Note: Descriptions are shown in the official language in which they were submitted.
W O 95/34666 ~ 219 2 9 4 2
PCTIDK95100249
1
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
CA 02192942 2004-05-28
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 $,,cerevisiae NFal
signal/leader sequence at the 5~ end of the gene for the
3o desired protein secretion and processing of the desired protein
was demonstrated.
R'O 95/34666 ~ 219 2 9 4 2 PCTIDK95100249
3
EP 206 783 discloses a system for the secretion of polypeptides.
from ,~ cerevisiae using an a-factor leader sequence which has
been truncated to eliminate the four a-factor units present on
the native leader sequence so as to leave the leader peptide
itself fused to a heterologous polypeptide via the a-factor
processing site LysArgGluAlaGluAla. This construction is
indicated to lead to an efficient processing of smaller
peptides (less than 50 amino acids). For the secretion and
processing of larger polypeptides, the native a-factor leader
sequence has been truncated to leave one or two of the a-factor
units between the leader peptide and the polypeptide.
A number of secreted proteins are routed so as to be exposed to
a proteolytic processing system which can cleave the peptide
bond at the carboxy end of two consecutive basic amino acids.
This enzymatic activity is in ~ cerevisiae encoded by the KEX
2 gene (Julius, D.A. et al., Cell ~7 (1984bj 1075). Processing
of the product by the KEX 2 protease is needed for the
secretion of active S,~ ~erevisia~ mating factor ai (MFa1 or a-
facto;) whereas KEX 2 is not involved in the secretion of ac-
tive Sue. cerevisiae mating factor a.
Secretion and correct processing of a polypeptide intended to
be secreted is obtained in some cases when culturing a yeast
organism which is transformed with a vector constructed as
indicated in the references given above. In many cases, how-
ever, the level of secretion is very low or there is no se-
cretion, or the proteolytic processing may be incorrect or
incomplete. It is therefore the object of the present invention
to provide leader peptides which ensure a more efficient
expression and/or processing of polypeptides.
SUMMARY OF T$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
4
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.
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wo>s2192942
6
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
7
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
8
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
WO 95/34666 219 2 9 4 2 PCTIDK95100249
9
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-
WO 95/34666 219 2 9 4 2 PCTIDK95100249
11
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:
WO 95/34666 PCT1DK9S100249
- 2.~ 92942
12
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:
CA 02192942 2004-05-28
~ wo» ~ 2192942
13
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
14
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
16
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
17
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).
CA 02192942 2004-05-28
wo 9sr3~s. . ~ ~ ' v~ 19 2 9 4 2 rcrmxssioe~
1
18
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
CA 02192942 2004-05-28
PCTID1C95100149
2192942 .
19
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.
ERAMPLE 2
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
21
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.
ERAMPLE 3
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
22
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.
EBAMPLE 4
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
23
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.
EBAMPLE 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
WO 95/34666 Z PCTIDK95100249
24
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.
EBAMPLE 6
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
W O 95/34666
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.
EXAMPLE 7
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
WO 95134666 219 2 9 4 2 FCTIDEC95/00249
a
26
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.
2192942
R'O 95/34666 PCTIDK95100249
27
ESAMPLE 9
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
EBAMPLE 10
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
VVO 95134666 219 2 9 4 2 PCTIDK95100249
29
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
CA 02192942 2004-05-28
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
2192942
W0 95/34666 PCTlDK95100249
31
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
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
32
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
(iii) NUMBER OF SEQUENCES: 73
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Novo Nordisk A/S
Corporate Patents
(B) STREET: Novo Alle
(C) CITY: DK-2880 Bagsvaerd
(E) COUNTRY: Denmark
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
{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
33
(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
15
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
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:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
G1n Pro I1e Asp Asp G1u Ser Asn Thr Thr Ser Va1 Asn Leu Pro A1a
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
G1n Pro I1e Asp Asp Glu Asn Thr Thr Ser Yat Asn Leu Pro Ya1
I 5 10 15
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
R'O 95134666 219 2 9 4 2 PCT~~S/00149
34
(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
1 5 10 15
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
G1n Pro I1e Asp Asp Thr G1u Ser Asn Thr Thr Ser Va1 Asn Leu Pro
1 5 10 15
Ala
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
G1n Pro I1e Asp Asp Glu Asn Thr Thr Ser Yal Asn Leu Met Ala
I 5 10 15
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
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
1 5 10 15
G1y A1a
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
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:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
i1n Pro I1e Asp 5sp Thr G1u Ser Asn Thr Thr Ser Ya1 Asn Va1 Pro
10 15
Thr
(2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
WO 95/34666 219 2 9 4 2 PCTIDK95100249
36
G1n Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Leu Ya1 Asn Ya1 Pro
1 5 10 15
Thr
(2) INFORMATION FOR SEQ ID N0:12:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
G1n Pro I1e Asp Asp Thr G1u Ser Asn Thr Thr Ser Ya1 Asn Leu Pro
1 5 10 15
Thr
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1B amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
G1n Pro I1e Asp Asp Thr G1u Ser Asn Thr Thr Leu Ya1 Asn Ya1 Pro
1 5 10 15
61y A1a
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: Linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
WO 95/34666 219 2 9 4 2 PCT11~K95100249
37
G1n Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Ser Va1 Asn Leu Met
1 5 10 15
A1a Pro A1a Va1 A1a
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Gln Pro I1e Asp Asp Thr G1u Ser Asn Thr Thr Ser Val Asn Leu Met
1 5 10 15
Asp Leu A1a Va1 Gly Leu Pro G1y A1a
20 25
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
G1n Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Ser Va1 Asn Leu Met
1 5 10 15
Ala Asp Asp Thr Glu Ser I1e Asn Thr Thr Leu Yal Asn Leu Pro Gty
20 25 30
A1a
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
R'O 95/34666 . 219 2 9 4 2 p~~gg~p0249
38
G1n Pro I1e Asp Asp Thr G1u Ser I1e Asn Thr Thr Leu Ya1 Asn Leu
1 5 10 15
Pro Gly A1a
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
G1n Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Leu Yal Asn Leu Pro
1 5 10 15
G1y A1a
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
G1n Pro IIe Asp Asp Thr G1u Ser Asn Thr Thr Ser Ya1 Asn Leu Met
1 5 10 15
A1a Asp Asp Thr G1u Ser Arg Phe Ala Thr Asn Thr Thr Leu Ya1 Asn
20 25 30
Leu Pro Leu
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
WO 95/34666 219 2 9 4 2 PCT1DK95100249
39
i1n Pro I1e Asp 5sp Thr G1u Ser Asn Thr Thr Ser Va1 Asn Leu Met
15
A1a Asp Asp Thr G1u Ser I1e Asn Thr Thr Leu Ya1 Asn Leu A1a Asn
25 30
Ya1 A1a Met Ala
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(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
10 15
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
10 15
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
1 5 10 15
Met A1a Asp Asp Thr Glu Ser Arg Phe A1a Thr Asn Thr Thr Leu Ya1
20 25 30
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
1 5 10 15
Met A1a Asp Asp Thr G1u Ser Arg Phe A1a Thr Asn Thr Thr Leu Asp
20 25 30
Va1 Va1 Asn Leu Pro G1y A1a
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(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
1 5 10 15
Asn Leu Pro G1y Ala
(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 amino acids
2192942
WO 95/34666 PCT1DK95100249
41
(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
1 5 10 15
Ala Asp Asp Thr Glu Ser Arg Phe A1a Thr Asn Thr Thr Leu Va1 Asn
20 25 30
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
1 5 10 15
Ata Asp Asp Thr G1u Ser Arg Phe A1a Thr Asn Thr Thr Leu Asp Va1
20 25 30
Val Asn Leu I1e Ser Met A1a
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
G1n Pro IIe Asp Asp Thr G1u Ser Asn Thr Thr Ser Ya1 Asn Leu Met
1 5 10 15
Ata Asn Thr Thr G1u Ser Arg Phe A1a Thr Asn Thr Thr Leu Asp Va1
20 25 30
WO 95134666 2 ~ ~ 9 2 9 4 2 PCTI1DK95I00249
42
Ya1 Asn Leu I1e Ser Met Ata
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
TAAATCTATA ACTACAAAAA ACACATA 27
(2) INFORMATION FOR 5EQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
6ACTCTCTTA ACTGGCAAGT TGACA 25
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(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:
AAGTACAAAG CTTCAACCAA GTGAGAACCA CACAAGTGTT GGTTAACGAA TCTCTT 56
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
WO 95/34666 219 2 9 4 2 pCTIDK95100?A9
43
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
CATACACAAT ATAAACGACG G 21
(2) INFORMATION FOR SEQ ID N0:33:
(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:
GAATCTCTTA GCTGGCAAGT TGACAGAAGT AGTGTTAGTT TCAGAGTCGT CAATT 55
(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(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:
AACGAATCTC TTAGCACCTG GCAAGTTGAC AGAAGT 36
(2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(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:
AACGAATCTC TTAGCACCTG GCAAGTTGAC CAAAGTAGTG TTGATAGATT CAGTGTCGTC 60
(2) INFORMATION FOR SEQ ID N0:36:
S
W O 95Y34666 219 2 9 4 2 PCT~~~00249
44
(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:
AACGAATCTC TTAGCACCTG GCAAGTTAAC CAAAGTAGTG TTGATAGATT CAGTGTCGTC 60
AGCCATCAAG TTGAC 75
(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:
AACGAATCTC TTCAATGGCA AGTTAACCAA AGTAGTGTTA GTAGCGAATC TAGATTCAGT 60
6TCGTCAGCC AT ~ 72
(2) INFORMATION FOR SEQ ID N0:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
AACGAATCTC TTAGCCATGG CAACGTTAGC CAAGTTAACC AAAGT 45
(2) INFORMATION FOR SEQ ID N0:39:
(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:
AACGAATCTC TTAGCACCTG GCAAGTTGAC CAAAGTAGTG TTGATAGCAG ATTCAGTGTC 60
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
60
CAAACAAGAA
GATTACAAAC
TAAACGACGG GTACCAAAAT ATG AAACTGAAA ACTGTAAGATCT GCGGTC 111
A
Met LysLeuLys ThrYalArgSer A1aYa1
1 5 10
CTTTCG CTC TTTGCATCT CAGGTCCTT GGCCAACCAATA GACGAA 159
TCA
LeuSer Leu PheA1aSer GtnYa1Leu G1yGtnProIle AspG1u
Ser
15 20 25
GACAAC ACT TCTTCCATG GCTAAGAGA TTCGTTAACCAA CACTTG 207
GAC
AspAsn Thr SerSerMet AtaLysArg PheYa1AsnGtn HisLeu
Asp
30 35 40
TGCGGT CAC TTGGTTGAA GCTTTGTAC TTGGTTTGCGGT GAAAGA 255
TCC
CysGly His LeuVa1G1u A1aLeuTyr .LeuVa1CysG1y G1uArg
Ser
45 50 55
GGTTTC TAC ACTCCTAAG GCTGCTAAG GGTATTGTCGAG CAATGC 303
TTC
G1yPhe Tyr ThrProLys AtaAlaLys G1yI1eYa1G1u G1nCys
Phe
60 65 70
TGTACC ATC TGCTCCTTG TACCAATTG GAAAACTACTGC AACTAGACGCAGC
TCC
358
CysThr I1e CysSerLeu TyrG1nLeu G1uAsnTyrCys Asn
Ser
75 80 85 90
CCGCAGGCTC 372
TAGA
(2)INFORMATION FORSEQID :
N0:41
(i) CHARACTERIST ICS:
SEQUENCE
R'O 95134666 2 , ~ 9 2 9 4 2 PCT~~~I00249
46
(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
1 5 10 15
Ser G1n Ya1 Leu G1y G1n Pro I1e Asp Glu Asp Asn Asp Thr Ser Ser
20 25 30
Met Ala Lys Arg Phe Va1 Asn G1n His Leu Cys G1y Ser His Leu Ya1
35 40 45
G1u A1a Leu Tyr Leu Va1 Cys G1y G1u Arg G1y Phe Phe Tyr Thr Pro
5D 55 60
Lys A1a A1a Lys Gty I1e Val G1u G1n Cys Cys Thr Ser I1e Cys Ser
65 70 75 80
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:
CAA CCA ATT GAC GAC GAA AAC ACT ACT TCT GTC AAC TTG CCA GTT 45
Gln Pro I1e Asp Asp Gtu Asn Thr Thr Ser Ya1 Asn Leu Pro Va1
1 5 10 15
(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
47
(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
1 5 10 15
(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:
ATGAAACTGAAA GTAAGATCT GCGGTCCTT TCGTCACTCTTT GCA 48
ACT
MeiLysLeuLys Th Va1ArgSer AlaVa1Leu SerSerLeuPhe Ata
5 10 15
TCTCAGGTCCTT GGCCAACCAATT GACGACGAA AACACTACTTCT GTC 96
SerG1nVa1Leu GlyGlnProI1e AspAspGlu AsnThrThrSer Va1
20 25 30
AACTTGCCAGTT AAGAGATTCGTT AACCAACAC TTGTGTGGTTCT CAC 144
AsnLeuProVa1 LysArgPheYa1 AsnG1nHis LeuCysG1ySer His
35 40 45
TTGGTTGAAGCT TTGTACTTGGTT TGCGGTGAA AGAGGTTTCTTC TAC 192
LeuVa1G1uA1a LeuTyrLeuYat CysG1yG1u ArgGlyPhePhe Tyr
50 55 60
ACTCCTAAGGCT GCTAAGGGTATT GTCGAACAA TGCTGTACCTCC ATC 240
ThrProLysA1a A1aLysG1yIle YalGtuGtn GysCysThrSer I1e
65 70 75 80
TGCTCCTTGTAC CAATTGGAAAAC TACTGCAAC TAGACGCAGC 293
CCGCAGGGTC
CysSerLeuTyr G1nLeuG1uAsn TyrCysAsn
85 90
TAGA
297
(2)INFORMAT ION FORSEQID
N0:45:
( i) EQUENCE CHARACTE RISTICS:
S
(A) LENGTH:91 amino ids
ac
(B) TYPE: aci d
amino
W0 95134666 PCTNK95/00249
2192942
48
(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
1 5 10 15
Ser G1n Va1 Leu Gty Gtn Pro I1e Asp Asp G1u Asn Thr Thr Ser Ya1
20 25 30
Asn Leu Pro Va1 Lys Arg Phe Ya1 Asn Gln His Leu Cys G1y Ser His
35 40 45
Leu Va1 Gtu Ala Leu Tyr Leu Va1 Cys G1y G1u Arg G1y Phe Phe Tyr
50 55 60
Thr Pro Lys A1a A1a Lys G1y I1e Va1 G1u Gtn Cys Cys Thr Ser I1e
65 70 75 80
Cys Ser Leu Tyr G1n Leu G1u Asn Tyr Cys Asn
85 90
(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:
CAA CCA ATT GAC GAC ACT GAA TCT AAC ACT ACT TCT GTC AAC TTG CCA 48
G1n Pro I1e Asp Asp Thr Glu Ser Asn Thr Thr Ser Va1 Asn Leu Pro
1 5 10 15
GCT 51
A1a
(2) INFORMATION FOR SEQ ID N0:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: Linear
2192942
R'O 95134666 PC"lYDK95100249
49
(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
1 5 10 15
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:
CAA CCA ATT GAC GAC ACT GAA TCT AAC ACT ACT TCT GTC AAC TTG CCA 48
Gln Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Ser Yal Asn Leu Pro
1 5 10 15
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:
CAA CCA ATT GAC GAC ACT GAA TCT ATC AAC ACT ACT TTG GTC AAC TTG 48
61n Pro I1e Asp Asp Thr Glu Ser I1e Asn Thr Thr Leu Va1 Asn Leu
1 5 10 15
2x92942
WO 95134666 PCTIDK95100249
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:
CAA CCA ATT GAC GAC ACT GAA TCT AAC ACT ACT TCT GTC AAC TTG ATG 48
G1n Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Ser Vat Asn Leu Met
1 5 10 15
GCT GAC GAC ACT GAA TCT ATC AAC ACT ACT TTG GTT AAC TTG CCA GGT 96
Ata Asp Asp Thr G1u Ser I1e Asn Thr Thr Leu Va1 Asn Leu Pro G1y
20 25 30
GCT 99
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:
CAA CCA ATT GAC GAC ACT GAA TCT AAC ACT ACT TCT GTC AAC TTG ATG 48
G1n Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr Ser Va1 Asn Leu Met
1 5 10 15
WO 95134666 219 2 9 4 2 pCTIDK95100249
51
GCT GAC GAC ACT GAA TCT AGA TTC GCT ACT AAC ACT ACT TTG 6TT AAC 96
A1a Asp Asp Thr G1u Ser Arg Phe Ata Thr Asn Thr Thr Leu Ya1 Asn
20 25 30
TTG CCA TTG 105
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:
CAA CCA ATT GAC GAC ACT GAA TCT AAC ACT ACT TCT GTC AAC TTG ATG 48
G1n Pro I1e Asp Asp Thr G1u Ser Asn Thr Thr Ser Va1 Asn Leu Met
1 5 10 15
GCT GAC GAC ACT GAA TCT ATC AAC ACT ACT TTG GTT AAC TTG GCT AAC 96
A1a Asp Asp Thr G1u Ser I1e Asn Thr Thr Leu Ya1 Asn Leu A1a Asn
' 20 25 30
GTT GCC ATG GCT 108
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
52
CAA CCA ATT GAC GAC ACT GAA TCT GCT ATC AAC ACT ACT TTG GTC AAC 48
G1n Pro I1e Asp Asp Thr G1u Ser A1a Ite Asn Thr Thr Leu Yal Asn
1 5 10 15
TTG CCA GGT GCT 60
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:
TTAAATCTAT GGAATTCATT CAAGAATAGT 60
AACTACAAAA TCAAACAAGA
AACACATACA
AGATTACAAA ATAAACGACG GGTACCAAAA 115
CTATCAATTT TA
CATACACAAT ATG
Met
1
AAACTGAAAACTGTA TCT GTCCTT TCA CTCTTTGCA TCT 163
AGA GCG TCG
LysLeuLys Ya1 Ser Ya1Leu Ser LeuPheA1a Ser
Thr Arg A1a Ser
5 10 15
CAGGTCCTT CAA ATT GACGAA ACT ACTTCTGTT AAC 211
GGC CCA GAC AAC
GlnYa1Leu Gln I1e AspG1u Thr ThrSerYal Asn
G1y Pro Asp Asn
20 25 30
TTGCCAGCT AGA GTT CAACAC TGC GGTTCCCAC TTG 259
AAG TTC AAC TTG
LeuProA1a Arg Va1 G1nHis Cys GlySerHis Leu
Lys Phe Asn Leu
35 40 45
GTTGAAGCT TAC 276
TTG TT
YalGluA1a Tyr
Leu
50
(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
WO 95/34666 219 2 9 4 2 PCTIDK95100249
53
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:55:
Met Lys Leu Lys Thr Va1 Arg Ser Ala Yal Leu Ser Ser Leu Phe A1a
1 5 10 15
Ser G1n Ya1 Leu Gly G1n Pro I1e Asp Asp Gtu Asn Thr Thr Ser Ya1
20 25 30
Asn Leu Pro A1a Lys Arg Phe Yal Asn G1n His Leu Cys Gty Ser His
35 40 45
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
60
AACACATACA
GGAATTCATT
AGATTACAAA CTATCAATTT GGTACCAAAA
115
CATACACAAT TA
ATAAACGACG ATG
Met
1
AAACTG AAAACTGTA AGATCT GCG CTTTCGTCA CTCTTT6CATCT 163
GTC
LysLeu LysThrYa1 ArgSer A1a LeuSerSer LeuPheA1aSer
Va1
5 10 15
CAGGTC CTTGGCCAA CCAATT GAC ACTGAATCT AACACTACTTCT 211
GAC
G1nYal LeuG1yGln ProI1e Asp ThrGtuSer AsnThrThrSer
Asp
20 25 30
GTCAAC TTGCCAGCT AAGAGA TTC AACCAACAC TTGTGCGGTTCC 259
GTT
YalAsn LeuProA1a LysArg Phe AsnG1nHis LeuCysG1ySer
Ya1
35 40 45
CACTTG GTTGAAGCT TTGTAC TT 2g2
HisLeu Va1G1uA1a LeuTyr
50 55
(2)INFORMATION FOR SEQID N0:57:
W095134666 - - w-21 9 2 9 4 2 P~'~''~~s~~~'''9
1
54
(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
1 5 10 15
Ser Gtn Ya1 Leu G1y G1n Pro I1e Asp Asp Thr G1u Ser Asn Thr Thr
20 25 30
Ser Va1 Asn Leu Pro A1a Lys Arg Phe Ya1 Asn Gtn His Leu Cys G1y
35 40 45
Ser His Leu Va1 G1u A1a Leu Tyr
50 55
(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:
TTAAATCTAT AACTACAAAA AACACATACA GGAATTCATT CAAGAATAGT TCAAACAAGA 60
AGATTACAAA CTATCAATTT CATACACAAT ATAAACGACG GGTACCAAAA TA ATG 115
Met
1
AAA CTG AAA ACT GTA AGA TCT GCG GTC CTT TCG TCA CTC TTT GCA TCT 163
Lys Leu Lys Thr Ya1 Arg Ser A1a Ya1 Leu Ser Ser Leu Phe Ala Ser
10 15
CAG GTC CTT GGC CAA CCA ATT GAC GAC ACT GAA TCT AAC ACT ACT TCT 211
Gln Yai Leu Gly Gln Pro Ile Asp Asp Thr Gtu Ser Asn Thr Thr Ser
20 25 30
GTC AAC TTG ATG GCT AAG AGA TTC GTT AAC CAA CAC TTG TGC GGT TCC 259
Ya1 Asn Leu Met A1a Lys Arg Phe Ya1 Asn G1n His Leu Cys G1y Ser
- 35 40 45
WO 95/34666
219 2 9 4 2 PCTIDK95100249
CAC TTG GTT GAA GCT TTG TAC TT 282
His Leu Ya1 Glu A1a Leu Tyr
50 55
(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
10 15
Ser Gln Va1 Leu Giy G1n Pro I1e Asp Asp Thr Gtu Ser Asn Thr Thr
20 25 30
Ser Val Asn Leu Met A1a Lys A4g0 Phe Ya1 Asn G1n His Leu Cys G1y
35 45
Ser His Leu Ya1 G1u A1a Leu Tyr
50 55
(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:
TTAAATCTAT AACTACAAAA AACACATACA GGAATTCATT CAAGAATAGT TCAAACAAGA 60
AGATTACAAA CTATCAATTT CATACACAAT ATAAACGACG GGTACCAAAA TA ATG 115
Met
1
AAA CTG AAA ACT GTA AGA TCT GCG GTC CTT TCG TCA CTC TTT GCA TCT 163
Lys Leu Lys Thr Ya1 Arg Ser Ata Va1 Leu Ser Ser Leu Phe A1a Ser
5 10 15
WO 95134666 219 2 9 4 2 PCT~~5100249
i
56
CAGGTCCTTGGCCAA CCAATTGAC GACACTGAATCT AACACTACTTCT 211
GlnYa1LeuG1yG1n ProI1eAsp AspThrG1uSer AsnThrThrSer
2D 25 30
GTCAACTTGATGGCT GACGACACT GAATCTATCAAC ACTACTTTGGTT 259
Va1AsnLeuMetA1a AspAspThr G1u5erI1eAsn ThrThrLeuYa1
35 40 45
AACTTGCCAGGTGCT AAGAGATTC GTTAACCAACAC TTGTGCGGTTCC 307
AsnLeuProGtyA1a LysArgPhe YatAsnGtnHis LeuCysG1ySer
50 55 60 65
CACTTGGTTGAAGCT TTGTACTT 330
HisLeuVa1GtuA1a LeuTyr
70
(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
1 5 10 15
Ser G1n Yal~Leu G1y Gtn Pro Ite Asp Asp Thr G1u Ser Asn Thr Thr
20 25 30
Ser Ya1 Asn Leu Met A1a Asp Asp Thr Gtu Ser Ite Asn Thr Thr Leu
35 40 45
Va1 Asn Leu Pro G1y A1a Lys Arg Phe Ya1 Asn G1n His Leu Cys G1y
50 55 60
Ser His Leu Va1 G1u A1a Leu Tyr
65 70
(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
57
(B) LOCATION: 113..286
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:62:
TTAAATCTAT AACTACAAAA AACACATACA GGAATTCATT CAAGAATAGT TCAAACAAGA 60
AGATTACAAA CTATCAATTT CATACACAAT ATAAACGACG GGTACCAAAA TA ATG 115
Met
1
AAA CTG AAA ACT GTA AGA TCT GCG 6TC CTT TCG TCA CTC TTT GCA TCT 163
Lys Leu Lys Thr Va1 Arg Ser A1a Ya1 Leu Ser Ser Leu Phe A1a Ser
10 15
CAG GTC CTT GGC CAA CCA ATT GAC GAC ACT GAA TCT ATC AAC ACT ACT 211
61n Va1 Leu Gly G1n Pro I1e Asp Asp Thr G1u Ser Ile Asn Thr Thr
20 25 30
TTG GTC AAC TTG CCA GGT GCT AAG AGA TTC GTT AAC CAA CAC TTG TGC 259
Leu Ya1 Asn Leu Pro G1y A1a Lys Arg Phe Va1 Asn G1n His Leu Cys
35 40 45
GGT TCC CAC TTG GTT GAA GCT TTG TAC TT 2gg
G1y Ser His Leu Ya1 61u A1a Leu Tyr
50 55
(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
1 5 10 15
Ser Gln Yal Leu G1y Gln Pro Ile Asp Asp Thr G1u Ser I1e Asn Thr
20 25 30
Thr Leu Va1 Asn Leu Pro G1y A1a Lys Arg Phe Ya1 Asn Gtn His Leu
35 40 45
Cys G1y Ser His Leu Ya1 G1u A1a Leu Tyr
50 55
(2) INFORMATION FOR SEQ ID N0:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 82 base pairs
W095134666 ' ' " PCTIDK95100?A9
2192942
58
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D} TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:64:
GGTTAACGAA CTTTGGAGCT TCAGCTTCAG CTTCTTCTCT CTTAGCCATG GAGATCAAGT 60
TAACAACATC CAAAGTAGTG TT B2
(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:
CAAGTACAAA GCTTCAACCA AGTGGGAACC GCACAAGTGT TGGTTAACGA ACTT 54
(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:
CAACCAATTG ACGACACTGA ATCTAACACT ACTTCTGTCA ACTTGATGGC TGACGACACT 60
GAATCTAGAT TCGCTACTAA CACTACTTTG GATGTTGTTA ACTTGATCTC CATGGCT 117
(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
59
iln Pro I1e Asp 5sp Thr G1u Ser Asn Thr Thr Ser Va1 Asn Leu Met
15
A1a Asp Asp Thr G1u Ser Arg Phe A1a Thr Asn Thr Thr Leu Ata Leu
25 30
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:
TCTCTTAGCC ATGGAGATCA AGTTAACAAC ATCCAAAGCC AAAGTAGTGT T 51
(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:
CAACCAATTG ACGACACTGA ATCTAACACT ACTTCTGTCA ACTTGATGGC TGACGACACT 60
GAATCTAGAT TCGCTACTAA CACTACTTTG GCTTTGGATG TTGTTAACTT GATCTCCATG 120
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
1 5 10 15
His Leu Cys Gly Ser His Leu Vat Glu A1a Leu Tyr Leu Va1 Cys Gty
20 25 30
G1u Arg G1y Phe Phe Tyr Thr Pro Lys A1a A1a Lys G1y I1e Va1 61u
35 40 45
Gln Cys Cys Thr Ser I1e Cys Ser Leu Tyr Gln Leu G1u Asn Tyr Cys
50 55 60
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:
AAGAGAGAAG AAGCTGAAGC TGAAGCTGAA CCAAAGTTCG TTAACCAACA CTTGTGTGGT 60
TCTCACTTGG TTGAAGCTTT GTACTTGGTT TGCGGTGAAA GAGGTTTCTT CTACACTCCT 120
AAGGCTGCTA AGGGTATTGT CGAACAATGC TGTACCTCCA TCTGCTCCTT GTACCAATTG 180
GAAAACTACT GCAACTAGAC GCAGCCCGCA GGCTCTAGA 219
(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:
TTAAATCTAT AACTACAAAA AACACATACA GGAATTCCAT TCAAGAATAG TTCAAACAAG 60
AAGATTACAA ACTATCAATT TCATACACAA TATAAACGAC GGTACCAAAA TAATGAAACT 120
6AAAACTGTA AGATCTGCGG TCCTTTCGTC ACTCTTTGCA TCTCAGGTCC TTGGCCAACC 180
2192942
WO 95/34666 PCTIDK95100249
61
AATTGACGACACTGAATCTAACACTACTTCTGTCAACTTGATGGCTGACG ACACTGAATC240
TAGATTCGCTACTAACACTACTTTGGTTAACTTGGCTAACGTTGCCAACC AACACTTGTG300
TGGTTCTCACTTGGTTGAAGCTTTGTACTTATGGCTAAGAGATTCGTT 348
(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:
TTAAATCTATAACTACAAAAAACACATACAGGAATTCCATTCAAGAATAGTTCAAACAAG 60
AAGATTACAAACTATCAATTTCATACACAATATAAACGACGGTACCAAAATAATGAAACT 120
GAAAACTGTAAGATCTGCGGTCCTTTCGTCACTCTTTGCATCTCAGGTCCTTGGCCAACC 180
AATTGACGACACTGAATCTAACACTACTTCTGTCAACTTGATGGCTGACGACACTGAATC 240
TAGATTCGCTACTAACACTACTTTGGATGTTGTTAACTTGATCTCCATGGCTAAGAGAGA 300
AGAAGCTGAAGCTGAAGCTGAACCAAAGTTCGTTAACCAACACTTGTGTGGTTCTCACTT 360
GGTTGAAGCTTTGTACTTG 37g
