DNA fragment was subcloned into the ~ cerevisiae expression plasmid as described in Example 1.
The selected plasmid, pAK559, was shown by DNA sequencing analysis, as described in Example 1, to contain a DNA sequence encoding the leader SEQ ID No. 16. For the DNA sequence encoding the leader SEQ ID No. 16, see Fig. 9. The plasmid pAK559 was transformed into ~ cerevisiae strain MT663 as described in European patent application No. 86306721.1 and the resulting strain was named yAK559. The DNA sequences encoding the signal peptide and the insulin precursor MI3 were the same as those shown in Fig. 5.
Synthesis of the leader SEQ ID No. 19 for expression of the MI3 insulin precursor in ~ cerevisiae (strain yAK580).
The leader SEQ ID No. 19 has the following amino acid sequence:
GlnProIleAspAspThrGluSerAsnThrThrSerValASnLeuMetAlaAspAspThr-GluSerArgPheAlaThrAsnThrThrLeuValAsnLeuProLeu The folloiaing oligonucleotide was synthesised:
# 384 5'-AACGAATCTCTTCAATGGCAAGTTAACCAAAGTAGTGT
TAGTAGCGAATCTAGATTCAGTGTCGTCAGCCAT-3' 8EQ ID No. 37 The polymerase chain reaction was performed as described in Example 1 with the expection that oligonucleotide # 384 was used insted of oligonucleotide # 333 and plasmid pAK559 (Fig.
16) was used as template.
The Asp 718/Hind III DNA fragment of 228 by was subjected to electrophoresis on agarose gel and purified as described in Example 1. The Asp 718/Hind III DNA fragment was subcloned into the ~ cerevisiae expression plasmid as described in Example 1.
The selected plasmid, pAK580, was shown by DNA sequencing analysis, as described in Example 1, to contain a DNA sequence 219 2 9 4 2 pCT~~S/00249 encoding the leader SEQ ID No. 19. For the DNA sequence.
encoding the leader SEQ ID No. 19, see Fig. 10. The plasmid pAK580 was transformed into ,~ cerevisiae strain MT663 as described in European patent application No. 86306721.1 and the 5 resulting strain was named yAK580. The DNA sequences encoding the signal peptide and the insulin precursor MI3 were the same as those shown in Fig. 5.
Synthesis of the leader SEQ ID No. 20 for expression of the MI3 10 insulin precursor in ~ s sa (strain yAK583).
The leader SEQ ID No. 20 has the following amino acid sequence:
GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMetAlaAspAspThr-GluSerIleAsnThrThrLeuValAsnLeuAlaAsnValAlaMetAla 15 The following oligonucleotide was synthesised:
# 390 5'-AACGAATCTCTTAGCCATGGCAACGTTAGCCAAGTTAA
~CCAAAGT-3' SEQ ID No. 38 The polymerase chain reaction was performed as described in Example 1 with the expection that oligonucleotide # 390 was 20 used insted of oligonucleotide # 333 and plasmid pAK559 (Fig.
16) was used as template.
The Asp 718/Hind III DNA fragment of 231 by was subjected to electrophoresis on agarose gel and purified as described in Example 1. The Asp 718/Hind III DNA fragment was subcloned into 25 the ~ cerevisiae expression plasmid as described in Example 1.
The selected plasmid, pAK583, was shown by DNA sequencing analysis, as described in Example 1, to contain a DNA sequence encoding the leader SEQ ID No. 20. For the DNA sequence encoding the leader SEQ ID No. 20, see Fig. 11. The plasmid pAK583 was transformed into Sue. ~erevisiae strain MT663 as a described in European patent application No. 86306721.1 and the resulting strain was named yAK583. The DNA sequences encoding the signal peptide and the insulin precursor MI3 were the same as those shown in Fig. 5.
E%AMPLE 8 Synthesis of the leader SEQ ID No. 21 for expression of the MI3 insulin precursor in ~ cerevisiae (strain yAK586).
The leader SEQ ID No. 21 has the following amino acid sequence:
GlnProIleAspAspThrGluSerAlaIleAsnThrThrLeuValAsnLeuProGlyAla The following oligonucleotide was synthesised:
# 401 5'-AACGAATCTCTTAGCACCTGGCAAGTTGACCAAAGTAG
TGTTGATAGCAGATTCAGTGTCG-3° SEQ ID No. 39 The palymerase chain reaction was performed as described in Example 1 with the exception that oligonucleotide # 401 was used insted of oligonucleotide # 333 and plasmid pAK562 (Fig.
17) was used as template.
The Asp 718/Hind III DNA fragment of 183 by was subjected to electrophoresis on agarose gel and purified as described in Example 1. The Asp 718/Hind III DNA fragment,was subcloned into the ~ cerevisiae expression plasmid as described in Example 1.
The selected plasmid, pAK586,. was shown by DNA sequencing analysis, as described in Example 1, to contain a DNA sequence encoding the leader SEQ ID No. 21, see Fig. 12. The plasmid pAK586 was transformed into ~ cerevisiae strain MT663 as described in European patent application No. 86306721.1 and the resulting strain was named yAK586. The DNA sequences encoding , the signal peptide and the insulin precursor MI3 were the same as those shown in Fig. 5.
R'O 95/34666 PCTIDK95100249 Expression of the MI3 insulin precursor using selected leader sequences according to the present invention.
Yeast strains harbouring plasmids as described above, were grown in YPD medium (Sherman, F. et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, 1981). For each strain 6 individual 5 ml cultures were shaken at 30°C for 72 hours, with a final OD6oo 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 Snel, L. et al. Chromatoaraphia ~ (1987) 329-332.
In Table 1 the expression levels of the insulin precursor, MI3, obtained by use of selected leader sequences according to the present invention, are given as a percentage of the level obtained with transformants of pMT742, utilizing the MFa(1) leader of ~ cerevisiae.
Table 1 ~ Leader Expression level, %
MT748 a-leader 100 SEQ ID No. 15 87 SEQ ID No. 16 215 SEQ ID No. 17 157 SEQ ID No. 19 166 SEQ ID No. 20 86 SEQ ID No. 21 145 SEQ ID No. 22 137 SEQ ID No. 23 121 WO 95134666 219 2 9 4 2 PCT~~5100249 Synthesis of the leader SEQ ID No. 27 for expression of the extended MI3 insulin precursor in S~ cerevisiae (strain yAK677).
The leader SEQ ID No. 27 has the following amino acid sequence:
GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMetAlaAspAspThr-GluSerArgPheAlaThrAsnThrThrLeuASpValValASnLeuIleSerMetAla The following oligonucleotides were synthesised:
# 440 5'-GGTTAACGAACTTTGGAGCTTCAGCTTCAGCTTCTTCTCTCTTAGCCAT
GGAGATCAAGTTAACAACATCCAAAGTAGTGTT-3' BEQ ID No. 64 and # 441 5'-CAAGTACAAAGCTTCAACCAAGTGGGAACCGCACAAGTGTTGGTTAACG
AACTT-3' SEQ ID No. 65 Polymerise chain reactions were performed as described in Example in 1 with the exception that oligonucleotide # 440 was used instead of oligonucleotide # 333 and plasmid pAK614 was used as template. For the second polymerise chain reaction, oligonucleotide # 441 was used instead of oligonucleotide #
312.
The purified PCR DNA fragment was isolated and digested with the restriction endonucleases Asp 718 and Hind III as described in Example 1. The Asp 718/Hind III DNA fragment of 268 by was subjected to electrophoresis on agarose gel and purified as descYibed in Example 1. The Asp 718/Hind III DNA
fragment was subcloned into the ~ cerevisiae expression plasmid as described in Example 1, with the exception that the 140 by Hind III/Xba I DNA fragment was derived from pAK602 and encodes Asp$28 human insulin. The selected plasmid, pAK616, was shown by DNA sequencing analysis, as described in Example 1, to contain the DNA sequence encoding the leader SEQ ID No. 27. For the DNA sequence, SEQ ID No.
66, encoding the leader SEQ ID No. 27, see Fig. 18. The Asp 718/Hind III DNA fragment of 268 by from pAK616 was isolated and ligated with the 10986 by Asp 718/Xba I DNA fragment from pAK601 and the 140 by DNA fragment Hind III/Xba I from pAK464 (encoding an extended version of AspB28 human insulin) and named pAK 625. The 180 by Asp 718/Nco I DNA fragment from pAK625 was isolated and ligated with the 221 by Nco I/Xba I
DNA fragment from pJB146 (encoding and extended version of the insulin precursor) and the 10824 by Asp 718/Xba I DNA
fragment from pAK601 and the resulting plasmid was named pAK677. The plasmid pAK677 was transformed into S~ cerevisiae strain MT663 as described in European patent application 86306721.1 and the resulting strain was named yAK677. With the exception of the DNA sequence encoding the leader, the DNA sequence encoding the signal peptide is as described in Fig. 5. The DNA sequence coding for the extended MI3 insulin precursor is as described in Fig. 19.
EgAMPLE ~1 Synthesis of the leader SEQ ID No. 67 for expression of the extended MI3 insulin precursor in Sue. cerevisiae (yAK680) The leader SEQ ID No. 67 has the following amino acid sequence:
GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMetAlaAspAspThr GluSerArgPheAlaThrAsnThrThrLeuAlaLeuAspValValAsnLeuIleSerMet Ala The following oligonucleotide was synthesised:
# 577 5'-TCTCTTAGCCATGGAGATCAAGTTAACAACATCCAAAG
CCAAAGTAGTGTT-3' SEQ ID No. 68 wo>s~ . . 2192942 so The PCR vas performed es daecribed in Example fn 1 with the exception that oligonucleotlde ~ 577 vas need instead of oligonucleotide ~ 933 and plasmid puC625 vas used as te~lata and the second PcR vas not perforaad. The FC7t lragment vas 3 digested with the restriction endonuclaases Asp 718 and Nco I
as described in Exeaplo 1:
The Asp 71~%ll0o I DNA fragsent o! 19o by vas subjected to electrophoresis on agarose gei and puriliad as described in Example 1 expect that the 10824 by Asp 718/Xba I vector DNA
iragsem 'vas isolated iron and tros pA1~601. Tba 190 by Asp 718/Nco I DNA fragment vas subclonsd into the ~ ~YifiiA!
expression plasmid as described in Exaaple 1, expect that the 221 by DNA fragosnt Nco I/Xba I (encoding an extended version of the !!I3 insulin precursor) vas isolated lram pAR677 and used instead of the Nind III/Xba I DNA lrsgsant. The selected plasmid vas shown by DNA sequencing analysis as described in Example I to contain the DNA sequence encoding the leader SEQ
ID No. 67 a~ named pA1C680. For the DNA saquanoe, BEQ ID No.
69, encoding the feeder SEQ ID No. 67, sea Fig. 20. Tha plasmid pA1C680 vas transforsed into $z cereviaiae strain 1~T663 as' described in European patent Tlo. 0214 $26.
and the resulting strain vas named yAR68o. lPith the exception of the DNA sequence encoding the leader, the DNA sequence encoding the signal peptide is as described in Fig. 5 and the extended insulin precursor liI3 DNA saqusnae is as described 1n rig. 19. _ LIIUIPLE l2 Exprasslon of N-terminally extended 1~I3 insulin precursors using the leader sequences SEQ ID No. 27 and SEQ ID No. 67 3o according to flee preset invention.
Yeast strains harbouring plasmids as described above, Ware grown in YPD medium (Shernan, F. at al., Methodg,in Yeast W0 95/34666 PCTlDK95100249 Genetics, Cold Spring Harbor Laboratory Press, 1981). For each strain 6 individual 5 ml cultures were shaken at 30'C
for 72 hours, with a final oDboo of approximately 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 Snel, L. et al. Chromatog!raphia ,fig (1987) 329-332.
In Table 2 the expression levels of some N-terminally extended MI3 insulin precursors, obtained by use of the leader sequences SEQ ID No. 27 and SEQ ID No. 67 according to the present invention, are given as a percentage of the level obtained with transformants of pMT742, utilizing the MFa(ij leader of S_s Cerevisiae.
Table 2 Strain Signal peptideLeader Extension Relative to MT748 a a yAK675 YAP3 SEQ ID EEAEAEAP 251%
No.27 K
yAK677 YAP3 SEQ ID EEAEAEAE 224%
No.27 PK
yAK681 YAP3 SEQ ID EEAEAEAP 248%
No.67 K
2o yAK680 YAP3 SEQ ID EEAEAEAE 362%
No.67 PK
WO 95134666 219 2 9 4 2 PCT~~SI00249 SEQUENCE LISTING
(1) GENERAL INFORMATION:
{i) APPLICANT:
(A) NAME: Novo Nordisk A/S
(B) STREET: Novo Alle (C) CITY: DK-2880 Bagsvaerd (E) COUNTRY: Denmark (G) TELEPHONE: +45 44448888 (H) TELEFAX: +45 44490555 (I) TELEX: 37173 (ii) TITLE OF INVENTION: SYNTHETIC LEADERS PEPTIDE SEQUENCES
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{A) APPLICATdON NUMBER: DK 0705/94 and US 08/282,852 (B) FILING DATE: 16-JUN-1994 and 29-JUL-1994 (viii} ATTORNEY/AGENT INFORMATION:
A) NAME: Jorgensen, Dan et a1.
~C) REFERENCE/DOCKET NUMBER: 4085-WO, DJ
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: +45 44448888 (B) TELEFAX: +45 44493256 (2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear WO 95/34666 219 2 9 4 2 PC'I'~I~95100249 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
itn Pro I1e Asp 51u Asp Asn Asp Thr Ser Va1 Asn Leu Pro A1a (2) INFORMATION FOR SEQ ID N0:2:
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G1n Pro I1e Asp Asp G1u Asn Thr Thr Ser Val Asn Leu Pro Ala i 5 10 15 (2) INFORMATION FOR SEQ ID N0:3:
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G1n Pro I1e Asp Asp Glu Asn Thr Thr Ser Yat Asn Leu Pro Ya1 (2) INFORMATION FOR SEQ ID N0:5:
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R'O 95134666 219 2 9 4 2 PCT~~S/00149 (A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5: -Gtn Pro I1e Asp Asp Thr G1u Asn Thr Thr Ser Ya1 Asn Leu Pro A1a (2) INFORMATION FOR SEQ ID N0:6:
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G1n Pro I1e Asp Asp Thr G1u Ser Asn Thr Thr Ser Va1 Asn Leu Pro Ala (2) INFORMATION FOR SEQ ID N0:7:
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G1n Pro I1e Asp Asp Glu Asn Thr Thr Ser Yal Asn Leu Met Ala (2) INFORMATION FOR SEQ ID N0:8:
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WO 95/34666 219 2 9 4 2 PCTJDK95l00249 Gln Pro Ile Asp Asp Thr GIu Ser Asn Thr Thr Ser Va1 Asn Leu Pro G1y A1a (2) INFORMATION FOR SEQ ID N0:9:
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G1n Pro I1e Asp Asp Thr G1u Ser Asn Thr Thr Ser Ya1 Asn Leu Met i 5 10 15 A1a (2) INFORMATION FOR SEQ ID N0:10:
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i1n Pro I1e Asp 5sp Thr G1u Ser Asn Thr Thr Ser Ya1 Asn Va1 Pro Thr (2) INFORMATION FOR SEQ ID N0:11:
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G1n Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Leu Ya1 Asn Ya1 Pro Thr (2) INFORMATION FOR SEQ ID N0:12:
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G1n Pro I1e Asp Asp Thr G1u Ser Asn Thr Thr Leu Ya1 Asn Ya1 Pro 61y A1a (2) INFORMATION FOR SEQ ID N0:14:
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WO 95/34666 219 2 9 4 2 PCT11~K95100249 G1n Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Ser Va1 Asn Leu Met A1a Pro A1a Va1 A1a (2) INFORMATION FOR SEQ ID N0:15:
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Gln Pro I1e Asp Asp Thr G1u Ser Asn Thr Thr Ser Val Asn Leu Met Asp Leu A1a Va1 Gly Leu Pro G1y A1a (2) INFORMATION FOR SEQ ID N0:16:
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G1n Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Ser Va1 Asn Leu Met Ala Asp Asp Thr Glu Ser I1e Asn Thr Thr Leu Yal Asn Leu Pro Gty A1a (2) INFORMATION FOR SEQ ID N0:17:
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R'O 95/34666 . 219 2 9 4 2 p~~gg~p0249 G1n Pro I1e Asp Asp Thr G1u Ser I1e Asn Thr Thr Leu Ya1 Asn Leu Pro Gly A1a (2) INFORMATION FOR SEQ ID N0:18:
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G1n Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Leu Yal Asn Leu Pro G1y A1a (2) INFORMATION FOR SEQ ID N0:19:
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G1n Pro IIe Asp Asp Thr G1u Ser Asn Thr Thr Ser Ya1 Asn Leu Met A1a Asp Asp Thr G1u Ser Arg Phe Ala Thr Asn Thr Thr Leu Ya1 Asn Leu Pro Leu (2) INFORMATION FOR SEQ ID N0:20:
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i1n Pro I1e Asp 5sp Thr G1u Ser Asn Thr Thr Ser Va1 Asn Leu Met A1a Asp Asp Thr G1u Ser I1e Asn Thr Thr Leu Ya1 Asn Leu A1a Asn Ya1 A1a Met Ala (2) INFORMATION FOR SEQ ID N0:21:
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(A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
i1n Pro I1e Asp 5sp Thr Glu Ser A1a Ile Asn Thr Thr Leu Yal Asn Leu Pro G1y A1a (2) INFORMATION FOR SEQ ID N0:22:
(i) 5~QUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
i1n Pro I1e Asp 5sp Thr GIu Ser Phe A1a Thr Asn Thr Thr Leu Yal Asn Leu Pro Gly A1a (2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide WO 95!34666 219 2 9 4 2 PCT~~5100249 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
G1n Pro I1e Asp Asp Thr G1u Ser Ite Asn Thr Thr Leu Ya1 Asn Leu Met A1a Asp Asp Thr Glu Ser Arg Phe A1a Thr Asn Thr Thr Leu Ya1 Asn Leu Pro Leu (2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 amino acids (B) TYPE: amino acid (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
G1n Pro I1e Asp Asp Thr G1u Ser I1e Asn Thr Thr Leu Vat Asn Leu Met A1a Asp Asp Thr G1u Ser Arg Phe A1a Thr Asn Thr Thr Leu Asp Va1 Va1 Asn Leu Pro G1y A1a (2) INFORMATION FOR SEQ ID N0:25:
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(A) LENGTH: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide {xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
G1n Pro I1e Asp Asp Thr Glu Ser Ata A1a Ite Asn Thr Thr Leu Va1 Asn Leu Pro G1y Ala (2) INFORMATION FOR SEQ ID N0:26:
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(A) LENGTH: 39 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
G1n Pro Ile Asp Asp Thr G1u Ser Asn Thr Thr Ser Yat Asn Leu Met Ala Asp Asp Thr Glu Ser Arg Phe A1a Thr Asn Thr Thr Leu Va1 Asn Leu A1a Asn Yat Ala Met Ala (2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 amino acids (B) TYPE: amino acid (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
Gln Pro I1e Asp Asp Thr Glu Ser Asn Thr Thr Ser Va1 Asn Leu Met Ata Asp Asp Thr G1u Ser Arg Phe A1a Thr Asn Thr Thr Leu Asp Va1 Val Asn Leu I1e Ser Met A1a (2) INFORMATION FOR SEQ ID N0:28:
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G1n Pro IIe Asp Asp Thr G1u Ser Asn Thr Thr Ser Ya1 Asn Leu Met Ata Asn Thr Thr G1u Ser Arg Phe A1a Thr Asn Thr Thr Leu Asp Va1 WO 95134666 2 ~ ~ 9 2 9 4 2 PCTI1DK95I00249 Ya1 Asn Leu I1e Ser Met Ata (2) INFORMATION FOR SEQ ID N0:29:
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
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(A) LENGTH: 56 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
(2) INFORMATION FOR SEQ ID N0:34:
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(A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
(2) INFORMATION FOR SEQ ID N0:35:
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(A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:
(2) INFORMATION FOR SEQ ID N0:36:
S
W O 95Y34666 219 2 9 4 2 PCT~~~00249 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 75 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:
(2) INFORMATION FOR SEQ ID N0:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 72 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
6TCGTCAGCC AT ~ 72 (2) INFORMATION FOR SEQ ID N0:38:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
W O 95/34666 219 2 9 4 2 PCTlDK95100249 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
G sl (2) INFORMATION FOR SEQ ID N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 372 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 82..351 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:
GAATTCATTC AAGAATAGTT TATCAATTTC ATACACAATA
CAAACAAGAA
GATTACAAAC
A
Met LysLeuLys ThrYalArgSer A1aYa1 TCA
LeuSer Leu PheA1aSer GtnYa1Leu G1yGtnProIle AspG1u Ser GAC
AspAsn Thr SerSerMet AtaLysArg PheYa1AsnGtn HisLeu Asp TCC
CysGly His LeuVa1G1u A1aLeuTyr .LeuVa1CysG1y G1uArg Ser TTC
G1yPhe Tyr ThrProLys AtaAlaLys G1yI1eYa1G1u G1nCys Phe TGTACC ATC TGCTCCTTG TACCAATTG GAAAACTACTGC AACTAGACGCAGC
TCC
CysThr I1e CysSerLeu TyrG1nLeu G1uAsnTyrCys Asn Ser TAGA
(2)INFORMATION FORSEQID :
N0:41 (i) CHARACTERIST ICS:
SEQUENCE
R'O 95134666 2 , ~ 9 2 9 4 2 PCT~~~I00249 (A) LENGTH: 89 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:
Met Lys Leu Lys Thr Ya1 Arg Ser Ala Ya1 Leu Ser Ser Leu Phe A1a Ser G1n Ya1 Leu G1y G1n Pro I1e Asp Glu Asp Asn Asp Thr Ser Ser Met Ala Lys Arg Phe Va1 Asn G1n His Leu Cys G1y Ser His Leu Ya1 G1u A1a Leu Tyr Leu Va1 Cys G1y G1u Arg G1y Phe Phe Tyr Thr Pro Lys A1a A1a Lys Gty I1e Val G1u G1n Cys Cys Thr Ser I1e Cys Ser Leu Tyr G1n Leu G1u Asn Tyr Cys Asn (2) INFORMATION FOR SEQ ID N0:42:
(i) SEQUENCE CHARACTERISTICS:
~(A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..45 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:
Gln Pro I1e Asp Asp Gtu Asn Thr Thr Ser Ya1 Asn Leu Pro Va1 (2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear WO 95/34666 2-192 9 4 2 PCT~1~95/0.0249 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
G1n Pro I1e Asp Asp G1u Asn Thr Thr Ser Va1 Asn Leu Pro Va1 (2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 297 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..276 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:
ACT
MeiLysLeuLys Th Va1ArgSer AlaVa1Leu SerSerLeuPhe Ata SerG1nVa1Leu GlyGlnProI1e AspAspGlu AsnThrThrSer Va1 AsnLeuProVa1 LysArgPheYa1 AsnG1nHis LeuCysG1ySer His LeuVa1G1uA1a LeuTyrLeuYat CysG1yG1u ArgGlyPhePhe Tyr ThrProLysA1a A1aLysG1yIle YalGtuGtn GysCysThrSer I1e CCGCAGGGTC
CysSerLeuTyr G1nLeuG1uAsn TyrCysAsn TAGA
(2)INFORMAT ION FORSEQID
N0:45:
( i) EQUENCE CHARACTE RISTICS:
S
(A) LENGTH:91 amino ids ac (B) TYPE: aci d amino (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:
Met Lys Leu Lys Thr Ya1 Arg Ser Ata Ya1 Leu Ser Ser Leu Phe A1a Ser G1n Va1 Leu Gty Gtn Pro I1e Asp Asp G1u Asn Thr Thr Ser Ya1 Asn Leu Pro Va1 Lys Arg Phe Ya1 Asn Gln His Leu Cys G1y Ser His Leu Va1 Gtu Ala Leu Tyr Leu Va1 Cys G1y G1u Arg G1y Phe Phe Tyr Thr Pro Lys A1a A1a Lys G1y I1e Va1 G1u Gtn Cys Cys Thr Ser I1e Cys Ser Leu Tyr G1n Leu G1u Asn Tyr Cys Asn (2) INFORMATION FOR SEQ ID N0:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..51 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:46:
G1n Pro I1e Asp Asp Thr Glu Ser Asn Thr Thr Ser Va1 Asn Leu Pro A1a (2) INFORMATION FOR SEQ ID N0:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: Linear R'O 95134666 PC"lYDK95100249 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:
Gln Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Ser Ya1 Asn Leu Pro A1a (2) INFORMATION FOR SEQ ID N0:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..54 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:
Gln Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Ser Yal Asn Leu Pro GGT GCT ~ 54 G1y A1a (2) INFORMATION FOR SEQ ID N0:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
~ (A) NAME/KEY: CDS
(B) LOCATION: 1..57 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:
61n Pro I1e Asp Asp Thr Glu Ser I1e Asn Thr Thr Leu Va1 Asn Leu 2x92942 ccA GGT Gcr Pro G1y A1a (2) INFORMATION FOR SEQ ID N0:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 99 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..99 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:
G1n Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Ser Vat Asn Leu Met Ata Asp Asp Thr G1u Ser I1e Asn Thr Thr Leu Va1 Asn Leu Pro G1y A1a (2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 105 base pairs B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATIDN: 1..105 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:
G1n Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Ser Va1 Asn Leu Met WO 95134666 219 2 9 4 2 pCTIDK95100249 A1a Asp Asp Thr G1u Ser Arg Phe Ata Thr Asn Thr Thr Leu Ya1 Asn Leu Pro Leu (2) INFORMATION FOR SEQ ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 108 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..108 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:52:
G1n Pro I1e Asp Asp Thr G1u Ser Asn Thr Thr Ser Va1 Asn Leu Met A1a Asp Asp Thr G1u Ser I1e Asn Thr Thr Leu Ya1 Asn Leu A1a Asn ' 20 25 30 Ya1 Ata Met A1a (2) INFORMATION FOR SEQ ID N0:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..60 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:53:
WO 95134666 ~ s w219 2 9 4 2 PCT~~5100249 G1n Pro I1e Asp Asp Thr G1u Ser A1a Ite Asn Thr Thr Leu Yal Asn Leu Pro G1y Ata (2) INFORMATION FOR 5EQ ID N0:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 276 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 113..274 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:54:
AACTACAAAA TCAAACAAGA
AACACATACA
CTATCAATTT TA
CATACACAAT ATG
Met AGA GCG TCG
LysLeuLys Ya1 Ser Ya1Leu Ser LeuPheA1a Ser Thr Arg A1a Ser GGC CCA GAC AAC
GlnYa1Leu Gln I1e AspG1u Thr ThrSerYal Asn G1y Pro Asp Asn AAG TTC AAC TTG
LeuProA1a Arg Va1 G1nHis Cys GlySerHis Leu Lys Phe Asn Leu TTG TT
YalGluA1a Tyr Leu (2) INFORMATION FOR SEQ ID N0:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 amino acids (B) TYPE: amino acid (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:55:
Met Lys Leu Lys Thr Va1 Arg Ser Ala Yal Leu Ser Ser Leu Phe A1a Ser G1n Ya1 Leu Gly G1n Pro I1e Asp Asp Gtu Asn Thr Thr Ser Ya1 Asn Leu Pro A1a Lys Arg Phe Yal Asn G1n His Leu Cys Gty Ser His Leu Ya1 G1u Ala Leu Tyr (2) INFORMATION FOR SEQ ID N0:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 282 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 113..280 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:56:
TTAAATCTAT AACTACAAAA CAAGAATAGT TCAAACAAGA
AACACATACA
GGAATTCATT
AGATTACAAA CTATCAATTT GGTACCAAAA
CATACACAAT TA
ATAAACGACG ATG
Met GTC
LysLeu LysThrYa1 ArgSer A1a LeuSerSer LeuPheA1aSer Va1 GAC
G1nYal LeuG1yGln ProI1e Asp ThrGtuSer AsnThrThrSer Asp GTT
YalAsn LeuProA1a LysArg Phe AsnG1nHis LeuCysG1ySer Ya1 CACTTG GTTGAAGCT TTGTAC TT 2g2 HisLeu Va1G1uA1a LeuTyr (2)INFORMATION FOR SEQID N0:57:
W095134666 - - w-21 9 2 9 4 2 P~'~''~~s~~~'''9 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 56 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:57:
Met Lys Leu Lys Thr Ya1 Arg Ser A1a Ya1 Leu Ser Ser Leu Phe A1a Ser Gtn Ya1 Leu G1y G1n Pro I1e Asp Asp Thr G1u Ser Asn Thr Thr Ser Va1 Asn Leu Pro A1a Lys Arg Phe Ya1 Asn Gtn His Leu Cys G1y Ser His Leu Va1 G1u A1a Leu Tyr (2) INFORMATION FOR SEQ ID N0:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 282 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single {D) TOPOLOGY: linear {ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 113..280 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:
Met Lys Leu Lys Thr Ya1 Arg Ser A1a Ya1 Leu Ser Ser Leu Phe Ala Ser Gln Yai Leu Gly Gln Pro Ile Asp Asp Thr Gtu Ser Asn Thr Thr Ser Ya1 Asn Leu Met A1a Lys Arg Phe Ya1 Asn G1n His Leu Cys G1y Ser His Leu Ya1 Glu A1a Leu Tyr (2) INFORMATION FOR SEQ ID N0:59:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 56 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:59:
Mei Lys Leu Lys Th5 Yat Arg Ser A1a Ya1 Leu Ser Ser Leu Phe A1a Ser Gln Va1 Leu Giy G1n Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Ser Val Asn Leu Met A1a Lys A4g0 Phe Ya1 Asn G1n His Leu Cys G1y Ser His Leu Ya1 G1u A1a Leu Tyr (2) INFORMATION FOR SEQ ID N0:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 330 base pairs {B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 113..328 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:60:
Met Lys Leu Lys Thr Ya1 Arg Ser Ata Va1 Leu Ser Ser Leu Phe A1a Ser WO 95134666 219 2 9 4 2 PCT~~5100249 i GlnYa1LeuG1yG1n ProI1eAsp AspThrG1uSer AsnThrThrSer Va1AsnLeuMetA1a AspAspThr G1u5erI1eAsn ThrThrLeuYa1 AsnLeuProGtyA1a LysArgPhe YatAsnGtnHis LeuCysG1ySer HisLeuVa1GtuA1a LeuTyr (2) INFORMATION FOR SEQ ID N0:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 72 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:61:
Met Lys Leu Lys Thr Ya1 Arg Ser Ata Ya1 Leu Ser Ser Leu Phe A1a Ser G1n Yal~Leu G1y Gtn Pro Ite Asp Asp Thr G1u Ser Asn Thr Thr Ser Ya1 Asn Leu Met A1a Asp Asp Thr Gtu Ser Ite Asn Thr Thr Leu Va1 Asn Leu Pro G1y A1a Lys Arg Phe Ya1 Asn G1n His Leu Cys G1y Ser His Leu Va1 G1u A1a Leu Tyr (2) INFORMATION FOR SEQ ID N0:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 288 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
WO 95/34666 ~ 19 2 9 4 2 p~~g9g100249 (B) LOCATION: 113..286 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:62:
Met Lys Leu Lys Thr Va1 Arg Ser A1a Ya1 Leu Ser Ser Leu Phe A1a Ser 61n Va1 Leu Gly G1n Pro I1e Asp Asp Thr G1u Ser Ile Asn Thr Thr Leu Ya1 Asn Leu Pro G1y A1a Lys Arg Phe Va1 Asn G1n His Leu Cys GGT TCC CAC TTG GTT GAA GCT TTG TAC TT 2gg G1y Ser His Leu Ya1 61u A1a Leu Tyr (2) INFORMATION FOR SEQ ID N0:63:
(i) SEQUENCE CHARACTERISTICS:
~ (A) LENGTH: 58 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:63:
Met Lys Leu Lys Thr Val Arg Ser A1a Va1 Leu Ser Ser Leu Phe Ala Ser Gln Yal Leu G1y Gln Pro Ile Asp Asp Thr G1u Ser I1e Asn Thr Thr Leu Va1 Asn Leu Pro G1y A1a Lys Arg Phe Ya1 Asn Gtn His Leu Cys G1y Ser His Leu Ya1 G1u A1a Leu Tyr (2) INFORMATION FOR SEQ ID N0:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 82 base pairs W095134666 ' ' " PCTIDK95100?A9 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D} TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:64:
(2) INFORMATION FOR SEQ ID N0:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
{xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:
(2) INFORMATION FOR SEQ ID NO:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 117 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:66:
(2) INFORMATION FOR SEQ ID NO:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 amino acids {B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:67:
WO 95/34666 219 2 9 ~ 2 pCTIDK95100249 iln Pro I1e Asp 5sp Thr G1u Ser Asn Thr Thr Ser Va1 Asn Leu Met A1a Asp Asp Thr G1u Ser Arg Phe A1a Thr Asn Thr Thr Leu Ata Leu Asp Ya1 Yal Asn Leu Ile Ser Met Ala 35 . 40 (2) INFORMATION FOR SEQ ID N0:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:68:
(2) INFORMATION FOR SEQ ID N0:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 123 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:69:
GCT . 123 (2) INFORMATION FOR SEQ ID N0:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 65 amino acids (B) TYPE: amino acid {D) TOPOLOGY: Linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:70:
WO 95!34666 219 2 9 4 2 PCT~~5~00249 Lys Arg Gtu G1u Ala G1u A1a Gtu A1a G1u Pro Lys Phe Va1 Asn 61n His Leu Cys Gly Ser His Leu Vat Glu A1a Leu Tyr Leu Va1 Cys Gty G1u Arg G1y Phe Phe Tyr Thr Pro Lys A1a A1a Lys G1y I1e Va1 61u Gln Cys Cys Thr Ser I1e Cys Ser Leu Tyr Gln Leu G1u Asn Tyr Cys Asn (2) INFORMATION FOR SEQ IO N0:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 219 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID N0:71:
(2) INFORMATION FOR SEQ ID NO:72:
(1) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 348 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:72:
(2) INFORMATION FOR SEQ ID N0:73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 379 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:73:
GGTTGAAGCTTTGTACTTG 37g
Claims (27)
1. A DNA expression cassette comprising the following sequence:
5'-P-SP-LS-PS-*gene*-(T)i-3' wherein P is a promoter sequence, SP is a DNA sequence encoding a signal peptide, LS is a DNA sequence encoding a leader peptide with the general formula I:
GlnProIle(Asp/Glu)(Asp/Glu)X1(Glu/Asp)X2AsnZ(Thr/Ser)X3 (I) wherein X1 is a peptide bond or a codable amino acid;
X2 is a peptide bond, a codable amino acid or a sequence of up to 4 codable amino acids which may be the same or different;
Z is a codable amino acid except Pro;
and X3 is a sequence of from 4 to 30 codable amino acids which may be the same or different;
PS is a DNA sequence encoding a processing site;
*gene* is a DNA sequence encoding a polypeptide;
T is a terminator sequence;
and i is 0 or 1.
5'-P-SP-LS-PS-*gene*-(T)i-3' wherein P is a promoter sequence, SP is a DNA sequence encoding a signal peptide, LS is a DNA sequence encoding a leader peptide with the general formula I:
GlnProIle(Asp/Glu)(Asp/Glu)X1(Glu/Asp)X2AsnZ(Thr/Ser)X3 (I) wherein X1 is a peptide bond or a codable amino acid;
X2 is a peptide bond, a codable amino acid or a sequence of up to 4 codable amino acids which may be the same or different;
Z is a codable amino acid except Pro;
and X3 is a sequence of from 4 to 30 codable amino acids which may be the same or different;
PS is a DNA sequence encoding a processing site;
*gene* is a DNA sequence encoding a polypeptide;
T is a terminator sequence;
and i is 0 or 1.
2. The expression cassette according to claim 1, wherein X1, in general formula I, is Ser, Thr or Ala.
3. The expression cassette according to claim 1, wherein X2, in general formula I, is Ser, Thr or Ala.
4. The expression cassette according to claim 1, wherein X2, in general formula I, is SerIle.
5. The expression cassette according to claim 1, wherein X2, in general formula I, is SerAlalle.
6. The expression cassette according to claim 1, wherein X2, in general formula I, is SerPheAlaThr.
7. The expression cassette according to claim 1, wherein X3, in general formula I, is an amino acid sequence of the general formula II
X4-Xs-X6 (II) wherein X4 is a sequence of from 1 to 21 codable amino acids;
X5 is Pro or an amino acid sequence including the amino acid sequence ValAsnLeu, LeuAlaAsnValAlaMetAla, LeuAspValValAsnLeuProGly, or LeuAspValValAsnLeuIleSerMet;
and X6 is a sequence of from 1 to 8 codable amino acids.
X4-Xs-X6 (II) wherein X4 is a sequence of from 1 to 21 codable amino acids;
X5 is Pro or an amino acid sequence including the amino acid sequence ValAsnLeu, LeuAlaAsnValAlaMetAla, LeuAspValValAsnLeuProGly, or LeuAspValValAsnLeuIleSerMet;
and X6 is a sequence of from 1 to 8 codable amino acids.
8. The expression cassette according to claim 7, wherein X4, in general formula II, is an amino acid sequence including one or more of the motifs LeuValAsnLeu, SerValAsnLeu, MetAlaAsp, ThrGluSer, ArgPheAlaThr and ValAlaMetAla.
9. The expression cassette according to claim 7, wherein X4, in general formula II, is an amino acid sequence including the sequence AsnSerThr or AsnThrThr.
10. The expression cassette according to claim 7, wherein X4, in general formula II, is an amino acid sequence including the sequence (Ser/Leu) ValAsnLeu, (Ser/Leu) ValAsnLeuMetAlaAsp, (Ser/Leu) ValAsnLeuMetAlaAspAsp, (Ser/Leu) ValAsnLeuMetAlaAspAspThrGluSer, (Ser/Leu) ValAsnLeuMetAlaAspAspThrGluSerIle, or (Ser/Leu) ValAsnLeuMetAlaAspAspThrGluSerArgPheAlaThr.
11. The expression cassette according to claim 7, wherein X4 in general formula II, is an amino acid sequence including the sequence Asn(Thr/Ser)ThrLeu, Asn(Thr/Ser)ThrLeuAsnLeu, or Asn(Thr/Ser)ThrLeuValAsnLeu.
12. The expression cassette according to claim 7, wherein X5, in general formula II, is Pro.
13. The expression cassette according to claim 7, wherein X5, in general formula II, is the amino acid sequence ValAsnLeu.
14. The expression cassette according to claim 7, wherein X5, in general formula II, is the amino acid sequence LeuAlaAsnValAlaMetAla.
15. The expression cassette according to claim 7, wherein X5, in general formula II, is the amino acid sequence LeuAspValValAsnLeuProGly.
16. The expression cassette according to claim 7, wherein X5, in general formula II, is the amino acid sequence LeuAspValValAsnLeuIleSerMet.
17. The expression cassette according to claim 7, wherein X6, in general formula II, is Ala, Gly, Leu, Thr, Val, or Ser.
18. The expression cassette according to claim 7, wherein X6, in general formula II, is GlyAla or SerAla.
19. The expression cassette according to claim 7, wherein X6, in general formula II, is AlaValAla.
20. The expression cassette according to claim 7, wherein X6, in general formula II, is GlyAlaAspSerLysThrValGlu.
21. The expression cassette according to claim 1, wherein the leader peptide coded for by the DNA sequence LS is selected from the group comprising:
SEQ ID No. 1 GlnProIleAspGluAspAsnAspThrSerValAsnLeuProAla;
SEQ ID No. 2 GlnProIleAspAspGluAsnThrThrSerValAsnLeuProAla;
SEQ ID No. 3 GlnProIleAspAspGluSerAsnThrThrSerValAsnLeuProAla;
SEQ ID No. 4 GlnProIleAspAspGluAsnThrThrSerValAsnLeuProVal;
SEQ ID No. 5 GlnProIleAspAspThrGluAsnThrThrSerValAsnLeuProAla;
SEQ ID No. 6 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuProAla;
SEQ ID No. 7 GlnProIleAspAspGluAsnThrThrSerValAsnLeuMetAla;
SEQ ID No. 8 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuProGlyAla;
SEQ ID No. 9 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMetAla;
SEQ ID No. 10 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnValProThr;
SEQ ID No. 11 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnValProThr;
SEQ ID No. 12 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuProThr;
SEQ ID No. 13 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnValProGlyAla;
SEQ ID No. 14 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMetAlaPro AlaValAla;
SEQ ID No. 15 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu MetAspLeuAlaValGlyLeuProGlyAla;
SEQ ID No. 16 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu MetAlaAspAspThrGluSerIleAsnThrThrLeuValAsnLeuProGlyAla;
SEQ ID No. 17 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsnLeuProGlyAla;
SEQ ID No. 18 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnLeuProGlyAla;
SEQ ID No. 19 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuV alAsnLeuProLeu;
SEQ ID No. 20 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu MetAlaAspAspThrGluSerIleAsnThrThrLeuValAsnLeuAlaAsnValAlaMetAla;
SEQ ID No. 21 GlnProIleAspAspThrGluSerAlaIleAsnThrThrLeuVal AsnLeuProGlyAla;
SEQ ID No. 22 GlnProIleAspAspThrGluSerPheAlaThrAsnThrThr LeuValAsnLeuProGlyAla;
SEQ ID No. 23 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsn LeuMetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuValAsnLeuProLeu;
SEQ ID No. 24 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsnLeuMetAlaAspAsp ThrGluSerArgPheAlaThrAsnThrThrLeuAspValValAsnLeuProGlyAla;
SEQ ID No. 25 GlnProIleAspAspThrGluSerAlaAlaIleAsnThrThrLeu ValAsnLeuProGlyAla;
SEQ ID No. 26 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMetAlaAsp AspThrGluSerArgPheAlaThrAsnThrThrLeuValAsnLeuAlaAsnValAlaMetAla;
SEQ ID No. 27 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuAspVal-ValAsnLeuIleSerMetAla;
SEQ ID No. 28 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu MetAlaAsnThrThrGluSerArgPheAlaThrAsnThrThrLeuAspValValAsnLeuIleSerMetAla;
and SEQ ID No. 67 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuAlaLeuAspValValAsnLeuIleSer MetAla.
SEQ ID No. 1 GlnProIleAspGluAspAsnAspThrSerValAsnLeuProAla;
SEQ ID No. 2 GlnProIleAspAspGluAsnThrThrSerValAsnLeuProAla;
SEQ ID No. 3 GlnProIleAspAspGluSerAsnThrThrSerValAsnLeuProAla;
SEQ ID No. 4 GlnProIleAspAspGluAsnThrThrSerValAsnLeuProVal;
SEQ ID No. 5 GlnProIleAspAspThrGluAsnThrThrSerValAsnLeuProAla;
SEQ ID No. 6 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuProAla;
SEQ ID No. 7 GlnProIleAspAspGluAsnThrThrSerValAsnLeuMetAla;
SEQ ID No. 8 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuProGlyAla;
SEQ ID No. 9 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMetAla;
SEQ ID No. 10 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnValProThr;
SEQ ID No. 11 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnValProThr;
SEQ ID No. 12 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuProThr;
SEQ ID No. 13 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnValProGlyAla;
SEQ ID No. 14 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMetAlaPro AlaValAla;
SEQ ID No. 15 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu MetAspLeuAlaValGlyLeuProGlyAla;
SEQ ID No. 16 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu MetAlaAspAspThrGluSerIleAsnThrThrLeuValAsnLeuProGlyAla;
SEQ ID No. 17 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsnLeuProGlyAla;
SEQ ID No. 18 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnLeuProGlyAla;
SEQ ID No. 19 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuV alAsnLeuProLeu;
SEQ ID No. 20 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu MetAlaAspAspThrGluSerIleAsnThrThrLeuValAsnLeuAlaAsnValAlaMetAla;
SEQ ID No. 21 GlnProIleAspAspThrGluSerAlaIleAsnThrThrLeuVal AsnLeuProGlyAla;
SEQ ID No. 22 GlnProIleAspAspThrGluSerPheAlaThrAsnThrThr LeuValAsnLeuProGlyAla;
SEQ ID No. 23 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsn LeuMetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuValAsnLeuProLeu;
SEQ ID No. 24 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsnLeuMetAlaAspAsp ThrGluSerArgPheAlaThrAsnThrThrLeuAspValValAsnLeuProGlyAla;
SEQ ID No. 25 GlnProIleAspAspThrGluSerAlaAlaIleAsnThrThrLeu ValAsnLeuProGlyAla;
SEQ ID No. 26 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMetAlaAsp AspThrGluSerArgPheAlaThrAsnThrThrLeuValAsnLeuAlaAsnValAlaMetAla;
SEQ ID No. 27 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuAspVal-ValAsnLeuIleSerMetAla;
SEQ ID No. 28 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu MetAlaAsnThrThrGluSerArgPheAlaThrAsnThrThrLeuAspValValAsnLeuIleSerMetAla;
and SEQ ID No. 67 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuAlaLeuAspValValAsnLeuIleSer MetAla.
22. The expression cassette according to claim 1, wherein SP is a DNA sequence encoding .alpha.-factor signal peptide, the signal peptide of mouse salivary amylase, carboxypeptidase signal peptide, yeast aspartic protease 3 signal peptide, or yeast BAR1 signal peptide.
23. The expression cassette according to claim 1, wherein PS is a DNA sequence encoding LysArg, ArgLys, ArgArg, LysLys or IleGluGlyArg.
24. The expression cassette according to claim 1, wherein the polypeptide is selected from the group consisting of aprotinin, tissue factor pathway inhibitor, or other protease inhibitors, insulin or insulin precursors, insulin-like growth factor I, insulin-like growth factor II, human or bovine growth hormone, interleukin, glucagon, glucagon-like peptide 1, tissue plasminogen activator, transforming growth factor .alpha. or .beta., platelet-derived growth factor, enzymes, or a functional analogue thereof.
25. A yeast expression vector comprising the expression cassette according to any of claims 1 to 24.
26. A yeast cell which is capable of expressing a polypeptide and which is transformed with the yeast expression vector according to claim 25.
27. A process for producing a polypeptide in yeast, the process comprising culturing a yeast cell which is transformed with the yeast expression vector according to claim 25, in a suitable medium to obtain expression and secretion of the polypeptide, after which the polypeptide is recovered from the medium.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
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DK70594 | 1994-06-16 | ||
DK0705/94 | 1994-06-16 | ||
US28285294A | 1994-07-29 | 1994-07-29 | |
US08/282,852 | 1994-07-29 | ||
PCT/DK1995/000249 WO1995034666A1 (en) | 1994-06-16 | 1995-06-16 | Synthetic leader peptide sequences |
Publications (2)
Publication Number | Publication Date |
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CA2192942A1 CA2192942A1 (en) | 1995-12-21 |
CA2192942C true CA2192942C (en) | 2006-03-14 |
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CA002192942A Expired - Fee Related CA2192942C (en) | 1994-06-16 | 1995-06-16 | Synthetic leader peptide sequences |
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CA (1) | CA2192942C (en) |
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